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53rd Symposium of the Society for Histochemistry
Current Role of Histochemistry
in Preclinical and Clinical Research
12 – 15 October 2011 · Munich · Germany
Table of Contents 1
Table of Contents
Table of Contents................................................................................... 1
Welcome Message ................................................................................. 2
Society for Histochemistry .................................................................... 3
Scientific Program Overview .............................................................. 13
Scientific Program ............................................................................... 17
Abstracts of Plenary Lectures .............................................................. 21
Posters .................................................................................................. 66
Abstracts of Poster Presentation .......................................................... 71
General Information .......................................................................... 133
Access to Venues ............................................................................... 136
Social Events ..................................................................................... 139
Exhibitors and Sponsors .................................................................... 140
Map of Exhibition Area ..................................................................... 142
Welcome Message
2
Dear Members of the Society for Histochemistry,
Dear Scientists and Colleagues,
On behalf of the Organizing Committee we are delighted to welcome you to the 53rd
Symposium of the Society for Histochemistry 2011. The Symposium will bring
together leading international experts and provide a critical mass in the field of
molecular histology, molecular medicine and cell biology. Join us to enjoy great
science and Munich, one of the most beautiful cities in Germany.
The 53rd Symposium of the Society for Histochemistry will provide insight into the
Current role of Histochemistry in Preclinical and Clinical Research. The “tissue is the
issue” remains an important principle for further progress in a frontier research in
molecular medicine, molecular and cell biology and pathology. The conference will
focus on latest morphology-based methodological developments as well as state-of-
the-art technology and applications in research and diagnostics. Particular emphasis
will be on Imaging Mass Spectrometry in biomedical imaging and in life science
research.
We are confident that you will enjoy the Scientific Program of this international
symposium.
Just enjoy it!
Axel Walch Ioannis Mylonas
Local organizer Local organizer
Treasurer of the Society for Histochemistry
Society for Histochemistry
3
The Society for Histochemistry is an international association of scientists which was
founded in 1952. Membership of the Society is open to scientists of all countries. The
Society organizes annual scientific symposia, sponsors the Robert-Feulgen Lecture
and awards the Robert-Feulgen Prize. The activities of the Society are connected with
histochemistry and all related fields, especially cell and tissue biology, molecular
biology, pathology, anatomy, and microscopy. The official journal of the Society is
Histochemistry and Cell Biology.
President: Marco Biggiogera
Symposium Scientific Committee
Local Organizers
Axel Walch
Ioannis Mylonas
Local Organizing Committee
Ansgar Brüning
Annette Feuchtinger
Heinz Höfler
Ioannis Mylonas
Sandra Rauser
Axel Walch
http://histochemistry.eu/index.html
Marco Biggiogera Pavia, Italy
Paul Debbage Innsbruck, Austria
Heinz Höfler Munich, Germany
Pavel Hozák Prague, Czech Republic
Ioannis Mylonas Munich, Germany
Axel Walch Munich, Germany
Society for Histochemistry
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Why to become a Member of the Society
Prestigious prizes, awards and stipends as well as competitive student packages
for attending symposia of the Society.
Personal subscription to the Society’s official "Histochemistry and Cell
Biology" at only ten percent of the regular rate. Based on the ISI-impact factor,
"Histochemistry and Cell Biology" is the leading journal in the field.
Interactions with senior scientists resulting in multiple career opportunities.
Selected top scientists in the field can be met and contacts and discussions with
them are encouraged in the informal atmosphere of the symposia.
Symposia are conveniently scheduled (usually in September) at reasonable
prices.
Besides oral presentations by top scientists in the field, the program of the
symposia allows for selected presentations on the basis of submitted abstracts.
"Free communications" and presentation of posters receive ample attention at
the symposia and are considered to be important elements of the symposia.
Access to the "members only" section of the Society´s highly informative
homepage (http://www.histochemistry.eu).
Annual Newsletter for members only.
An annual membership fee of only 25 EUR, which entitles a reduction of the
registration fee for the symposia of the Society.
Society for Histochemistry
5
List of Robert Feulgen Lectures
2011 Munich, Germany
Richard M. Caprioli, Nashville, TN, USA
Molecular imaging of tissue sections by mass spectrometry: Providing information
beyond the microscope
2010 Prague, Czech Republic
Stefan W. Hell, Göttingen, Germany
Nanoscopy with focused light
2009 Fulpmes, Austria
P. J. Peters, Amsterdam, The Netherlands
Cellular organelles as nanomachines
2008 Interlaken, Switzerland
K. Takata, Maebashi, Japan
Localization and trafficking of aquaporin 2 in the kidney
2007 Freiburg, Germany
M. Frotscher, Freiburg, Germany
New ways of looking at synapses
2006 Stresa, Lake Maggiore Italy
D. Hernandet-Verdun, Paris, France
The nucleolus: a model for the organization of nuclear functions
2005 Noordwijkerhout, Netherland
M. Dahan, Paris, France
From analog to digital: exploring cell dynamics with single quantum dots
Society for Histochemistry
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2004 Prague, Czech Republic
St. Fakan, Lausanne, Switzerland
The functional architecture of the nucleus as analyzed by ultrastructural cytochemistry
2003 Les Diablerets, Switzerland
A. Engel, Basel, Switzerland
Structure and function of membrane channels
2002 Vlissingen, The Netherlands
T. Misteli, Bethesda, MD, USA
New views of the cell: Genomics, proteomics and dynamic networks
2001 Vienna, Austria
R. G. W. Anderson, Dallas, TX, USA
Caveolae spatially organize signal transduction at the cell surface
2000 Les Diablerets, Switzerland
J. Lippincott-Schwartz, Bethesda, MD, USA
Dynamic fluorescence imaging of living cells
1999 Gargellen, Austria
A. Willie, Cambridge, UK
Apoptosis in the genesis and treatment of cancer
1998 Giessen, Germany
D. Vestweber, Münster, Germany
Molecular mechanisms that control leukocyte extravasation
1997 Jena, Germany
K. Simons, Heidelberg, Germany
Biogenesis of a polarized cell surface in epithelial cells
Society for Histochemistry
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1996 Gargellen, Austria
M. Trendelenburg, Heidelberg, Germany
Novel insights into the nucleolar structural complexity and function
1995 Rigi-Kaltbad, Switzerland
D. Shotton, Oxford, UK
Electronic light microscopy: Past, present, future
1994 Heidelberg, Germany
M. J. Karnovsky, Boston, MA, USA
Cytochemistry and oxy radicals
1993 Gargellen, Austria
S. Rosen, San Francisco, CA, USA
L-selection and its endogenous ligands
1992 Munich, Germany
G. Klein, Stockholm, Sweden
The contribution of encogenes and tumor supressor genes to the multistep
development of cancer
1991 Ghent, Belgium
J. E. Dumont, Brusels, Belgium
The surface receptors in the model of the thyroid cell
1990 Gargellen, Austria
M. N. Moore, Plymouth, UK
Environmental distress signals: cellular reactions to marine pollution
1989 Gargellen, Austria
W. W. franke, Heidelberg, Germany
The intermediate filament cytoskeleton and its association with other structures
Society for Histochemistry
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1988 Gargellen, Austria
L. I. Larsson, Copenhagen, Denmark
Cytochemical detection of regulatory peptides and of mRNA molecules coding for
peptide precursors
1987 Basel, Switzerland
W. J. Gehring, Basel, Switzerland
The generation of the body plan as studied by in situ hybridization in the developing
embryo
1986 Gargellen, Austria
G. C. Bennet, Montreal, Canada
Radioautographic and cytochemical studies of the synthesis and intracellular transport
of glycoproteins
1985 Göttingen, Germany
K. Weber, Göttingen, Germany
Cytoskeletal proteins: structure, function, pathology
1984 Maastricht, The Netherlands
I. B. Black, New York, USA
Phenotypic plasticity in the nervous system
1983 Gargellen, Austria
A. G. E. Pearse, London, UK
The phylogeny of the diffuse neuroendocrine system
1982 Gargellen, Austria
W. E. Stumpf, Chapel Hill, NC, USA
Histochemical characteristics and significance of cell receptors in biology and
pathology
Society for Histochemistry
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1981 Münster, Germany
G. Pfefferkon, Münster, Germany
Histochemische Analyse mit Licht - und Elektronenstrahlen
1980 Würzburg, Germany
L. A. Sternberger, Rochester, NY, USA
Immunocytochemistry - Past, present, future
1979 Gargellen, Austria
O. Eränkö, Helsinki, Finland
Histochemical observations on the distribution of catecholamines and catecholamine-
synthesizing enzymes in the nerve cells and SIF cells of the sympathetic ganglion
Society for Histochemistry
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List of Robert Feulgen Prize Laureates
2010
H. de Wit
Amsterdam, The Netherlands
2009
P. F. Lenne
Marseille, France
2008
B.N.G. Giepmans
Groningen, The Netherlands
2007
A. Pombo
London, United Kingdom
2006
P. J. Verschure
Amsterdam, The Netherlands
M. Nilsson
Uppsala, Sweden
2005
Th. Misgeld
Cambridge, USA
2003
J. Priller
Berlin, Germany
2002
F.J. Iborra
Oxford, UK
2001
J. Lippincott-Schwartz
Bethesda, MD, USA
2000
K. König
Jena, Germany
1999
E.J. Speel
Zürich, Switzerland
1997
Eveline Baumgart
Heidelberg, Germany
1996
R.W. Dirks
Leiden, The Netherlands
1995
M. Thiry
Liege, Belgium
1994
J. Oberdick
Columbus, OH, USA
1993
J.-L. Carpentier
Genève, Switzerland
Society for Histochemistry
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1992
C.R. Green
London, UK
N.J. Severs
London, UK
1991
M.P. Bachmann
Mainz, Germany
1990
W.J. Streit
Gainsville, FL, USA
1989
R. Gebhardt
Tübingen, Germany
D.J. Taatjes
Burlington, VT, USA
1988
S. Thanos
Tübingen, Germany
1987
J.E. Scott
Manchester, UK
C.F.J. Van Noorden
Amsterdam, The Netherlands
R.G. Butcher
London, UK
1986
J. Gerdes
Berlin, Germany
1985
P. Brandtzaeg
Oslo, Norway
1984
M. Bendayan
Montreal, Canada
1983
J.G.J. Bauman
Leiden, The Netherlands
1982
J. Roth
Genève, Switzerland
1981
A.C. Cuello
Oxford, UK
1980
H.F. Teutsch
Freiburg/Br., Germany
1979
H. Korr
Würzburg, Germany
Society for Histochemistry
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1978
G. Moyne
Villejuif, France
1977
P.T. Kjellstrand
Lund, Sweden
1976
W.D. Kuhlmann
Heidelberg, Germany
1975
D. Grube
Heidelberg, Germany
1974
J. Winckler
Frankfurt a. M., Germany
1973
G. Thiessen
Hannover, Germany
1972
J. Nolte
Freiburg/Br., Germany
D. Pette
Konstanz, Germany
Scientific Program Overview
13
Scientific Program Overview
Wed
nesd
ay
14:00 - 18:30 Registration desk open
17:30 - 18:30 Welcome drink
18:30 - 19:00 Symposium opening, awarding the Young Histochemist Travel Awards
19:00 - 19:45 The Robert Feulgen lecture: Richard M. Caprioli (USA)
19:45 Get Together
Th
ursd
ay
08:30 - 10:10 Workshop I: MALDI Imaging for protein analysis of tissues - Part 1
08:30 -08:50 Bernhard Spengler (Germany)
08:50 - 09:10 Michael Volný (Czech Republic)
09:10 - 09:30 Fred Hamprecht (Germany)
09:30 - 09:50 Peter Maaß (Germany)
09:50 - 10:10 Andreas Römpp (Germany)
10:10 - 10:25 Coffee break
10:25 - 12:05 Workshop II: Drug imaging in tissues and living systems
10:25 - 10:45 Jens Siveke (Germany)
10:45 - 11:05 Jonathan Stauber (France)
11:05 - 11:25 Peter Marshall (United Kingdom)
11:25 - 11:45 Brendan Prideaux (Switzerland)
11:45 - 12:05 Walter Stumpf (USA)
12:05 - 13:00 Lunch break
13:00 - 14:20 Workshop I: MALDI Imaging for protein analysis of tissues - Part 2
13:00 - 13:20 Isabelle Fournier (France)
13:20 - 13:40 Mareike Elsner (Germany)
13:40 - 14:00 Liam McDonnell (The Netherlands)
14:00 - 14:20 Malcolm Clench (United Kingdom)
14:20 - 16:00 Poster session with refreshments
16:00 - 17:00 Industry Workshop: Bruker Daltonik GmbH
16:00 - 16:15 Sören-Oliver Deininger (Bruker Daltonik GmbH)
16:15 - 16:30 Kristina Schwamborn (Germany)
16:30 - 16:45 Benjamin Balluff (Germany)
16:45 - 17:00 Detlev Suckau (Bruker Daltonik GmbH)
17:00 - 17:45 Keynote Lecture: Gerd Binnig (Nobel Laureate, Germany)
17:45 - 18:45 Business meeting
Scientific Program Overview
14
09:00 - 11:45 Workshop III: Ultrastructural studies in biomedicine
Frid
ay
09:00 - 09:20 Margit Pavelka (Austria)
09:20 - 09:40 Pavel Hozák (Czech Republic)
09:40 - 10:00 Marco Biggiogera (Italy)
10:00 - 10:20 Jürgen Roth (South Korea)
10:20 - 10:45 Coffee break
10:45- 11:05 Marlene Thaler (Carl Zeiss NTS GmbH)
11:05 - 11:25 Paul Debbage (Austria)
11:25 - 11:45 Ben Giepmans (The Netherlands)
11:45 - 13:00 Lunch break
13:00 Transfer to Helmholtz Zentrum München - Registration obligatory! - Meeting point: Registration desk Frauenklinik Maistraße
14:00 - 17:00 parallel sessions
Guided tour at Helmholtz Zentrum München
- Registration obligatory! -
Industry Workshop (Carl Zeiss NTS GmbH)
- Registration obligatory! -
17:00 Transfer to Frauenklinik Maistraße Meeting point: Lecture room 052 / building 57
19:00 Symposium banquet - Ratskeller München
09:00 - 10:40 Workshop IV: Novel techniques in histochemistry, pathology and microscopy
Satu
rd
ay
09:00 - 09:20 Vasilis Ntziachristos (Germany)
09:20 -09:40 Olga Ilina (The Netherlands)
09:40 - 10:00 Savvas Damaskinos (Intas Science Imaging & HuronTechnologies)
10:00 - 10:20 Ralf Schönmeyer (Definiens AG)
10:20 - 10:40 Jan-Erik Heil (Carl Zeiss NTS GmbH)
10:40 - 11:00 Coffee break
11:00 - 12:20
parallel sessions Workshop V: Microscopy and
image analysis - DNA/RNA/protein analyses of tissues
Industry Hands-On Workshop
(Intas Science Imaging & Huron Technologies) lecture hall 1+2
Immunhistochemistry or Fluorescence
probes can be scanned in that workshop
- Registration obligatory! -
11:00 - 11:20 Niels Grabe (Germany)
11:20 - 11:40 Stefanie Hauck (Germany)
11:40 - 12:00 Michaela Aubele (Germany)
12:00 - 12:20 Sibylle Gündisch (Germany)
12:20 - 13:00 Lunch
13:00 - 14:20 Free topics communication
13:00 - 13:20 Stefan Maier (Germany)
13:20 - 13:40 Julia Hess (Germany)
13:40 - 14:00 Gudrun C. Thurner (Austria,Young Histochemist Travel Award)
14:00 - 14:20 Rémi Longuespée (France, Young Histochemist Travel Award)
14:20 - 14:40 Closing of the Symposium and Poster Awards
Scientific Program
15
Scientific Program
Wednesday, 12 October 2011
14:00 - 18:30 Registration desk open
17:30 - 18:30 Welcome drink
18:30 - 19:00 Symposium opening, awarding the Young Histochemist Travel
Awards
19:00 - 19:45 The Robert Feulgen lecture
(Chair: Marco Biggiogera)
Richard M. Caprioli (USA): Molecular imaging of tissue sections by mass spectrometry: Providing information beyond
the microscope
19:45 Get Together
Thursday, 13 October 2011
08:30 - 10:10 Workshop I: MALDI Imaging for protein analysis of tissues - Part 1
(Chair: Bernhard Spengler)
08:30 - 08:50 Bernhard Spengler (Germany): High resolution in mass and
space: AP-MALDI FTMS imaging of biological tissue
08:50 - 09:10 Michael Volný (Czech Republic): Visualization of Small Molecules in Tissues by Laser Desorption Ionization Mass
Spectrometry Imaging
09:10 - 09:30 Fred Hamprecht (Germany): Automated analysis of (MS)
images
09:30 - 09:50 Peter Maaß (Germany): MALDI imaging: clustering, super-
resolution and applications
09:50 - 10:10 Andreas Römpp (Germany): A new dimension of histological information: Highly specific molecular imaging at cellular
resolution
10:10 - 10:25 Coffee break
Scientific Program
16
10:25 - 12:05 Workshop II: Drug imaging in tissues and living
systems (Chairs: Isabelle Fournier / Walter Stumpf)
10:25 - 10:45 Jens Siveke (Germany): MALDI molecular imaging for translational approaches in pancreatic cancer
10:45 - 11:05 Jonathan Stauber (France): Quantification of drug candidates by Label free Mass Spectrometry Imaging
11:05 - 11:25 Peter Marshall (United Kingdom): Combining Histology and
MALDI-Mass Spectrometry Imaging in Drug Discovery
11:25 - 11:45 Brendan Prideaux (Switzerland): Mass spectrometry imaging: A powerful tool in drug discovery and development
11:45 - 12:05 Walter Stumpf (USA): Drugs in the brain - cellular imaging and discoveries with receptor microscopic autoradiography
12:05 - 13:00 Lunch break
13:00 - 14:20 Workshop I: MALDI Imaging for protein analysis of
tissues - Part 2
(Chair: Richard M. Caprioli)
13:00 - 13:20 Isabelle Fournier (France): MALDI imaging mass
spectrometry for studying cancer diseases
13:20 - 13:40 Mareike Elsner (Germany): MALDI imaging reveals mitochondrial dysfunction as strong predictor for response to
neoadjuvant chemotherapy in Barrett's Cancer
13:40 - 14:00 Liam McDonnell (The Netherlands): Differentiating between morphologically identical tumors using imaging MS-based
molecular histology
14:00 - 14:20 Malcolm Clench (United Kingdom): MALDI-MS imaging and
profiling of pancreatic and stomach cancer tissue microarrays
14:20 - 16:00 Poster session with refreshments
16:00 - 17:00 Industry Workshop: Bruker Daltonik GmbH
16:00 - 16:15 Sören-Oliver Deininger (Bruker Daltonik GmbH): Tools for MALDI imaging based histology and data interpretation
16:15 - 16:30 Kristina Schwamborn (Germany): Prostate Cancer - What
can we learn from MALDI imaging
Scientific Program
17
16:30 - 16:45 Benjamin Balluff (Germany): MALDI imaging mass
spectrometry in gastric cancer
16:45 - 17:00 Detlev Suckau (Bruker Daltonik GmbH): Strategies for
identification and characterization of Protein biomarkers for MALDI imaging studies
17:00 - 17:45 Keynote Lecture
(Chairs: Axel Walch / Ioannis Mylonas)
Gerd Binnig (Nobel Laureate, Germany): Present and Future Impact of Image Analysis and Image Mining on Histochemistry
17:45 - 18:45 Business meeting
Friday, 14 October 2011
09:00 - 11:45 Workshop III: Ultrastructural studies in biomedicine
(Chairs: Margit Pavelka / Jürgen Roth)
09:00 - 09:20 Margit Pavelka (Austria): Fine structural 3D-analyses of
cellular reorganizations induced by metabolic stress
09:20 - 09:40 Pavel Hozák (Czech Republic): Nanoparticle-based
Immunocytochemistry reveals PIP2-containing microarchitecture of the cell nucleus
09:40 - 10:00 Marco Biggiogera (Italy): Tracing the movement of mRNA towards the nuclear pore
10:00 - 10:20 Jürgen Roth (South Korea): Microscopic analysis of protein misfolding diseases and the effect of therapeutic synthetic
chaperones
10:20 - 10:45 Coffee break
10:45- 11:05 Marlene Thaler (Carl Zeiss NTS GmbH): Energy Filtering TEM
applications in medical diagnostics
11:05 - 11:25 Paul Debbage (Austria): Ultrastructure and drug delivery:
gateways and barriers
11:25 - 11:45 Ben Giepmans (The Netherlands): Correlated microscopy and
nanotomy to analyze Islets of Langerhans in Type I diabetes
11:45 - 13:00 Lunch break
Scientific Program
18
13:00 Transfer to Helmholtz Zentrum München Meeting point: Registration desk Frauenklinik Maistraße - Registration obligatory! -
14:00 - 14:30 Lecture room 052
Introduction to the Helmholtz Zentrum München (Christian Langebartels, Department Program Planning and
Management) - Registration obligatory! -
14:30 - 17:00 - Registration obligatory! -
parallel sessions
Guided tour at Helmholtz Zentrum
München
Industry Workshop
(Carl Zeiss NTS GmbH)
German Mouse Clinic (Helmut Fuchs) Introduction to Energy Filtering
TEM" - Live practical session on the microscope
Research Unit Medical Radiation Physics and
Diagnostics (Christoph Hoeschen)
Genome Analysis Center (Jerzy Adamski)
17:00 Transfer to Frauenklinik Maistraße Meeting point: Lecture building 57/room 052 (Helmholtz Zentrum
München)
19:00 Symposium banquet - Ratskeller München
Saturday, 15 October 2011
09:00 - 10:40 Workshop IV: Novel techniques in histochemistry, pathology and microscopy
(Chairs: Vasilis Ntziachristos / Pavel Hozák)
09:00 - 09:20 Vasilis Ntziachristos (Germany): Illuminating biomedical discovery with advanced photonic imaging
09:20 - 09:40 Olga Ilina (The Netherlands): Tissue structures guiding
collective breast carcinoma invasion
09:40 - 10:00 Savvas Damaskinos (Intas Science Imaging & Huron Technologies): Scanning Laser Imaging of Macroscopic
Samples
10:00 - 10:20 Ralf Schönmeyer (Definiens AG): Automated co-analysis of MALDI- and H&E-images of retinal tissue for an
improved spatial MALDI-resolution
10:20 - 10:40 Jan-Erik Heil (Carl Zeiss NTS GmbH): Superresolution
Technologies from Carl Zeiss: Fluorescence Imaging beyond the Resolution Limit
Scientific Program
19
10:40 - 11:00 Coffee break
11:00 - 12:20 Workshop V Industry Hands-On
Workshop parallel sessions
11:00 - 12:20 Workshop V: Microscopy and image analysis - DNA/RNA/protein analyses of tissues
(Chair: Heinz Höfler)
11:00 - 11:20 Niels Grabe (Germany): Towards systems-pathology by integrating whole-slide imaging, high-dimensional data and
biological networks
11:20 - 11:40 Stefanie Hauck (Germany): Quantitative Tissue Proteomics for Deciphering Molecular Processes in
Autoimmune Uveitis
11:40 - 12:00 Michaela Aubele (Germany): In situ quantification of protein-protein complexes in paraffin-sections using
Proximity Ligation Assay (PLA)-technique
12:00 - 12:20 Sibylle Gündisch (Germany): Evaluation of PAXgene-fixed, paraffin-embedded tissues for morphological and
molecular analysis
11:00 - 12:20 parallel in lecture
hall 1+2
Industry Hands-On Workshop (Intas Science Imaging & Huron Technologies):
“Laser Confocal Slide Scanning System”
Scan your small or big Tissue section with the unique TissueScope4000 - Laser Confocal Slide Scanning System.
Slide Dimensions: 25x75mm up to 150x200mm Immunhistochemistry or Fluorescence probes can be
scanned in that workshop. - Registration obligatory! -
12:20 - 13:00 Lunch
Scientific Program
20
13:00 - 14:20 Free topics communication
(Chair: Marco Biggiogera)
13:00 - 13:20 Stefan Maier (Germany): Building the MALDI Imaging Protein Biomarker List from the Bottom Up
13:20 - 13:40 Julia Hess (Germany): Gain of chromosome band 7q11 in papillary thyroid carcinomas of young patients is associated
with exposure to low-dose irradiation
13:40 - 14:00 Gudrun C. Thurner (Austria, Young Histochemist Travel
Award): MRI molecular imaging with targeted albumin-based nanoparticles: conceptual design strategies to create
the Magic Bullet
14:00 - 14:20 Rémi Longuespée (France, Young Histochemist Travel Award): MALDI MSI for ovarian cancer biomarkers
research: latest developments of the technology for screening and tracking
14:20 - 14:40 Closing of the Symposium and Poster Awards
Abstracts of Plenary Lectures
21
Abstracts of Plenary Lectures
Molecular imaging of tissue sections by mass spectrometry: Providing information beyond the microscope Caprioli R.M.
1
1Vanderbilt University, Biochemistry, Nashville, United States
Imaging MALDI MS (matrix-assisted laser desorption ionization mass spectrometry)
produces molecular images of peptides, proteins, lipids and metabolites present in
intact tissue sections. It employs desorption of molecules by direct laser irradiation to
map the location of specific molecules from fresh frozen and formalin fixed tissue
sections without the need of target specific reagents such as antibodies. Molecular
maps can be directly correlated to known histological regions within the tissue. A high
density of spots (pixels) ablated by the laser over the entire tissue produces many
hundreds of molecular images or density maps with spatial resolutions from 5-200
microns. Images are produced in specific m/z (mass-to-charge) values, or ranges of
values, typically covering the MW range 1000-100,000. Individual m/z values derived
from each pixel can then be assembled to produce selected molecular images.
Similarly, the approach has also been applied to a protocol termed histology-directed
molecular analysis whereby only selected areas of cells in the tissue are of interest are
ablated and analyzed based on studies performed by microscopy and other histology
protocols. Both fresh frozen and formalin fixed tissues can be analyzed. The
technology is extraordinarily high throughput with high molecular specificity, easily
lending itself to the analysis of tissue microarrays. Sections obtained from any tissue
type can be imaged, including sections through whole organs or animals.
We have employed Imaging MS in studies of a variety of diseases, including several
types of cancers, neurodegenerative diseases and kidney diseases, comparing proteins
differentially expressed in diseased tissue with those in the corresponding normal
tissue. From such comparisons, molecular signatures are developed that differentiate
these tissues, typically consisting of 10-20 or more different proteins. Imaging MS has
been applied to drug targeting and metabolic studies following drug administration.
Whole animal sagittal sections have been imaged to measure molecular changes in
Abstracts of Plenary Lectures
22
proteins in multiple organs and correlating this with drug concentrations in these same
organs. 3-D images may also be generated from serial tissue sections after registration
and volume rendering.
This presentation will focus on recent technological advances both in sample
preparation and instrumental performance to achieve images at high spatial resolution
and at high speeds so that a typical sample tissue (e.g., a whole mouse section) can be
imaged in less than 10 min. Some selected examples will include studies of tumor
bearing tissues and normal developmental processes in mouse. Other aspects of the
technology, such as 3-D imaging, will also be discussed.
High resolution in mass and space: AP-MALDI FTMS imaging of
biological tissue Spengler B.
1, Römpp A.
1, Günther S.
1, Schulz O.
1, Köstler M.
1, Bouschen W.
1, Leisner
A.1, Hinz K.-P.
1
1Justus Liebig University, Analytical Chemistry, Giessen, Germany
MALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1],
has only recently turned into a routine method of highest performance for the
molecular histology of biological tissue [2]. The method, providing high resolution in
mass and space, has been developed for a reliable identification and localization of
individual tissue components, and has been applied to a number of research areas in
pathology, cancer diagnostics, metabolic pathway analysis and plant research.
Targeted compound classes include phospholipids, peptides, proteins, drug compounds
and metabolites.
The home-built atmospheric pressure ion source is based on a dedicated microoptical
setup, designed for highest optical resolution under mass analytical sampling
conditions, providing a spatial resolution on tissue of 3 to 5 micrometer. The ion
source is coupled to a Fourier transform mass spectrometer (ion cyclotron resonance or
orbital trapping), at a mass accuracy of better than 1 ppm RMS. Ion images can be
acquired with an acquisition speed of more than 2 pixels per second. The presentation
will describe technical principles of the new instrumentation and its availability,
including examples for imaging biomolecules in mammalian tissue and plant tissue at
Abstracts of Plenary Lectures
23
high spatial resolution in relation to classical histological staining exeriments.
Combining high spatial resolution down to 3 µm with high mass resolution, high mass
accuracy, high imaging selectivity, MSn capability and an improved sample
preparation technology resulted in an unprecedented quality of imaging data in our
experiments. It shows that imaging mass spectrometry in particular requires highly
accurate raw data from FT instruments, while low accuracy data contain a high risk of
creating non-reliable, invalid, non-authentic or ambiguous information in imaging
experiments of complex biological tissue samples. Fundamental and technical aspects
of high performance UV/IR MALDI imaging, its properties and its prospects will be
described, including optical focusing, ion formation and transfer at atmospheric
pressure, image acquisition, data analysis and substance identification.
The high specificity in mass and space of the method results in an unprecedented
information quality and depth which can be advantegeous in a large number of
clinical, pharmacological or fundamental applications in the future.
References:
[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion
Imaging with a new Scanning UV-Laser Microprobe, Proceedings of the 42nd Annual
Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3,
1994, pp 1041.
[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B.
Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization
Obtained from High-Accuracy Bioanalytical Imaging. Angewandte Chemie
International Edition. 49(22): p. 3834-3838.
Abstracts of Plenary Lectures
24
Visualization of small molecules in tissues by laser desorption
ionization mass spectrometry imaging Strohalm M.
1, Pól J.
1, Faltýsková H.
1, Novák P.
1, Havlíček V.
1, Vidová V.
1, Volný
M.1,2
1Academy of Sciences of the Czech Republic, Institute of Microbiology, Prague, Czech
Republic, 2University of Washington, Department of Chemistry, Seattle, United States
Laser Desorption Ionization (LDI) Mass Spectrometry Imaging (MSI) can be used to
investigate the spatial distribution of lipids and other small molecules in tissue
sections. In LDI MSI, compounds are ionized directly from the tissue slice, which is
attached on a conductive surface moving in set raster steps under a laser beam. Mass
spectra collected from each point within the raster are used to assemble the molecular
images in correlation with their x- and y- positions. In matrix assisted LDI (MALDI),
which is the most common variant of the LDI experiment, the tissue surface is
uniformly coated by the deposition of a matrix aerosol, which facilitates desorption
and ionization. The coating process is computer controlled to avoid excessive over-
wetting, which would deteriorate the spatial distribution of the molecules in the tissue.
While the presence of matrix is essential for ionization of proteins, it can be sometimes
avoided for small molecules if specially designed surfaces and/or special sample
treatment procedures are used. Ions desorbed from the tissue are accelerated into the
mass spectrometer for separation based on the mass-to-charge ratios (m/z) of the ions
and detected. Sampling of the ions directly from the tissue into the mass spectrometer
provides very rich mass spectra with respect to the number of ions with different m/z,
so a high resolution mass analyzer is often an advantage.
In the presented work LDI MSI images were acquired using APEX Ultra 9.4 T FT-
ICR mass spectrometer or Ultraflex III TOF/TOF mass spectrometer (both Bruker
Daltonics, Germany). Both standard mass spectrometers were upgraded to be equipped
with a Smart beam laser (200 Hz). The molecular images of the analytes were
analyzed using an open source program mMass (www.mMass.org) and visualized by
the FlexImaging software. Lipid Maps database (www.lipidmaps.org ) was used for
identification
Abstracts of Plenary Lectures
25
Three main examples of LDI MSI applications for tissue imaging are presented and
discussed 1) In situ detection of globotriaosylceramide (GL3) by LDI MSI in a murine
lysosomal alpha-Gal A gene knock-out (Fabry disease model). The nature of the
accumulation in the mouse model is investigated by the determination of the spatial
distribution of GL3 in kidney tissue sections from wild-type and Fabry mice.
Individual isoforms can be distinguished by MSI and overall distribution is compared
with the antibody staining. 2) Visualization of spatial distribution of
glycerophospholipids and sphingomyelins in the ocular lenses 3) Imaging of
phospholipids and other small molecules in tissue imprints by LDI-MSI without
matrix addition.
Acknowledgment: The work was supported by the Czech Science Foundation
(Project P206/10/P018). M.V.´s research was supported by a MC IR Grant within the
7th EC Framework Program. Other support was provided by the Ministry of
Education, Youth, and Sports of the Czech Republic (MSMT LC07017, LC545 and
ME10013).
Automated analysis of (MS) images
Hamprecht F.A.1
1Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany
I will present ilastik, the Interactive Learning and Segmentation Toolkit. ilastik is a
user-friendly tool for image classification and segmentation in up to three spatial and
one spectral dimension which requires no experience in image processing.
In its basic form, ilastik uses local image or spectral features along with user
annotations to train a nonlinear classifier that allows to distinguish and predict an
arbitrary number of classes. The program provides real-time feedback of the current
classifier predictions and thus allows for targeted training and overall reduced labeling
time. Once the classifier has been trained on a representative subset of the data, it can
be exported and used to automatically process a very large number of images.
Recent developments include a framework that allows to combine modularized
operators to sophisticated workflows. Functionality such as unsupervised learning
Abstracts of Plenary Lectures
26
(probabilistic latent semantic analysis, pLSA) are particularly useful for MS images
which can now be read in the Analyze 7.5 format.
Besides MS images, I will demonstrate application to high throughput screening data
and to the segmentation of 3D neural tissue data.
ilastik is open source and available from http://ilastik.org
MALDI imaging: clustering, super-resolution and applications Alexandrov T.
1, Maass P.
2, Thiele H.
3, Trede D.
1
1Steinbeis Innovation Center for Scientific Computing in Life Sciences, Bremen,
Germany, 2University of Bremen, Center for Industrial Mathematics, ZeTeM, Bremen,
Germany, 3Bruker Daltonik GmbH, Bremen, Germany
Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry
(MALDI-imaging) is a label-free technology used for spatial molecular analysis. Over
the last decade MALDI Imaging has enjoyed a tremendous development. By now
MALDI Imaging is regarded as a most promising innovative measurement technology
in biochemistry, pharmaceutical research and medical applications.
Nevertheless, the information content of a MALDI Imaging data set has not yet been
fully exploited. This is particularly the case for 3D MALDI Imaging applications. Due
to the high experimental costs and due to the enormous computational challenges
posed by the shear size of full 3D MALDI Imaging data sets, this field of application
has not seen much progress beyond a recent proof-of-principle, where a 3D model
corresponding to a single m/z-value was reconstructed from consecutive slices and co-
registered.
Even the most basic task of discriminating different sample areas with distinctly
different molecular structure requires highly efficient and robust computational
methods, which moreover need to be adapted to the medical task under investigation.
We will discuss the potential and limitations of advanced computational methods for
spatial segmentation of MALDI-imaging data sets. In addition we propose a
computational tool for improving the resolution in visualizing MALDI Imaging
results. We will highlight the procedures with 2D applications (rat brain coronal
Abstracts of Plenary Lectures
27
section highlighting anatomical and functional structures of the brain; neuroendocrine
tumor invading the small intestine) as well as first results for 3D spatial segmentation
of a real-life 3D MALDI-imaging data set .
A new dimension of histological information: Highly specific
molecular imaging at cellular resolution Roempp A.
1, Guenther S.
1, Schober Y.
1, Takats Z.
1, Spengler B.
1
1Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry,
Giessen, Germany
Mass spectrometry imaging (MS imaging) has become a method of outstanding
importance in life sciences. The information content of the generated MS images
greatly depends on the quality of the underlying mass spectral data. Mass
spectrometers with high mass resolution are routinely used in many bioanalytical
areas, but only few applications were reported for MS imaging so far. Here we applied
a mass spectrometry imaging method that combines accurate mass measurements and
spatial resolution in the low micrometer range for the analysis of biological tissue.
These experiments were performed with a home-built atmospheric-pressure imaging
source attached to a 'LTQ Orbitrap' or 'Exactive Orbitrap' mass spectrometer (Thermo
Scientific GmbH, Bremen) [1]. All measurements were done with a mass accuracy
better than 3 ppm (root mean square), i.e. compounds were identified with high
confidence. MS images were generated with a bin size of Δm/z = 0.01, which largely
eliminates interferences from neighboring peaks.
Phospholipids, peptides and drug compounds were imaged in a wide range of
biological samples at an effective spatial resolution of 5 to 10 µm. Tissue types of
mouse urinary bladder were discriminated at the cellular level based on molecular
information. The internal structure and surrounding area of metastases in human brain
sections were characterized by their phospholipid pattern. This provided structural
features which were not visible in the histological staining experiments.
Lysophospholipids were specifically detected in the necrotic zone of lung carcinoma.
Phospholipids are less specific than proteins, but their pattern can be used to
distinguish different tumor types and also to determine primary tumors form
Abstracts of Plenary Lectures
28
measurement of the metastases. Tissue sections were stained (H&E or toluidine) after
MS analysis. MS image analysis for all these cases showed excellent agreement with
histological structures.
MS imaging of proteins remains a challenging task. Direct detection and identification
of proteins on tissue is limited by a number of factors including limited mass range
and fragmentation efficiency as well as incompatibility with formalin-fixed samples.
On-tissue digestion of proteins and detection of the resulting peptides can overcome
some of these limitations. In the current study we used a spraying device to apply
trypsin on tissue sections. With this setup we were able to obtain a spatial resolution of
50 µm for tryptic peptides. Initial experiments were done on mouse brain sections, but
the method has also been applied to human tumor tissue in the meantime.
In all experiments the high resolution and mass accuracy proved to be essential for
specific image generation and reliable identification of analytes. This significantly
improves the quality of molecular and structural information that can be obtained from
tissue sections.
[1] Römpp et al. (2010) Angewandte Chemie International Edition 49(22): 3834.
MALDI imaging mass spectrometry for marker discovery and drug
imaging in pancreatic cancer
Siveke J.1, Grüner B.
1, Winkelmann I.
2, Hahne H.
3, Esposito I.
4, Maier S.
3,
Feuchtinger A.2, Trajkovic-Arsic M.
1, Mazur P.
1, Rauser S.
2, Schmid R.
1, Küster B.
3,
Walch A.2
1II. Med. Klinik, Klinikum rechts der Isar der TU München, Munich, Germany,
2Institute of Pathology, Helmholtz Center Munich, Helmholtz Center Munich, Munich,
Germany, 3Department of Bioanalytics, Technical University of Munich, Munich,
Germany, 4Institute of Pathology, Technische Universität München, Munich, Germany
Pancreatic ductal adenocarcinoma (PDAC) remains a fatal disease despite tremendous
therapeutic efforts. The devastating clinical course is due to late detection and high
chemoresistance of PDAC. Recent evidence points to inefficient drug delivery as an
important contributor to the insensitivity of PDAC to therapy. Here we demonstrate
Abstracts of Plenary Lectures
29
the applicability of MALDI IMS for (i) diagnostic marker discovery in preneoplastic
lesions and (ii) drug distribution imaging using genetically engineered mice (GEM)
with endogenous PDAC.
For detection of potentially meaningful biomarkers of early neoplastic disease, various
GEM of PDAC were analyzed by MALDI IMS to investigate the peptide/protein-
expression pattern of preneoplastic lesions in comparison to normal pancreas, chronic
pancreatitis (CP) and PDAC with cellular resolution. Statistical analysis revealed
several discriminative m/z-species between normal and diseased tissue. Proteins from
candidate m/z-species were identified using liquid chromatography and tandem mass
spectrometry (LC-MS/MS). Further validation of two proteins was performed in
murine and human tissue and serum. In conclusion we show that GEM of endogenous
PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS
analysis, allowing in situ analysis of small precursor lesions and identification of
potentially meaningful preneoplastic biomarkers.
In a second approach we utilized MALDI IMS for drug imaging in PDAC. In MALDI
imaging of drug molecules and metabolites one faces the challenge to measure the
intensity of targeted drug signals in a complex background of matrix signals and
endogenous metabolites. The necessary specificity in such an analysis is usually
achieved by the measurement of a specific fragment of the targeted drug in MS/MS
mode (single reaction monitoring SRM). For in tissue analysis of drug molecules and
metabolites, we established and evaluated a novel sensitive and robust MS-based assay
(FAST-SRM) for the small molecule tyrosine kinase inhibitor erlotinib, which targets
the EGF receptor and is an approved therapy in pancreatic and lung cancer. FAST-
SRM measurement of erlotinib and des-methyl-erlotinib demonstrated detection of
both drugs with spatial resolution and an unambiguous correlation of the signal
intensity with the administered dose. Interestingly, signal intensity was lower in PDAC
than in normal exocrine tissue and preneoplastic lesions. In conclusion, the FAST-
SRM mode described here is suitable to analyze drugs and metabolites in tissue. It is
easy to set up and turns fast reflectron MALDI-TOF instruments such as the autoflex
speed into universal imaging instruments. The obtained results support the hypothesis
Abstracts of Plenary Lectures
30
that poor drug delivery may be an important contributor to poor chemotherapeutic
efficacy in PDAC.
Quantification of drug candidates by Label free Mass Spectrometry
Imaging
Hamm G.1, Bonnel D.
1, Piveteau C.
2, Willand N.
2, Deprez B.
2, Delbos J.M.
3, Bouzom
F.3, Stauber J.R.
1
1IMABIOTECH, Villeneuve d'Ascq, France,
2INSERM U761, Biostructures and Drug
Discovery, Lille, France, 3SERVIER TECHNOLOGIES, Orléans, France
Unlike traditional imaging techniques such as autoradiography, magnetic resonance
imaging or positron emission tomography, mass spectrometry imaging (MSI) permits
the label-free study of several compounds of interest simultaneously on the same tissue
section. However, the difficulty of obtaining an absolute quantification of
experimental data remains one of MSI's major disadvantages. This quantification
requires reproducible and homogenous measurements (signal), as well as a calibration
curve with minimal variability and increased linearity. Several methods addressing this
issue are described in literature, but none have universal applications.
Using different examples of drug distribution studies on whole-body samples, we
report a new methodology intended to respond to the main obstacles in quantification
through MALDI imaging. ANd we compare the results of quantitative mass
spectrometry to others gold standards methods such as QWBA (quantitative Whole
body autoradiography) and LC-MS².
Combining histology and MALDI-Mass spectrometry imaging in drug discovery Marshall P.S.
GlaxoSmithKline, Stevenage, United Kingdom
The use of MALDI-MS Imaging in the drug discovery process serves as a valuable
tool to investigate normal and diseased tissues and allows for the visualisation of
Abstracts of Plenary Lectures
31
compound distribution in tissue. The technique is routinely used to determine the
spatial distribution of small molecules (drugs and metabolites) and larger
biopharmaceuticals molecules. The information obtained can add an extra dimension
of understanding to the location of compound in the selected tissue.
However, drug distribution profiles remain “just a pretty picture” unless the data is
correlated with the histological features of the tissue section examined. To demonstrate
this important aspect examples will be shown of the direct overlay of histology data
with MALDI MS imaging data performed on the same section. The presentation will
illustrate how this combined data forms an integral part of the drug discovery process
and provides a greater insight into pharmacological and toxicological findings of a
compound or drug.
Mass spectrometry imaging: A powerful tool in drug discovery and
development Prideaux B.
1, Staab D.
1, Morandi G.
1, Ehrhard N.
1, Stoeckli M.
1
1Novartis Institutes for BioMedical Research, Analytical Sciences, Basel, Switzerland
Mass spectrometric imaging (MSI) technologies enable label-free spatial analysis of
biological tissue samples. Since MSI bases the detection of the analytes on the
molecular weight and/or specific fragmentation pattern, it offers the possibility to
simultaneously measure compound and metabolite distribution without the
requirement to label the drug. In addition, the potential to simultaneously acquire
thousands of endogenous species of interest during the same experiment such as lipids,
peptides and proteins opens up the possibility of simultaneous monitoring of drug,
metabolites and markers of both disease and therapeutic response.
Application of MSI technologies to biomedical research will be discussed with a
particular focus upon contributions to drug discovery and development. Examples
from applications including absorption, distribution, metabolism and excretion
(ADME), pharmacokinetics, toxicology and biotherapy will be presented highlighting
the unique value offered to drug discovery in disease research areas such as
tuberculosis, oncology, acne, phototoxicity and animal health.
Abstracts of Plenary Lectures
32
Drugs in the brain - cellular imaging and discoveries with receptor
microscopic autoradiography
Stumpf W.E.
University of North Carolina, Chapel Hill, NC, United States
For cell and tissue localization of drugs, receptor microscopic autoradiography is
reviewed, including its development history, multiple testing, extensive applications
and significant discoveries. This sensitive high-resolution imaging method is based on
the use of radiolabeled compounds (esp. tagged with 3H or
125I), preservation through
freezing of in vivo localization of tissue constituents, cutting thin frozen sections, and
close contact with the recording nuclear emulsion.
After extensive testing of the utility of this method, the distribution of radiolabeled
compounds has been identified and characterized for estradiol, progestagens, adrenal
steroids, thyroid hormone, ecdysteroids, vitamin D, retinoic acid, metabolic indicators
glucose and 2-deoxyglucose, as well as extracellular space indicators. Target cells and
associated tissues have been characterized with special stains, fluorescing compounds,
or combined autoradiography-immunocytochemistry with antibodies to dopamine-
beta-hydroxylase, GABA, enkephalin, specific receptor proteins, or other cellular
products. Blood-brain barrier and brain entries via capillary endothelium, ependyma,
or circumventricular recess organs have been visualized for 3H-dexamethasone,
210Pb
lead, and 3
H-1,25(OH)2 vitamin D3.
Through this histopharmacologic approach, cellular details and tissue integrative
overviews can be assessed at the same time in the same preparation. As a result,
information has been gained that would have been difficult or impossible otherwise.
Maps of brain drug distribution have been developed and relevant target circuits have
been recognized. Examples include, the stria terminalis that links septal-amygdaloid-
thalamic-hypothalamic structures and telencephalic limbic system components which
extend as the periventricular neuroendocrine system into the mid- and hindbrain.
Discoveries challenged existing paradigms, engendering new concepts and providing
seminal incentives for further research toward understanding drug actions. Most
notable are discoveries made during the 1980s of over 50 target tissues of vitamin D
(reviewed 1995 in Histochem. Cell Biol.) that went beyond systemic calcium
Abstracts of Plenary Lectures
33
regulation and revealed strong nuclear receptor binding in specific cell populations in
the brain and spinal cord (rendered negative in biochemical radioassays), pituitary,
adrenal, skin, and multiple other tissues.
Such unexpected findings have been instrumental in recognizing the life sustaining
role of this 'sunshine hormone' (soltriol), beyond effects on bone growth and repair.
The vitamin D nuclear receptor data, reviewed in specific brain maps and in a
systemic-holistic 'Drug Homunculus ', are a source of important information for
biochemical and clinical follow-up in drug development. This methodology has been
crucial in enabling vitamin D-related CNS prophylaxis and therapies for multiple
sclerosis, parkinsonism, depression, memory loss, sleeping problems, neuroendocrine
disorders and more.
MALDI imaging mass spectrometry for studying cancer diseases Longuespée R.
1, Boyon C.
1, Vinatier D.
2, Salzet M.
1, Fournier I.
1
1University Lille, Fundamental and Applied Biological Mass Spectrometry - EA 4550,
Villeneuve d'Ascq Cedex, France, 2Hôpital Jeanne de Flandre, Clinique
Gynécologique, Lille, France
Early diagnostic and disease management is one of the most important challenges
facing modern medicine, which is particularly relevant in cancer. The lack of effective
assays measuring multiple blood-based biomarkers is absent in many types of cancer.
Moreover, transforming a biomarker into a useful clinical diagnostic test is a complex
process, which starts with identification, proceeds through validation. Identification
can be carried out by various means (gene arrays, purification procedures, proteomics),
that focus on observed changes of the marker correlated with the disease progression,
either in the solid tumour or in a body fluid. As the markers are identified within
extracts or in a non-spatial context, further validation is always required. Several
choices are then available, such as establishing specific antibodies, using protein
microarrays or including more refined techniques such as tissue laser micro-dissection.
A major new alternative that combines both biomarker identification and validation in
a single step is now possible at the tissue level with the development of MALDI Mass
Abstracts of Plenary Lectures
34
Spectrometry Imaging. In a single experiment, molecular information on hundreds of
molecules can be retrieved. By automation of this method and data processing,
molecular maps of compounds can be generated from tissue sections. The obvious
advantage of having the spatial localization of identified compounds is the predictive
potential of which markers are most likely to be successful at the clinical level.
MALDI-MSI is a non-targeted analysis, but due to its high data acquisition, permits
the establishment of a classification of cell phenotypic changes at the molecular level,
which can be used in complement to histology techniques. The correlation between
molecular images obtained by MALDI-MSI and the ones obtained by pathologists
using classical histochemistry can be inclusive of all grades, stages, cancer types, and
cell types. However, differently from classical histochemistry.
In this context, we present here data obtained on ovarian cancer using such a
technology. Many questions are being raised about the potential mechanisms of
ovarian cancer origin and progression. Our findings reflect that a global molecular
profile is more associated with the pathological states observed in endometrioid or
serous ovarian cancers compared to the benign stage, based on principal component
analysis (PCA) and Hierarchical clustering (HC) analyses of MALDI MS profiling
studies. We also identified several biomarkers related to immune response modulation
at early stages of the disease like the Cter part of PA28, “Reg Alpha” and HLA-G.
Based on our data we investigate more deeply the serous ovarian cancer origin, its
escape and invasion strategies. These data open the door of therapeutic strategy based
on collected data obtains by MALDI MSI.
Abstracts of Plenary Lectures
35
MALDI imaging reveals mitochondrial dysfunction as strong
predictor for response to neoadjuvant chemotherapy in Barrett's
Cancer
Elsner M.1, Rauser S.
1, Aichler M.
1, Ludyga N.
1, Maier S.
1, Balluff B.
1, Schöne C.
1,
Meding S.1, Sarioglu H.
2, Feuchtinger A.
1, Langer R.
3, Feith M.
4, Küster B.
5, Ueffing
M.2, Höfler H.
1,3, Walch A.
1
1Helmholtz Zentrum München, Institute of Pathology, Neuherberg, Germany,
2Helmholtz Zentrum München, Department of Protein Science, Neuherberg, Germany,
3Technische Universität München, Institute of Pathology, München, Germany,
4Klinikum rechts der Isar, Technische Universität München, Department of Surgery,
München, Germany, 5Technische Universität München, Chair of Proteomics and
Bioanalytics, München, Germany
Aims: In esophageal adenocarcinoma (Barrett's Cancer), neoadjuvant chemotherapy
improves survival in a subgroup of patients. To avoid unnecessary therapy, there is a
need for markers that predict therapy response. In order to find proteomic markers,
MALDI imaging was performed on pretherapeutic Barrett's Cancer biopsies.
Methods: Pretherapeutic endoscopic Barrett's Cancer biopsies (n=23) were obtained
during primary staging. After two cycles of cisplatin and 5-fluoroouracil therapy,
response to treatment was assessed in each patient. The tissue samples were measured
by MALDI imaging and proteomic profiles were correlated with the response data.
Candidate species discriminating between responders and non-responders were
identified by LC-MS/MS. Predictive impact of the mitochondrial protein COX7A2
was validated on an independent set of pretherapeutic, endoscopic biopsies (n=54).
Subsequently, the correlation between COX7A2 expression and mitochondria was
evaluated by electron microscopy.
Results: MALDI imaging revealed 22 m/z species correlating with therapy response in
Barrett's Cancer (p< 0.05). Hierarchical clustering showed that MALDI imaging
profiles could be used to accurately define responders to neoadjuvant therapy from
non-responders. By LC-MS/MS, a signature of 4 mitochondrial proteins with reduced
expression in responders was identified. Immunohistochemistry of the most
discriminating protein COX7A2 in the validation set confirmed the MALDI imaging
Abstracts of Plenary Lectures
36
results and revealed the predictive impact of COX7A2 (p=0.003). Electron microscopy
showed a destruction of mitochondria in cases with reduced-expression of COX7A2.
Conclusion: By MALDI imaging, a novel signature of mitochondrial proteins was
found, which plays a role in response to neoadjuvant chemotherapy in Barrett's
Cancer. Furthermore, a correlation between reduced expression of mitochondrial
proteins, mitochondrial dysfunction and therapy response could be shown for the first
time.
Differentiating between morphologically identical tumors using imaging MS-based molecular histology Jones E.A.
1, Waaijer C.
2, Schmitz N.
2, van Remoortere A.
1, van Zeijl R.J.
1, Deelder
A.M.1, Bovee J.V.M.G.
2, Mc Donnell L.A.
1
1Leiden University Medical Center, Department of Parasitology, Leiden, Netherlands,
2Leiden University Medical Center, Department of Pathology, Leiden, Netherlands
Introduction: Matrix assisted laser desorption/ionization mass spectrometry
(MALDI-MS) can generate profiles directly from tissue that contain hundreds of
distinct biomolecular ions. Spatially-correlated analysis, imaging MS, can
simultaneously reveal how the intensity of each of these biomolecular ions varies in
tissue samples. There is growing evidence that imaging MS is having an impact in
disease detection, particularly cancer. The differential profiles found in tumors can be
used to identify specific candidate biomarkers. One of the advantages of imaging MS
is that it can differentiate regions of tissue based on their MS profiles and thereby
distinguish biomolecularly different regions even if they are not distinct using
established histological tools.
Here we demonstrate how imaging MS-based molecular histology can help
differentiate between morphologically identical chondrosarcomas. Chondrosarcoma
are a heterogeneous group of bone tumors. The most common subtypes, conventional
central and secondary peripheral, are morphologically identical but exhibit different
clinical behavior, have different genetic backgrounds and different treatment targets
have been identified. They are largely insensitive to conventional chemo- and radio-
Abstracts of Plenary Lectures
37
therapy, and so surgery is the mainstay of treatment. It is thus vital to identify markers
that confidently differentiate central from peripheral chondrosarcoma.
Methods: MALDI-Imaging MS of proteins was performed using an Autoflex
MALDI-ToF and sinapinic acid as matrix. Automated feature identification and
extraction reduced the data load by >300X enabling a series of multivariate analyses to
be performed to identify the regions of tissue consistently highlighted as molecularly
distinct.
Results: Five central and five peripheral grade II chondrosarcoma samples, identical
morphology and grade, were analyzed. Pair-wise analyses revealed that the tumors
could be differentiated using a variety of multivariate methods. However, significant
intratumor heterogeneity led to variability in the MS features that differentiated
between the tumors.
Automated data reduction of the entire ten-tissue dataset enabled, for the first time,
molecular histology of the patient series, and thus allowed three sources of variance to
be distinguished.
intratumor variability within each patient tissue
inter-patient variability within each tumor class
variability between central and peripheral chondrosarcoma.
Variable stroma and intense blood-related protein signals from a subset of tissues
undermined initial unsupervised separation. Reducing the weight of these signals
enabled the entire patient series to be correctly assigned, and highlighted twelve
peptide and proteins biomarkers.
Conclusion: Imaging MS based molecular histology helped identify the first
biomarkers that distinguish between morphologically identical chondrosarcomas.
Abstracts of Plenary Lectures
38
MALDI-MS imaging and profiling of pancreatic and stomach cancer
tissue microarrays Djidja M.-C.
1, Turner A.J.
1, Scriven P.
2, Claude E.
3, Cole L.M.
1, Clench M.
1
1Sheffield Hallam University, Biomedical Research Centre, Sheffield, United Kingdom,
2The University of Sheffield, Academic Surgical Oncology Unit, University of
Sheffield, Sheffield, United Kingdom, 3Waters Corporation, Manchester, United
Kingdom
The development of tissue micro array (TMA) technologies provides insights into
high-throughput analysis of proteomics patterns from a large numbers of archived
tumour samples. In the work reported here, matrix assisted laser desorption/ionisation-
ion mobility separation-mass spectrometry (MALDI-IMS-MS) profiling and imaging
methodology has been used to visualise the distribution of several peptides and
identify them directly from TMA sections after on-tissue tryptic digestion. A novel
approach that combines MALDI-IMS-MSI and principal component analysis-
discriminant analysis (PCA-DA) is described, which has the aim of generating tumour
classification models based on protein profile patterns. The molecular classification
models obtained by PCA-DA have been validated by applying the same statistical
analysis to other tissue cores and patient samples. The ability to correlate proteomic
information obtained from samples with known and/or unknown clinical outcome by
statistical analysis is of great importance, since it may lead to a better understanding of
tumour progression and aggressiveness and hence improve diagnosis, prognosis as
well as therapeutic treatments. The selectivity, robustness and current limitations of
the methodology are discussed.
Tools for MALDI imaging based histology and data interpretation:
Deininger S.
Bruker Daltronik GmbH, Bremen, Germany
Often times in powerpoint presentations or papers on MALDI imaging the correlation
of some histological data with MALDI images is shown. What does not become clear
in this context is how the correlation is actually done: We will show in a live demo
Abstracts of Plenary Lectures
39
how the flexImaging software handles the integration of microscopic data into the
MALDI imaging workflow on selected examples. We will also show the benefit of the
implementation of new statistical methods for simplifying the analysis of MALDI
imaging data.
Prostate Cancer - What can we learn from MALDI imaging
Schwamborn K.1,2
, Wild P.3, Caprioli R.
2
1Technische Universität München, Institute of Pathology, Munich, Germany,
2Vanderbilt University, Department of Biochemistry, Nashville, United States,
3University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland
Prostate cancer (PCa) is the most common cancer in men in the United States and
Germany. Although highly curable at an early stage the overall death toll remains high
due to recurrence of “cured” cases and progression to hormone-refractory and/ or
metastatic disease. Notwithstanding the sizeable number of cancer-related deaths, only
a small proportion of cancers will progress to a life-threatening disease. Elucidating
changes at the protein level involved in PCa progression would provide invaluable
information. Imaging mass spectrometry (IMS) applied to prostate cancer tissue
biopsies enables the visualization of the spatial distribution of cancer specific
protein/peptide expression profiles in correlation with histological features that allow
disease staging and prediction of outcome.
Formalin-fixed and paraffin-embedded prostate samples (normal, 69; PCa, 506;
hormone-refractory PCa, 49 and metastases, 23) were subjected to on-tissue tryptic
digestion. Briefly, sections were mounted onto conductive glass slides and underwent
paraffin removal well as antigen retrieval. On-tissue digestion was achieved by
spotting trypsin onto the tissue in an array pattern using a Portrait 630 reagent multi-
spotter. Following digestion, CHCA was spotted directly onto the array of tryptic
spots. Samples were analyzed utilizing an Ultraflextreme MALDI-TOF/TOF mass
spectrometer. Additionally, MS/MS measurements of selected peptides were acquired.
Data analysis was performed by using the ClinProTools 2.2 and FlexImaging 2.1
software.
Abstracts of Plenary Lectures
40
On-tissue tryptic digestion of prostate tissue revealed on average 210 high abundance
peptides in the mass range from m/z 900-4000. When analyzing spectra from cancer
regions in comparison to spectra from regions without cancer distinctive differences in
peak patterns could be identified. For example, from the cancerous prostate regions
peptides detected at m/z 1515.64, 1570.82 and 2798.11 were at significantly higher
expression. Similarly, peptides from the non-cancer regions at m/z 2087.88 and
2402.79 were also seen with significantly higher abundances. Combining 23 peaks in a
support vector machine based model resulted in an overall cross validation of 95%
with a sensitivity of 99.3% and a specificity of 90.6% in the training set. Classification
of spectra from localized PCa and hormone-refractory PCa samples could be achieved
by combining 6 peaks in a genetic algorithm based model resulting in an overall cross
validation of 84.9% with a sensitivity of 79.3% and a specificity of 90.4%. The tryptic
peptide at m/z 1515.64 could be identified as a peptide from actin. Another trptic
peptide at m/z 2798.11 was sequenced as a peptide from tubulin beta. Further
identification of differentially expressed peptides and validation of these initial
promising findings on an independent second sample set are ongoing and could
facilitate the discovery of proteins involved in the progression of PCa.
MALDI imaging mass spectrometry in gastric cancer
Balluff B.1,2
, Ebert M.3, Walch A.
1
1Institute of Pathology, Helmholtz Zentrum München - German Research Center for
Environmental Health, Neuherberg, Germany; 2Department of Medicine II, Klinikum
rechts der Isar, Technische Universität München, Munich, Germany, 3 Department of
Medicine II, Universitätsklinikum Mannheim, University of Heidelberg, Mannheim,
Germany
Histology-driven matrix-assisted laser desorption/ionization (MALDI) imaging mass
spectrometry allows measuring the molecular content of tissues. We used MALDI
imaging in pre-clinical studies for the identification of clinical relevant proteins in
terms of prognosis and therapy response in gastric cancer patients. The results
Abstracts of Plenary Lectures
41
highlight the usefulness of MALDI imaging for providing novel and clinical relevant
information from tumor tissues, as well as its potential for tissue diagnostics.
Strategies for identification and characterization of Protein
biomarkers for MALDI imaging studies.
Suckau D.
Bruker Daltronik GmbH, Bremen, Germany
To identify the marker proteins that were discovered in clinical MALDI imaging
studies is crucial for their validation, e.g., by IHC, and for the further clinical use. We
present established procedures that provide access to protein identities that can be used
for both a) intact protein identification from fresh frozen tissue, and b) digest based
protein identification compatible with the use of FFPE tissue
Present and future impact of image analysis and image mining on
histochemistry Binnig G.
Definiens AG, München, Germany
The increasing spread of digital image acquisition hardware such as whole tissue slide
scanners provides the basis for a growing relevance of image analysis. Through
advanced context based image analysis complex images can be evaluated and objects
and structures therein can be quantified. Some of those derived data are not or hardly
accessible through the human visual system. Examples are multispectral
investigations, comprehensive analyses of a bundle of different or of multidimensional
images and the quantification of a large number of relevant objects. In particular the
local co-expression analysis of several immunohistochemical slides is nearly
impossible by visual inspection but conveniently achievable through image analysis of
co-registered tissue slides. The co-registration by itself is of high value and only
feasible in digital pathology in a meaningful way and enriched by sophisticated image
analysis opens the door for novel kind of investigations.
Abstracts of Plenary Lectures
42
Whereas image analysis provides rich useful information, image mining goes a step
further by extracting knowledge from large collections of images. Image analysis is
part of image mining and the more sophisticated the image analysis is the more
valuable the derived knowledge will be. The amount of data statistically analyzed in
image mining can be huge and therefore can only be handled by a computer. Very
valuable are correlations of health-related data of individuals with image based data.
Fine structural 3D-analyses of cellular reorganizations induced by metabolic stress Meisslitzer-Ruppitsch C.
1, Ranftler C.
1, Vetterlein M.
1, Ellinger A.
1, Neumüller J.
1,
Pavelka M.2
1Medical University of Vienna, Vienna, Austria,
2Medical University of Vienna, Cell
Biology and Ultrastructure Research, Vienna, Austria
Aims: The work presents 3D-analyses of fine structural changes in cells responding to
ATP-depletion and aims to receive improved insight into mechanisms cells use to
manage metabolic stress.
Methods: In cultured HepG2 hepatoma and endothelial cells, a reduction of the ATP-
pool was achieved by replacing in the culture media D-glucose by its non-
hydrolyzable analogue 2-deoxy-D-glucose (1,2). Peroxidase-labelled wheat germ
agglutinin (WGA) and BODIPY-ceramide (Cer) internalized into the cells prior to or
after ATP-depletion were visualized by oxidation and photooxidation of
diaminobenzidine (DAB), respectively. Three-dimensional analyses were performed
by electron tomography of 200-300nm thick sections of the Epon-embedded cell
cultures in a 200kV transmission electron microscope (Tecnai-20, FEI). For
reconstruction, the Inspect 3D software (FEI), the IMOD software, or the
“Tom_Release_2008 toolbox” software (kindly provided by W.Baumeister and
J.Plitzko, MPIB Martinsried) were used. The models were drawn with help of the
Amira 4.1 software (Mercury Computer Systems, Merignac Cedex, France).
Results: The results revealed that ATP-depletion leads to reversible reorganizations of
compartments of the secretory and endocytic systems. The Golgi apparatus stacks
loose their regular organization and become replaced by tubulo-glomerular bodies and
Abstracts of Plenary Lectures
43
networks. Initial membrane changes were apparent as early as 10 min after start of the
experiments. The 3D-reconstructions demonstrated that the regular stack organization
is already broken up at this time; hairpin bend-connections and twisted cisternae
occurred. After 45min ATP-depletion, regular Golgi stacks were almost absent and the
glomerular Golgi bodies and networks dominated. The combined endocytosis studies
showed that both endocytic and non-endocytic parts of the Golgi apparatus become
reorganized. The Golgi bodies formed in response to ATP-depletion contained
ceramide-concentrating subcompartments, supposed to be sites of synthesis of higher
lipids. Most interestingly, endocytosed Bodipy-ceramide was taken up and
concentrated in compartments of the Golgi bodies even, when they were formed prior
to the start of Cer-internalization indicating retained functionalities in the reorganized
Golgi apparatus.
Conclusions: The results showed that cells do not become damaged by metabolic
stress induced by ATP-depletion but respond with reversible reorganizations. It
became evident that the cells react rapidly to the changes of the ATP-levels; the
findings with both endocytosed WGA- and ceramide indicate maintained
functionalities, which may be significant for the cells´ management of metabolic stress
and their survival.
References:
1. M. Del Valle et al., J.Cell Sci. 112 (1999) p4017.
2. C. Meisslitzer-Ruppitsch, et al. Histochem. Cell Biol. 135 (2011) p15
The authors thankfully acknowledge the technical work and help of Mag.Beatrix
Mallinger, Mrs.Regina Wegscheider, Mr.Peter Auinger, Mr.Thomas Nardelli and
Mr.Ulrich Kaindl.
Abstracts of Plenary Lectures
44
Nanoparticle-based Immunocytochemistry reveals PIP2-
containing microarchitecture of the cell nucleus Hozak P.
1
1Institute of Molecular Genetics ASCR, Prague, Czech Republic
While fluorescent microscopy allows for simultaneous detection of multiple antigens,
the electron microscopy (EM) sensitive immunodetection is limited to only two
antigens. I will summarize the current possibilities of single molecule visualization
inside of cells and tissues, and discuss future needs of researches in biomedicine.
In order to overcome the current limitations of immunodetection, we prepared a set of
novel nanoparticles (NPs) which fulfill several criteria: size in the frame of 5-12 nm,
small size distribution, good contrast and stability in the electron microscope, stability
of colloidal solution during conjugation, and surface properties allowing for
conjugation with antibodies. The colloids were prepared from soluble metal salts by
controlled chemical reduction. Subsequently, conditions for conjugation of NPs with
antibodies were optimized and obtained conjugates were probed for the ability of
immunolabelling on ultrathin resin sections of cells. With the use of novel NPs,
various combinations with commercial gold NPs can be made to obtain a set for
simultaneous labelling. For the first time in ultrastructural histochemistry, up to five
molecular targets can be identified simultaneously. For conventional transmission
electron microscopy (TEM), NPs of different shapes can be included in addition to
spherical gold NPs. Different elemental composition of NPs can be used for their
discrimination by EFTEM, STEM/HAADF, TEM/EDX, or FESEM. Using our
previously developed tools of spatial statistics one could then map the regions of
distribution of multiple molecular targets within the cell, as well as to analyze a high
number of individual molecular interactions.
These methods allowed us to progress with understanding some novel molecular
interactions in the cell nucleus. I will discuss interactions of phosphatidylinositol-4,5-
bisphosphate (PIP2) and nuclear myosin I (NMI) which are involved in regulation of
gene expression. PIP2 resides in the nucleus in a different form than the classical
bilayer membrane, apparently forming specific nuclear protein complexes. Our data
Abstracts of Plenary Lectures
45
suggest that nucleolar PIP2 might serve as a transcription factor for ribosomal genes.
We therefore also investigated PIP distribution in cell nuclei with a special attention to
nucleoli by ultrastructural tomography, and mapped PIP colocalization with various
factor involved in RNA pol I transcription. We also showed that NM1 binds to PIP2 in
the cell nucleus, and this was further confirmed by electron microscopy. The results
will be discussed in the frame of the current nucleolar model and lipid functions in the
cell nucleus.
Acknowledgement: This research was supported by the Grant Agency of Czech
Republic (P305/11/2232, 204/09/H084), MSMT (LC545, LC06063 and 2B06063),
Academy of Sciences of the Czech Republic (KAN200520704) and by the IMG grant
AV0Z50390512.
Tracing the movement of mRNA towards the nuclear pore Basello D.
1, Cisterna B.
1, Biggiogera M.
1
1Università di Pavia, Dip. Biologia Animale - Lab. Biologia Cellulare, Pavia, Italy
Extranucleolar RNA synthesis takes place in the so called Perichromatin Area, a 100-
200 nm zone at the periphery of condensed chromatin (Albiez et al., 2006). There the
perichromatin fibrils (PF) are formed by the association of nascent transcripts with
hnRNPs and snRNPs. PF are cotranscriptionally spliced, RNA is cleaved and
polyadenylated and the pre-mRNA/mRNA moves towards the nuclear pore. In some
cases and for reasons so far not yet clarified, PF coil and form a Perichromatin Granule
(PG) which can be stored for some time in the nucleus and then can be exported into
the cytoplasm. PG are possibly a storage form for some mRNAs the cell would need in
time. Transcription, splicing and the movement of these structures involve several
proteins and factors, including actin and Nuclear Myosin I (see Visa and Percipalle,
2010) which are not only involved in transcription but also accompany PF in their
movements. So far, nuclear export of RNAs has been considered to follow a diffusion
mechanism through the interchromatin space, i.e. moving within a space physically
limited by macromolecular structures, and consequently undergoing an anomalous
diffusion process. In recent years, however, it has been proved that at least one
Abstracts of Plenary Lectures
46
macromolecular structure, the small ribosomal subunit, can be exported also by an
active mechanism which represent about 15% of the total export (Cisterna et al., 2006,
2009).
In this view, we have investigated by high resolution EM the movement of pre-
mRNA/mRNA by following the PF as well as the PG inside the cell nucleus after
immunolabelling of actin or nuclear myosin I in different experimental conditions on
HeLa cells. We have analyzed the number of transcripts present at the nuclear pore in
control cells, in cells after in vivo incorporation of anti-actin or anti-myosin antibodies,
or after blockade of de novo ATP synthesis. Immunolabelling with marker antibodies
as well as selective RNA staining with terbium has allowed a semiquantitative analysis
of the transcripts near the nuclear pore. In control cells, the presence of RNA in the
close vicinity of the pore is about 1.8% of the number of trascripts visible inside the
nuclear section, while after actin or ATP blockade the number rises over 6%. These
data suggest that a part of the movements of the non nucleolar transcripts seems to be
linked to an active mechanism. Moreover, PF contain a single RNA molecule which is
never coiled and clearly recognizable from the RNA contained inside PG after terbium
staining. Our preliminary data indicate that the (partial) blockade of energy-requiring
mechanism influence the transfer of one form of transcript more than the other. It is
tempting to hypothesize that cells may use diffusion for one RNP structure and ATP-
dependent movement for the other, thus adding a sort of priority to some mRNAs
instead of others.
Microscopic analysis of protein misfolding diseases and the effect of therapeutic synthetic chaperones Roth J.
1
1Yonsei University, World Class University Program of Yonsei University Graduate
School, Seoul, Korea, Republic of
Protein misfolding has been recognized as the cause of various human diseases. Non-
native proteins will be recognized and retained in the endoplasmic reticulum (ER) by a
sophisticated quality control mechanism and assisted by ER chaperones to achieve
Abstracts of Plenary Lectures
47
proper folding. Those proteins failing to reach a native conformation will become
dislocated to the cytosol for ER-associated degradation (ERAD) involving degradation
by proteasomes or macroautophagy (1, 2). Fabry disease is a lysosomal storage disease
caused by alpha-galactosidase A deficiency. Private point mutations cause enzyme
misfolding and retention in the ER, resulting in progressive multi-systemic
glycosphingolipid deposition, mainly globotriosylceramide Gb3. Fabry disease is the
prototype of a “loss-of-function” disease. Primary open-angle glaucoma represents the
prototype of a “pathological gain-of-function” disease and is caused by point
mutations of myocilin. Misfolded mutant myocilin forms heteromeric complexes with
wild-type myocilin in the ER, which result in Mallory body formation and apoptotic
cell death. Aspects of the molecular pathogenesis of both protein misfolding diseases
as revealed by microscopy will be presented to illustrate through which different
mechanisms cells manage the presence of misfolded proteins in the ER. Furthermore,
the use of synthetic chaperones to rescue the mutant proteins from the ER protein
quality control and to reverse the disease phenotype will be discussed.
Supported by the World Class University program through the National Research
Foundation of Korea funded by the Ministry of Education, Science and Technology
(R31-2008-000-10086-0), a grant from the National Research Foundation of Korea by
the Ministry of Education, Science and Technology (2010-0027736) and the Swiss
National Science Foundation.
1) Roth, J., Cho, J.W., Zuber, C., Park, S., Jang, I., Lee, Y., Gaplovska Kysela, K., Le
Fourn, V., Santimaria, R., Guhl, B.: Protein N-glycosylation, protein folding, and
protein quality control. Mol Cells 30: 497-506, 2010
2) Roth, J., Yam, G.H-F., Fan, J., Hirano, K., Gaplovska-Kysela, K., Le Fourn, V.,
Guhl, B., Santimaria, R., Torossi, T., Ziak, M. and Zuber, C.: Protein quality control:
the who's who, the where's and therapeutic escapes. Histochem Cell Biol 129, 163-
177, 2008
Abstracts of Plenary Lectures
48
Ultrastructure and drug delivery: gateways and barriers Thurner G.C.
1, Wallnöfer E.A.
1, Soelder E.
2, Kremser C.
1, Talasz H.
3, Helbok A.
4,
Klammsteiner N.5, Dietrich H.
6, Jaschke W.
1, Debbage P.
5
1Medical University of Innsbruck, Clinic for Radiology, Innsbruck, Austria,
2Medical
University of Innsbruck, Department of Obstetrics and Gynaecology, Innsbruck,
Austria, 3Medical University Innsbruck, Sektion für Klinische Biochemie, Biozentrum,
Innsbruck, Austria, 4Medical University of Innsbruck, Department of Nuclear
Medicine, Innsbruck, Austria, 5Medical University Innsbruck, Department of Anatomy,
Histology & Embryology, Innsbruck, Austria, 6Medical University Innsbruck, Central
Animal Research Facility, Innsbruck, Austria
Introduction: Size, charge and aqueous solubility are the major factors determining
whether a drug can access its target molecules in pathophysiologically altered tissue.
Focussing on size, most drugs even now are small, 1-5 nm in size. Most cellular
substructures are an order of magnitude larger, so by adjusting charge and solubility of
the drug it has generally been possible to access the target molecules. However, such
drug molecules also access a wide range of other (non-target) molecules because they
enter most functional compartments of the body. The development of macromolecule-
sized drugs has brought size relations at the nanoscale into prominence: these drugs
are excluded from accessing most potential targets by cellular structures which are
about the same size as the drug itself. Development of nanoparticles as drug vehicles
brings nanoscale size relationships into the very foreground of pharmaceutical interest,
and provides presently the most significant of several Grand Challenges in
Nanomedicine. In Innsbruck we examined these issues by synthesizing
macromolecules for use as drug vehicles, and by using these macromolecules to create
nanoparticles. Systemic application of these materials in laboratory animals revealed
barriers to their distribution in the body.
Methods: Albumin-DTPA-gadolinium conjugates were synthesized and injected
intravenously into mice and rats, and also into human placental cotyledons ex situ.
Albumin-DTPA-gadolinium or albumin-DTPA-indium111
conjugates were emulsified
with polylactic acid to create nanoparticles, which also were injected intravenously
into the test animals. The distribution of the albumin component was followed by
Abstracts of Plenary Lectures
49
immunohistochemistry of fixed and embedded organs, mainly using light microscopy.
The nanoparticles were visualized by use of transmission electron microscopy. The
gadolinium component was tracked by use of atomic absorption spectrophotometry.
Indium111
was tracked by PET/SPECT and by scintigraphy.
Results: Both nanoparticles and the conjugated protein localised rapidly into the liver,
spleen and kidneys. The conjugated protein failed to cross endothelial barriers and
therefore did not enter organ compartments. The stabilized albumin nanoparticles
crossed endothelial barriers but not the underlying epithelial barriers, and therefore
also did not enter most organ compartments.
Discussion: Our data reveal the numerous blood-tissue barriers, the test materials
being excluded from most compartments and localizing into unspecific uptake systems
(liver, spleen) or into an organ with albumin-uptake mechanisms (kidney). The data
also show significant differences in pharmacokinetics between the macromolecular
material (7-10 nm in size) and the nanoparticles (30 nm diameter). These results
elucidate the recent impressive failure of several major industrial research efforts to
develop nanoparticle-based formulations for delivery of highly promising new drugs.
Correlated microscopy and nanotomy to analyze Islets of Langerhans in Type I diabetes Giepmans B.N.G.
1, Kalicharan R.
1, Sjollema K.A.
1, Dijk F.
1, Visser J.T.
1, Algra A.
1,
Avramut M.C.2, Koster A.J.
2, Faas F.G.
2, Ravelli R.B.
2
1University Medical Center Groningen (UMCG), Dept. Cell Biology, Groningen,
Netherlands, 2Leiden University Medical Center (LUMC), Dept. Molecular Cell
Biology, Leiden, Netherlands
Electron microscopy (EM) is the method to visualize tissue composition, cellular
interactions and physiological conditions. However, EM only covers a small area and
usually lacks the context of the tissue. Moreover, EM precludes live-cell imaging and
protein-identification with ultrastructural preservation remains a hurdle. Here, we
present (1) novel probes for correlated microscopy; and (2) large-area EM imaging
that allows to characterize large cross-sections (up to millimeters) at unprecedented
resolution, called “nanotomy”. Islets of Langerhans during Type I diabetes onset have
Abstracts of Plenary Lectures
50
been studied with nanotomy. Our data not only shows the progressive destruction of
insulin-producing cells, but also small electron-dense particles that seem to be
associated with diabetes progression. We will discuss our data and present the
workflow of nanotomy, that will be of general use to other tissues and research
questions.
Illuminating biomedical discovery with advanced photonic imaging
Ntziachristos V.1
1Technische Universität München / Helmholtz Zentrum München, Neuherberg,
Germany
Optical imaging is unequivocally the most versatile and widely used visualization
modality in clinical practice and life sciences research. In recent years, advances in
photonic technologies and image formation methods have received particular attention
in biological research and the drug discovery process for non-invasively revealing
information on the molecular basis of disease and treatment. An increasing availability
of endogenous reporters such as fluorescent proteins and probes with physiological
and molecular specificity enable insights to cellular and sub-cellular processes through
entire small animals, embryos, fish and insects and have revolutionized the role of
imaging on the laboratory bench, well beyond the capability of conventional
microscopy. This talk describes current progress with instruments and methods for in-
vivo photonic tomography of whole intact animals and model biological organisms.
We show how new tomographic concepts are necessary for accurate and quantitative
molecular investigations in tissues and why it could be potentially a valuable tool for
accelerated investigations of therapeutic efficacy and outcome. We further
demonstrate that cellular function and bio-chemical changes can be detected in-vivo,
through intact tissues at high sensitivity and molecular specificity. Examples of
imaging enzyme up-regulation, carcinogenesis and gene-expression are given. The
potential for clinical translation is further outlined. Limitations of the method and
future directions are also discussed.
Abstracts of Plenary Lectures
51
Tissue structures guiding collective breast carcinoma invasion Ilina O.
1, Bakker G.-J.
1, Bult P.
2, van Krieken H.
2, Friedl P.
1
1Radboud University Nijmegen Medical Centre, NCMLS, Department of Cell Biology,
Nijmegen, Netherlands, 2Radboud University Nijmegen Medical Centre, Department
of Pathology, Nijmegen, Netherlands
Tumor cell invasion through surrounding tissue occurs by diverse modes, including
single-cell or collective migration patterns. Collective invasion results from the
motility of cell groups that retain cadherin-based cell-cell junctions, multicellular
front-rear polarity and coordinated acto-myosin contractility. Here we address how
pre-existing tissue structures guide cancer cell invasion and regulate a transition
between different invasion modes. To identify pro-invasive extracellular matrix
(ECM) patterns, 3D immunofluorescence and second harmonic generation imaging
from the tumor-stroma interface of thick sections from human ductal breast carcinoma
samples was performed. Fibrous tissue adjacent to normal lactiferous ducts was mainly
composed of a random network of collagen type I fibers that formed barrier-free tracks
with interfibre clefts measuring up to 20 µm in cross-section. Collagen fibers of the
peritumoral region were realigned to form densely packed parallel bundles laterally
bordering extensive tracks filled by single-cell files or multicellular carcinoma masses.
Invading carcinoma cells retained E-cadherin and beta-catenin along cell-cell
junctions, the functional role of which was confirmed using RNA interference against
E-cadherin in breast cancer cells collectively migrating within 3D organotypic
cultures. To validate the impact of barrier-free tracks formed by bundled collagen
fibres as guiding structures, cell-cell junctions, ECM conditions and the capacity to
proteolytically degrade ECM were altered in context. In loose or experimentally
patterned 3D collagen matrices, downregulation of E-cadherin dependent cell-cell
junctions was permissive for single-cell dissociation, whereas dense collagen
conditions imposed collective invasion irrespective of E-cadherin expression level.
Likewise, invasion into patterned 3D collagen mimicking realigned regions in vivo
supported MMP-independent collective invasion along fibrillar collagen interfaces,
whereas random collagen structures required MMP-dependent pericellular
collagenolysis and cell-generated path formation. These results suggest that the
Abstracts of Plenary Lectures
52
alignment and density of the ECM has major impact on the mechanisms of cell-cell
cohesion and MMP-dependence of breast cancer invasion.
Scanning Laser Imaging of Macroscopic Samples Damaskinos S
1
1Intas Science Imaging & Huron Technologies, Waterloo, Canada
Modern high resolution digital slide imagers compete well at resolutions once
dominated by traditional microscopes. Laser based point scanning through a high
numerical aperture lens is one means of creating such a device. These devices have
several advantages over other means of image acquisition, along with some draw
backs. They excel at producing confocal images in fluorescence and are also capable
of generating high resolution transmitted light images.
The TISSUEscope is a laser point scanning system based on patented imaging
technology. It uses an f-theta, telecentric laser scan lens instead of a standard
microscope objective lens in an imaging configuration. In this presentation the optical
arrangement of the TISSUEscope technology will be described and its
advantages/drawbacks relative to other imaging technologies will be discussed.
Applications of the technology in the field of large whole mount imaging will be
presented.
Automated co-analysis of MALDI- and H&E-images of retinal tissue for an improved spatial MALDI-resolution Schönmeyer R.
1, Schmidt G.
1, Meding S.
2, Walch A.
2, Binnig G.
1
1Definiens AG, München, Germany,
2Helmholtz Zentrum München, Institute of
Pathology, Neuherberg, Germany
MALDI imaging is a powerful technology to gain information with high mass
spectroscopic resolution for tissue slides. As its spatial resolution is lower than that of
optical microscopy, methods for improvements are desired. In this contribution we
present an approach to virtually improve MALDI's spatial resolution where the
relevant structures approximately exhibit a linear shape.
Abstracts of Plenary Lectures
53
Therefore a research prototype of a system based on Definiens Cognition Network
Technology fully automatically segments and classifies different regions of interest in
registered high resolution H&E-images. On an upper hierarchical analysis level tissue
types such as inner and outer nuclear layer as well as the layer of rods and cones are
found. On a lower analysis level the system also is capable of detecting individual
nuclei. We use the fact that cross-sections of retinal tissue show a defined sequence of
retinal layers. They can be seen as a one-dimensional system where centers of MALDI
spots have a certain distance to a reference line; in our case the border line between
outer nuclear layer and the layer of rods and cones. This line and all those
corresponding distances of all MALDI spots are automatically calculated and
projected into a one-dimensional system where the reference line represents the origin.
It is beneficial if the variation of distances is relatively large in contrast to them being
clustered around to a few values. Even for a regular sampling of MALDI spots this is
given for the retina due to its moderate curvature. In the one-dimensional system we
consider a range from -75µm to +75µm covering the layers of interest with enough
samples for binning with ~10µm resolution. For each bin ten to fifteen spectra are
available and averaged. This way spectral information with a five times higher spatial
resolution compared to MALDI and additionally an improved signal to noise ratio is
achieved.
Together with other quantitative measures from image analysis, like e.g. density of
nuclei in distinct nuclear layers, this constitutes valuable input data for an extended
MALDI analysis that will help to model the retinal function using a comprehensive
systems biology approach.
Parts of the work presented here are funded by the German Federal Ministry for
Education and Research (SysTec, grant 0315508).
Abstracts of Plenary Lectures
54
Towards systems-pathology by integrating whole-slide imaging,
high-dimensional data and biological networks Grabe N.
1
1Institute of Medical Biometry and Informatics and Hamamatsu Tissue Imaging and
Analysis Center, BIOQUANT University Hospital Heidelberg, Heidelberg, Germany
The paradigm of systems biology is currently transforming the search for mechanistic
explanations of complex biological processes. This is caused simply by technological
advances resulting in increasing amounts of quantitative and high-dimensional cell and
tissue data.
Such complex data sets can only be interpreted by network reaction models
comprising the RNA-, the protein- and other cellular levels.
Systems biology is thus not a scientific trend but the result of a technological
evolution. For pathology this creates the challenge to integrate spatial and quantitative
tissue data with systems biological methods into real systems pathological approaches.
Therefore, first the essential importance of histological, microscopic whole-slide
imaging and computational quantitative image analysis is outlined at examples from
our tumor and wound healing projects. From this it gets clear that the automatic and
objective quantitative spatial analysis of histological slides is the key stone of systems
pathology. It is then shown how quantitative tissue data could be linked to the
intracellular level via growth-factor-receptor network models. Such multi-scale models
will, when embedded in quantitative tissue data,form the conceptional basis of systems
pathology.
Abstracts of Plenary Lectures
55
Quantitative Tissue Proteomics for Deciphering Molecular
Processes in Autoimmune Uveitis Hauck S.M.
1, Deeg C.A.
2, Ueffing M.
1,3
1Helmholtz Zentrum München, Department of Protein Science, Neuherberg, Germany,
2Ludwig-Maximilians University, Institute of Animal Physiology, Department of
Veterinary Sciences, Munich, Germany, 3Centre of Ophthalmology, University
Medical Centre, Tübingen, Germany
Autoimmune Uveitis is an inflammatory disease of the eye, which is caused by
autoimmune processes and occurs in recurrent relapses resulting in irreversible
blindness. Autoreactive T-cells (CD4) transgress the blood-retinal barrier and enter the
inner eye where they destroy the normal tissue structures. However, the molecular
targets as well as the destructive mechanisms and factors triggering the relapses have
remained largely unknown. For elucidating molecular pathomechanisms of the
disease, we use the only available spontaneous animal model of this disease: the horse.
Horses develop equine recurrent uveitis (ERU) with a very high incidence and the
disease progression closely resembles the human form since it also occurs in remitting-
relapsing episodes.
Quantitative proteomics methods offer the unique possibility to directly assess changes
in disease tissues related to differential protein expression as well as identifying the
primary target molecules of autoagressive T-cells. Autoantigen identification was
achieved by mass spectrometric identification of proteins which are selectively
recognised by circulating antibodies from ERU horses and revealed CRALBP as a
novel target. CRALBP was then confirmed as disease-causative in two different
animal models (Lewis rats, horses: EAU) and was additionally shown to be an
important auto-antigen in human patients.
In a 2D-gel-based initial quantitative comparison of ERU and healthy retinal tissue, we
found increase of intermediate filaments, GFAP and vimentin, both indicating
reactivity of retinal Müller glial cells. Furthermore, in uveitic retina, Müller cells
expressed IFNg and lost expression of PEDF. However, disease progression is also
accompanied with a break-down of the blood-retinal barrier and consequently serum-
derived proteins mask the potential target tissue-related changes. To overcome this
Abstracts of Plenary Lectures
56
limitation, we recently used membrane-enriched retina fractions prepared from horses
suffering from equine recurrent uveitis (ERU), and compared expression levels by a
label-free LC-MSMS-based strategy to healthy control samples. We could readily
identify a total of 893 equine proteins with 57% attributed to the GO term
“membrane”. Of these, 179 proteins were found differentially expressed in ERU
tissue. Pathway enrichment analyses indicated an increase in proteins related to
antigen processing and presentation, TNF receptor signaling, integrin cell surface
interactions and focal adhesions. Additionally, loss of retina-specific proteins
reflecting decrease of vision was observed as well as an increase in Müller glial cell-
specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1,
integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry
and tissue staining patterns pointed to a significant increase of these proteins at the
level of the outer limiting membrane which is part of the outer blood-retinal barrier.
Taken together, quantitative proteomics, especially the membrane enrichment in
combination with LC-MSMS-based label-free quantification greatly increased the
sensitivity of the comparative tissue profiling and resulted in detection of novel
molecular pathways related to equine recurrent uveitis.
In situ quantification of protein-protein complexes in paraffin-
sections using Proximity Ligation Assay (PLA)-technique Aubele M.
1, Ludyga N.
1, Braselmann H.
2, Feuchtinger A.
1, Schmitt M.
3, Bartlett J.M.
4
1Helmholtz Zentrum München, Institute of Pathology, Neuherberg, Germany,
2Helmholtz Zentrum München, Department of Radiation Cytogenetics, Neuherberg,
Germany, 3Technische Universität München, Department of Obstetrics and
Gynecology, Munich, Germany, 4University of Edinburgh, Western General Hospital,
Edinburgh, United Kingdom
Nearest neigbor-analysis for the determination of protein-protein complexes are of
high clinical and therapeutical impact in breast cancer. The fairly new method of PLA
(proximity ligation assay technique) allows visualization and quantification of protein-
protein complexes in sections from formalin-fixed, paraffin-embedded (FFPE) human
tumor tissues.
Abstracts of Plenary Lectures
57
We employ this PLA technique on FFPE tissue sections from invasive breast
carcinomas to locate and analyse several different protein-protein complexes.
Examples will be shown for PTK6 (protein tyrosine kinase 6) - HER2, PTK6 - HER3,
for the HER2 - HER3 heterodimers, and for markers of the uPA system like uPA -
uPA-receptor and uPA - PAI-1. Images from the fluorescence labelled PLA-slides are
captered using a confocal laser scanning microscope (AxioImager, Zeiss, Jena,
Germany), and signals from protein complexes are evaluated using the Definiens
Enterprise Image Intelligence Suite software (Definiens, Munich, Germany). Protein
expression levels are then statistically analysed for their association with
histopathological parameters and the clinical follow-up of the disease. As an example,
PTK6 - HER2 complexes showed significant correlation with tumor size, and a low
PTK6 - HER2 signal frequency was associated with better prognosis of patients.
The PLA technique enables accurate visualization, subcellular localization and
quantitative evaluation of protein-protein-complexes in FFPE tissue.
Evaluation of PAXgene-fixed, paraffin-embedded tissues for
morphological and molecular analysis
Gündisch S.1, Schott C.
1, Reischauer B.
1, Meding S.
2, Langer R.
1, Kap M.
3, Viertler
C.4, Ferch U.
5, Riegman P.
3, Zatloukal K.
4, Walch A.
2, Becker K.-F.
1
1Institute of Pathology, Technische Universität München, München, Germany,
2Institute of Pathology, Helmholtz Center Munich, Munich, Germany,
3Department of
Pathology, Josephine Nefkens Institute, Rotterdam, Netherlands, 4Institute of
Pathology, Medical University of Graz, Graz, Austria, 5Third Medical Department,
Technische Universität München, Munich, Germany
Aims: For molecular diagnostics and personalized medicine protein biomarkers need
to be precisely measured in clinical tissue samples. In formalin-fixed and paraffin
embedded (FFPE) tissues protein analysis is still challenging. Within the European
project SPIDIA we evaluated the novel formalin-free tissue fixative “PAXgene Tissue
System” for better integration of morphological and molecular analysis, focussing on
protein approaches.
Abstracts of Plenary Lectures
58
Methods: Different murine and human tissue samples were either snap-frozen, fixed
with the novel tissue fixative, PAXgene tissue fixation and stabilization reagents, or
with formalin before paraffin-embedding. Proteins were analyzed by Coomassie
staining, two-dimensional gel electrophoresis, Western blotting, reverse phase protein
microarrays (RPPA) and matrix-assisted laser desorption/ionization imaging mass
spectrometry (MALDI-IMS). The preservation of the phosphoproteome was
investigated in a large-scale comparative study with 16 different non-malignant and 4
different malignant tissue entities with 11 phosphorylation-specific antibodies by
Western blot. Morphology and immunohistochemistry were evaluated and RNA
quality was assessed by PCR amplification assays.
Results: We were successful in extraction of non-degraded and immunoreactive
proteins from PAXgene-fixed tissue specimens. We analyzed for example E-cadherin,
Hsp70 and beta-actin and phosphorylated proteins, including p-Akt, p-Erk-1/2, and p-
NFkB. Recovered proteins showed very similar properties when compared to
cryopreserved samples by Western blotting or RPPA and were superior to proteins
from FFPE samples. Furthermore, the spectra of MALDI-IMS analysis were similar to
cryopreserved samples which were visualized by insulin and glucagon expression in
pancreatic tissue. Finally, morphology was comparable to FFPE samples whereas
RNA was far better preserved in PAXgene-fixed samples.
Conclusion: The PAXgene Tissue System preserves not only the proteome and most
importantly the phosphoproteome of a tissue sample but as well morphology and
nucleic acids. Thus it has great potential to serve as a novel multimodal fixative for
modern pathology, enabling widespread biomarker studies on clinical tissue samples.
Abstracts of Plenary Lectures
59
Building the MALDI Imaging Protein Biomarker List from the
Bottom Up Maier S.
1, Balluff B.
2, Elsner M.
2, Englert S.
2, Meding S.
2, Schöne C.
2, Walch A.
2,
Küster B.3
Institute(s):
1Technische Universität München, Department of Proteomics and Bioanalytics,
Freising, Germany, 2Helmholtz Zentrum München, Institute of Pathology, Neuherberg,
Germany, 3Technische Universität München, Department of Proteomics and
Bioanalytics, München, Germany
Introduction: MALDI imaging-MS allows merging morphological information with
the distribution of many different molecular features in-situ. In MALDI imaging
studies of proteins, many m/z values of candidate biomarkers remain unidentified but
the identification of the underlying proteins is mandatory as a starting point for further
biological and clinical assessment of these biomarkers. Here we used a bottom-up
approach to identify proteins from sinapinic acid coated tissue sections using trypsin
digestion and tandem mass spectrometry. While this bottom up strategy breaks the link
between the identified proteins and the m/z values of the intact species in the tissue,
the list of identified proteins serves as the pool of proteins from which all potential
biomarkers must be derived.
Methods: MALDI IMS samples were prepared from 10 different fresh frozen tissues.
The sinapinic acid layer was extracted first by 7.5% and second by 60% acetonitrile in
0.1% TFA. The remaining tissue slice was excised from the glass slide. All three
samples were, separated by SDS-PAGE, digested with trypsin, analyzed by LC-
MS/MS and proteins identified by sequence database searching.
Results: The rationale of our bottom up analysis of tissue sections is as follows: Only
proteins that are in the tissue can be embedded into the MALDI matrix layer and only
those proteins that are embedded into the layer can be ionized and detected in the
MALDI MS. Therefore, the bottom up list of proteins must necessarily represent the
pool of all potential protein markers from that tissue. From a single tissue, we
identified 2,100 proteins on average 400 of which were found in the low acetonitrile
and 700 in the high acetonitrile matrix extracts. Across the 10 analyzed tissues, 3738
Abstracts of Plenary Lectures
60
non redundant proteins were identified. With very few exceptions, the proteins
identified in the matrix extracts were almost always also identified in the total tissue
extract. Small proteins are enriched in the matrix layer, whereas structural proteins are
underrepresented. Most of the successfully identified m/z-species from the literature
are observed in our dataset with a high frequency. This shows the validity of this
approach and therefore focuses the number of possible markers down to a few hundred
that have to be considered further. Identification of the matrix proteome can also be
used to reduce the database search space for protein identification. Taken together, our
data shows that the task of identifying putative protein biomarkers from MALDI
imaging data can be greatly simplified and also lead to more confidence in the results
of targeted top- or middle down identification approaches.
Gain of chromosome band 7q11 in papillary thyroid carcinomas of
young patients is associated with exposure to low-dose irradiation Heß J.
1, Braselmann H.
1, Bogdanova T.
2, Thomas G.
3, Zitzelsberger H.
1, Unger K.
1,3
1Helmholtz Zentrum München, Research Unit of Radiation Cytogenetics, Neuherberg,
Germany, 2Institute of Endocrinology and Metabolism, Kiev, Ukraine,
3Imperial
College London, Human Cancer Studies Group, Department of Surgery and Cancer,
London, United Kingdom
The main consequence of the Chernobyl accident has been an increase in childhood
papillary thyroid carcinomas (PTC) in the contaminated areas. Although RET/PTC
rearrangements are prevalent in PTC it has been shown in previous studies that this
rearrangement does not reflect exposure to radiation. Consequently, this study aimed
to identify genomic radiation biomarkers.
We analysed a main (n=52) and a validation cohort (n=28) of PTC from patients that
were young at the time of exposure (median 1.5 years). DNA and RNA from the
tumours were provided by the Chernobyl Tissue Bank
(www.chernobyltissuebank.com). Both cohorts were matched for age at diagnosis, sex
and residence, and consisted of patients exposed (born before the Chernobyl reactor
accident) and not exposed (control group, born at least one year after the accident) to
Abstracts of Plenary Lectures
61
radioiodine fallout. Array CGH was performed to detect copy number alterations in
the tumours.
We found that gain of the chromosome band 7q11.22-11.23 was associated
(FDR=0.035) with exposure to radioiodine fallout. 39% from the exposed group and
none from the unexposed group showed the alteration. This finding was confirmed in
the validation set.
Since only a subgroup of cases in the exposed group exclusively showed gain of
chromosome band 7q11, it is likely that different molecular subgroups and routes of
radiation-induced carcinogenesis exist. The mRNA expression of the genes PMS2L11,
PMS2L3, and STAG3L3 correlated with gain of 7q11.22-11.23. The candidate gene
CLIP2 was specifically overexpressed in the exposed cases at the mRNA and protein
level (IHC). Candidate genes (n=56) from the gained region showed enrichment of GO
(Gene Ontology) terms associated with “DNA repair” (PMS2L3, PMS2L5), “response
to DNA damage stimulus” (BAZ1B, PMS2L3, PMS2L5, RFC2), and “cell-cell
adhesion” (CLDN3, CLDN4).
This study that shows a genomic radiation marker provides novel insights into the
radiation-related carcinogenesis of young onset PTC.
Abstracts of Plenary Lectures
62
MRI molecular imaging with targeted albumin-based
nanoparticles: conceptual design strategies to create the Magic
Bullet Thurner G.C.
1, Wallnöfer E.A.
1, Rohr I.
2, Talasz H.
3, Kremser C.
1, Abdelmoez A.A.
2,4,
Klammsteiner N.2, Dietrich H.
5, Matuszczak B.
6, Jaschke W.
1, Debbage P.
2
1Medical University of Innsbruck, Clinic for Radiology, Innsbruck, Austria,
2Medical
University Innsbruck, Department of Anatomy, Histology & Embryology, Innsbruck,
Austria, 3Medical University Innsbruck, Sektion für Klinische Biochemie, Biozentrum,
Innsbruck, Austria, 4Assiut University, Department of Pharmaceutical Organic
Chemistry, Assiut, Egypt, 5Medical University Innsbruck, Central Animal Research
Facility, Innsbruck, Austria, 6University of Innsbruck, Institute of Pharmaceutical
Chemistry, Innsbruck, Austria
Introduction: Paul Ehrlich in 1900 [1] described the idea of a targeted vehicle
carrying a drug. His notion of a „Magic Bullet“ changed the whole concept of medical
treatment. Richard Feynmann in 1959 [2] challenged scientists to create
nanomachines. Since then Nanomedicine has promised improved drug delivery,
reduced dosages and side-effects: "personalized medicine". Why has Nanomedicine
not delivered on this promise? Two major challenges hinder realisation of this dream:
1. identification of suitable biomarkers; 2. poor availability of nanoparticles capable of
homing to biomarker molecules hiding behind intact tissue barriers. We review the
reasons for these difficulties and suggest some approaches to overcome them.
Materials and methods: Albumin-based nanoparticles bearing gadolinium were
developed and extensively characterised. The lectin LEA was attached to the particles
to target oligolactosamines. During MR Imaging of living rats both imaging and
quantitative approaches were applied. Coordinated with the MRI, chemical and
immunohistochemical analyses tracked components of the nanoparticles in
longitudinal time series after injection, 15 minutes to 6 weeks, obtaining large runs of
quantitative data.
Results: Our nanoparticles were ~30 nm diameter. They were pure, contained no
starting materials and had good imaging properties with relaxivities ~1 • 107 1/Ms.
They were stable in various in vitro testings though not in SDS-gel-electrophoresis. In
Abstracts of Plenary Lectures
63
haemagglutination tests they agglutinated red blood cells; after intravenous injection
into living rats they gave high-resolution MR imaging of the vascular wall lasting >2
hours. The numbers of (lectin) targeting groups required for molecular imaging and of
gadolinium ions per nanoparticle necessary for high-resolution MRI were assayed in
relation to particle size and type of crosslinking.
Discussion: We determined critical parameters for MR Molecular Imaging by use of
nanoparticles. These particles require a second type of targeting group to migrate
across vascular walls and access subendothelial interstitial compartments, for
Molecular Imaging and Molecular Targeting of disease sites behind intact tissue
barriers. The concept of multiple targeting is new in Nanomedicine and represents the
hurdle that limits present-day techniques [3]. Assuming that quantitative aspects of
targeting will be similar for each of the multiple targeting groups, we already know
how to design nanoparticles for targeting both drugs and contrast agents to disease
lesions hiding behind blood-tissue barriers. Multiply-targeted nanoparticles are
Ehrlich´s "magic bullets".
Acknowledgements: Austrian Nano-Initiative (Project N201-NAN); Austrian
National Bank Jubilee Program (Projects 9273, 10844, 11574)
References:
1. Ehrlich, P. (1900) Proc R Soc Lond 66: 424 - 448.
2. Feynman RP. (1959) Miniaturization. Horace D. Gilbert, Ed. © Van Nostrand
Reinhold. New York
3. Debbage P., Thurner GC. (2010) Pharmaceuticals 3: 332 – 3416
Abstracts of Plenary Lectures
64
MALDI MSI for ovarian cancer biomarkers research: latest
developments of the technology for screening and tracking. Longuespée R.
1,2, Boyon C.
1,3, Kerdraon O.
4, Desmons A.
1, Vinatier D.
3, Fournier I.
1,
Day R.2, Salzet M.
1
1Laboratoire de Spectrométrie de Masse Biologique Fondamentale et
Appliquée/Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France,
2Institut de Pharmacologie de Sherbrooke/Université de Sherbrooke, Sherbrooke,
Canada, 3Service de Chirurgie Gynécologique/Hôpital Jeanne De Flandre/CHRU
Lille, Lille, France, 4Centre d'Anatomie et de Cytologie Pathologiques/CHRU Lille,
Lille, France
Ovarian cancer is the second major cause of gynecological death in Europe and USA.
Ovarian cancer has been studied at a proteomic level for biomarkers hunting with the
emergence of diverse techniques such as 2D electrophoresis and SELDI TOF MS. At
this time, CA125 is still the only biomarker used for the monitoring of the pathology.
Its sensitivity is 80% for stage III and stage IV cancers but only 30% for early stages
with high rates of false positives. Since its introduction in the early 90's, MALDI
Imaging Mass Spectrometry (IMS) has widely been used for biomarkers discovery in
many pathologies thanks to the possibility to discriminate the localization of
compounds in tissues of interest. Using this technology, we found the Cter part of
PA28, “Reg Alpha” present in the cancerous regions of ovarian biopsies. Recently, we
found this peptide in stage I epithelial cancer tissues, making it a potential marker for
wide screenings among women populations. Then, our recent developments in the
field of IMS allowed us to associate Histo Immuno Chemistry and IMS for the specific
tracking of this molecule in cancerous tissues. We indeed propose to incubate tissues
of interest with known anti-markers antibodies, then digest the surface of the tissues
with a solution of trypsin and finally perform the MALDI Image of the prepared
tissue, to reveal the presence of the tryptic peptides of the antibodies. It's now possible
to proceed to the quantitative detection of markers by directly analyzing the presence
of tryptic peptides of the antibodies targeting Reg Alpha. This procedure allows the
use of Immuno Histo Chemistry with the Mass Spectrometric devices as revealers
instead of a microscope, and would be strongly relevant for the tracking of cytotoxic
Abstracts of Plenary Lectures
65
antibodies in tumors. These developments also pave the way for new pharmacologic
applications such as the monitoring of the effect of treatments of the pathology by
screening the evolution of the presence of the compound in malignant tissues.
Posters
66
Poster
General Information
The poster session will be on Thursday at 14:20. The poster exhibition will take place
at the Frauenklinik Maistraße, lecture hall 1+2. Every poster board carries the poster
number given in the program on the upper left corner. Please refer to the program for
your poster number and ask at the registration desk for the exact placement of your
poster board. The material for mounting the posters will be supplied by the local
organizers. The posters should be mounted until Saturday. Please note that posters
which have not removed at the end of the meeting cannot be returned.
The presenting authors should be at their poster during the poster session on Thursday
for personal presentation.
Poster Prizes of the Society for Histochemistry
Three best student posters will be awarded during closing of the symposium with
100 € (conditions: First author, PhD student).
Poster Index
01 Balluff B. MALDI imaging identifies prognostic seven-protein
signature of novel tissue markers in intestinal-type
gastric cancer
02 Balluff B. Classification of HER2/neu status in gastric cancer using
a breast-cancer derived proteome classifier
03 Claude E. The advantages of coupling high efficiency ion mobility
separation with MALDI MS imaging of on-tissue
digested proteins
04 Crecelius A.C. Combining lipidomics and proteomics by MALDI-MSI
05 Deininger S.O. Normalization in MALDI-TOF imaging of proteins
Posters
67
06 Enthaler B. MALDI imaging of endogenous compounds directly in
human skin-tissue sections
07 Gerbig S. Desorption Electrospray Ionization (DESI) imaging of
tumor samples - using mass spectrometry to support
classical histology
08 Kobarg J.H. Spatial segmentation of hyperspectral 3D volume data
09 Krásný L. pHPMA-based tissue embedding medium compatible
with MALDI mass spectrometry imaging experiments
10 Longuespée R. MALDI MSI for ovarian cancer biomarkers research:
latest developments of the technology for screening and
tracking.
11 Meding S. Proteomic markers for regional lymph node metastasis in
primary colon cancer tissues
12 Nipp M. S100-A10, Thioredoxin, and S100-A6 as biomarkers of
papillary thyroid carcinoma with lymph node metastasis
identified by MALDI Imaging
13 Oetjen J. The MALDI-imaging/ MULTI-ARRAY core facility: A
new service platform at the University of Bremen
14 Schöne C. MALDI Imaging as a novel approach for the
systematical study of intratumoral heterogeneity
15 Strohalm M. In-tissue lipid profiling by mass spectrometry imaging
16 Suckau D. Spatial proteomics: A new LC-MS/MS tissue imaging
workflow providing protein identities and their
distribution in tissue
17 Trede D. Software for 3D MALDI imaging data: Visualization and
3D spatial segmentation
18 Wurlitzer M. MALDI-Imaging of frozen prostate tissue micro arrays
Posters
68
19 Acar N. The evaluation of expressions of FKBP52 and PRDX6
proteins and ultrastructure of wild type and Fkbp52
knockout mice uterus during pregnancy
20 Aunapuu M. Analysis of endometrial receptivity based on
immunohistochemical study
21 Bauer L. Differential expression of stem cell related genes in
neoadjuvant treated gastric cancer.
22 Bonin S. Cell mobility and metastatic spreading: a study on
human breast cancer cells using the nano-mechanical
approach
23 Brüning A. The HIV reverse transcriptase inhibitor efavirenz
selectively kills human cancer cells
24 Buchwalow I. Non-specific binding of antibodies in immunoassays:
fakes and facts
25 Buchwalow I. Signal amplification in immunoassays: heteropolymeric
HRP conjugates vs. polymer backbone conjugates
26 Csoka L. Polarization optical and histochemical characterization
and supramolecular structure of carbohydrate-protein
fibrils
27 Ebach K. Immunohistochemical investigations of keratin
expression for a characterization of epidermal stem cell
niches in bovine fetal skin
28 Emody L. Topo-optical follow up of phenothiazine induced charge
transfer reactions in the yeast cell wall
29 Epis S. The intramitochondrial bacterium Midichloria: novel
tools for its detection
30 Giagnacovo M. Ribonucleoprotein-containing foci increase in size upon
cell-cycle exit in cell nuclei from patients affected by
myotonic dystrophy type 2
Posters
69
31 Han K.-H. Renal ischemia-reperfusion injury causes intercalated
cell-specific disruption of occludin in the collecting duct
32 Heindl S. E-cadherin mutations contribute to gastric carcinogenesis
by modulating multiple EGFR-dependent downstream
signalling pathways
33 Houtkamp M.A. Immunohistochemical detection and non-invasive U-
SPECT imaging reveal distinct tumor tissue distribution
patterns of anti-EGFr antibody zalutumumab in a tumor
xenograft model
34 Kaneko T. Immune laser capture microdissection of macrophages in
engineered dental pulp tissues
35 Klappan A. Autophagosome formation by quercetin
36 Lee H.J. Cordycepin induces double strand breaks and the DNA
damage response in breast cancer cells
37 Luber B. Biomarker analysis of cetuximab plus
oxaliplatin/leucovorin/5-fluorouracil in first-line
metastatic gastric and oesophago-gastric junction cancer:
results from a phase II trial of the Arbeitsgemeinschaft
Internistische Onkologie (AIO) v
38 Makovitzky J. The topo-optical investigation of Alzheimer Amyloid
39 Makovitzky J. Late gastrointestinal metastases of invasive lobular
breast carcinoma mimicking Crohn´s disease
40 Markelic M. Role of macrophages in brown adipose tissue remodeling
in hyperinsulinemic rats
41 Matsingou C. Selective Induction of Inhibin beta E by the ER stress
reaction
42 Rodler D. Glycohistochemical characterization of the avian inner
perivitelline layer
43 Salovska J. Thyroid gland-commonness of hyperthyreodism and
hypothyreodism in the female and male population
Posters
70
44 Sinowatz F. Immunohistochemical localization of transit cells,
putative stem cells and stem cells in benign prostate
hyperplasia (BPH) and adenocarcinoma of the prostate
45 Stoemmer P. Cell adhesion molecules in melanocytic tumors
46 Strelkova O.S. Nuclear lamina in organization of DNA replication.
47 Thurner G.C. MRI molecular imaging with targeted albumin-based
nanoparticles: conceptual design strategies to create the
Magic Bullet
48 Timme S. EGFR, HER2 and HER3 expression and dimerization in
esophageal cancer
49 Ustunel I. Zonal distribution of CD105+/CD166+ cells in growing
rat humerus proximal epiphyseal cartilage
50 Weber E. Is CRIP1 a target gene of HER2?
51 Weipoltshammer K. Class I HDACs in the developing heart and limb of
chicken embryos
Abstracts of Poster Presentation
71
Abstracts of Poster Presentation
The evaluation of expressions of FKBP52 and PRDX6 proteins and ultrastructure of wild type and Fkbp52 knockout mice uterus
during pregnancy Acar N.
1, Hirota Y.
2, Daikoku T.
2, Dey S.K.
2, Ustunel I.
1
1Akdeniz University, School of Medicine, Histology and Embryology, Antalya, Turkey,
2University of Cincinnati College of Medicine Cincinnati Children's Hospital, Division
of Reproductive Sciences, Cincinnati, United States
Implantation is the nidation of blastocyst into mother's uterus. It starts on day 4 night.
An immunophilin FKBP52 (FK506 binding protein) serves as a cochaperone to govern
normal progesterone (P4)-P4 receptor (PR) signaling in mouse uterus. Previous studies
reported that Fkbp52 knock out (KO) females with reduced P4-PR signalling have
implantation failure, which is rescued by P4 supplementation in genetic background
and pregnant stage dependent manners (Tranguch S et al, J Clin Invest, 117 (2007)
1824; Tranguch S et al, Proc Nat Acad Sci USA, 102 (2005) 14326).
Our previous study showed that peroxiredoxin6 (PRDX6) is downregulated in Fkbp52
KO uterus. PRDX6 is an antioxidant molecule. Therefore we aimed to determine the
location and expression intensities of FKBP52 and PRDX6 proteins and to investigate
the ultrastructural characteristics of wild type and Fkbp52 KO mice uterus on certain
days of pregnancy.
For our purpose one male and two female mice were retained together in the same
cage overnight and next morning female mice with vaginal plug were accepted on day
1 of pregnancy. Wild type mice were sacrified on days 1,4,5,8 and 14 of pregnancy.
Fkbp52 KO mice were injected P4 until their sacrificatin on days 4,5,8 and 14. Uterus
horns or implantation sites were removed, processed for immunohistochemistry,
immunoflorescence and electron microscopic evaluation.
On days 1,4,5 of pregnancy in control group PRDX6 expression was observed
especially in apical side of lumen epithelium of endometrium, gland epithelium and in
stroma. On day 8 of pregnancy PRDX6 was expressed in antimesometrium and
primary decidual zone. On day 14 of pregnancy PRDX6 was expressed in labyrinth
and especially spongiotrophoblast of placenta and expression was weaker compared to
Abstracts of Poster Presentation
72
previous days. FKBP52 expression was colocalised with PRDX6 in the cytoplasm of
lumen epithelium on day 1; in cytoplasm of gland epithelium on day 4; in the stroma
of endometrium on day 5; in antimesometrium of uterus and primary decidual zone on
day 8; in labyrinth of placenta on day 14. On fourth day of pregnancy uterus
morphology of Fkbp52 KO mice without P4 injection was abnormal. PRDX6
expression was not continuous through the apical side of lumen epithelium. On the
same day in P4 injected Fkbp52 KO group uterus morphology was similar to control
group and PRDX6 expression was all through the apical side of lumen epithelium.
PRDX6 expression of Fkbp52 KO mice with P4 injection was similar to wild type
mice on other studied days
Electron microscopic observations done on day 4 of pregnancy showed although there
were differences between wild type and Fkbp52 KO mice without P4 injection;
ultrastructure of P4 injected Fkbp52 KO mice was similar to wild type mice.
By means of P4 injection lumen epithelium and stromal cells of Fkbp52 KO mice
become similar to wild type so implantation is succeeded. In addition to its
cochaperone function FKBP52 may cooperate with PRDX6 to overcome oxidative
stres.
Analysis of endometrial receptivity based on immunohistochemical
study Aunapuu M.
1, Arend A.
2
1University of Tartu, Anatomy, Tartu, Estonia,
2University of North CarolinaTartu,
Anatomy, Tartu, Estonia
Introduction: Infertility is a condition that affects a couple and is defined as the lack
of conception after an arbitrary period of 12 months with regular sexual intercourse
and without using any contraception. Infertility is a common medical problem present
in about 10% of couples in reproductive age. Tubal factor infertility (TFI) is one of the
leading causes of infertility in the world including Estonia. The most common method
treating infertility due to TFI is In Vitro Fertilization (IVF). In the IVF-procedure
oocytes are fertilized in in vitro conditions followed by incubation for 2-5 days.
Finally 1 to 3 embryos will be transplanted into the uterus. Occurrence of TFI
Abstracts of Poster Presentation
73
decreases receptivity of the uterine mucosa by lowering the expression of ανβ3
integrins, trophinin on the endometrial epithelium, which reduces the possibility of
implantation.
Materials and methods: Endometrial biopsies of patients with infertility problems were
carried out in Nova Vita Clinic (Tallinn, Estonia). Patients, who have had several
unsuccessful IVF procedures, were divided into two groups: Group I - younger than 34
years and Group II - older than 35 years. Material for light microscopy and
immunohistochemistry (IHC) were fixed in 10% buffered formalin solution and
embedded in paraffin with a vacuum processor Tissue-Tek®
VIPTM
5 Jr (Sakura,
USA). Specimens were cut with microtome Ergostar HM 200 (Microm, Germany) at
three-mm thickness and stained using H&E and van Gieson methods for general
orientation to sections. Histological methods were used to specify the stage of
menstrual cycle. IHC staining was used to detect integrin beta3 (CD61) and trophinin
expression in the endometrium. Slides were observed and ptohographed by a Zeiss
Axiophot 2 microscope (Zeiss, Germany).
Results: Light microscopy studies of biopsies revealed no pathologic changes in
endometric morphology. The ratio between glandular and stromal components varied
from 1:1 to 2:1. IHC staining with trophinin showed a weak reaction (grade 2) in two
patient's luminal epithelium and minimal (grade 1) in 4 patient's luminal epithelium
and two patient`s glandular epithelium. IHC staining with CD61 showed a very intense
reaction only in one patient biopsy. This result showed decreased receptivity of the
uterine mucosa by lowering the expression of β3 integrins on the endometrial
epithelium.
Conclusion: Although there are well characterised morphological and molecular
markers of implantation, the complete dynamics of the process as well as the
importance of each and every marker is still vague. Morphological studies showed that
TFI patients have decreased expression of β3 integrins and trophinin.
This research was supported by Estonian Science Foundation grant No. 7301.
Abstracts of Poster Presentation
74
MALDI imaging identifies prognostic seven-protein signature of
novel tissue markers in intestinal-type gastric cancer Balluff B.
1,2, Rauser S.
1, Meding S.
1, Elsner M.
1, Schöne C.
1, Feuchtinger A.
1,
Schuhmacher C.3, Novotny A.
3, Jütting U.
4, Maccarrone G.
5, Sarioglu H.
6, Ueffing
M.6, Braselmann H.
7, Zitzelsberger H.
7, Schmid R.M.
2, Höfler H.
1,8, Ebert M.P.
9,
Walch A.1
1Helmholtz Zentrum München, Institute of Pathology, Neuherberg, Germany,
2Klinikum rechts der Isar der TU München, 2. Medizinische Klinik und Poliklinik,
München, Germany, 3Klinikum rechts der Isar der TU München, Department of
Surgery, München, Germany, 4Helmholtz Zentrum München, Institute of
Biomathematics and Biometry, Neuherberg, Germany, 5Max Planck Institute of
Psychiatry, Department of Proteomics, München, Germany, 6Helmholtz Zentrum
München, Department of Protein Science, Neuherberg, Germany, 7Helmholtz Zentrum
München, Department of Radiation Cytogenetics, Neuherberg, Germany, 8Technische
Universität München, Institute of Pathology, München, Germany,
9Universitätsklinikum Mannheim, Department of Medicine II, Mannheim, Germany
Proteomics-based approaches allow us to investigate the biology of cancer beyond
genomic initiatives. We used histology-based matrix-assisted laser
desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that
predict disease outcome in gastric cancer after surgical resection.
A total of 181 intestinal-type primary resected gastric cancer tissues from two
independent patient cohorts were analyzed. Protein profiles of the discovery cohort
(n=63) were directly obtained from tumor tissue sections by MALDI imaging. A
seven-protein signature was found to be associated with an unfavorable overall
survival independent of major clinical covariates (HR=4.03; 95% CI: 1.69 - 9.61;
P=0.002). The prognostic significance of three individual proteins identified (CRIP1,
HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of
an independent validation cohort (n=118). While HNP-1 and S100-A6 were found to
further subdivide early (UICC-I) and late stage (UICC-II-III) patients into different
prognostic groups (P=0.024, P=0.013), CRIP1, a protein previously unknown in
Abstracts of Poster Presentation
75
gastric cancer, was confirmed as a novel and independent prognostic factor for all
patients in the validation cohort (HR=1.57; 95% CI: 1.01-2.44; P=0.044).
The protein pattern described here serves as a new independent indicator of patient
survival complementing the previously known clinical parameters in terms of
prognostic relevance. These results show that this tissue-based proteomic approach
may provide clinically relevant information that might be beneficial in improving risk
stratification for gastric cancer patients.
Classification of HER2/neu status in gastric cancer using a breast-cancer derived proteome classifier Balluff B.
1,2, Elsner M.
2, Kowarsch A.
3, Rauser S.
2, Meding S.
2, Schuhmacher C.
4,
Feith M.4, Herrmann K.
5, Röcken C.
6, Schmid R.M.
1, Höfler H.
2,7, Walch A.
2, Ebert
M.P.8
1Klinikum rechts der Isar der TU München, 2. Medizinische Klinik und Poliklinik,
München, Germany, 2Helmholtz Zentrum München, Institute of Pathology,
Neuherberg, Germany, 3Helmholtz Zentrum München, Institute of Bioinformatics and
Systems Biology, Neuherberg, Germany, 4Klinikum rechts der Isar der TU München,
Department of Surgery, München, Germany, 5Klinikum rechts der Isar der TU
München, Department of Nuclear Medicine, München, Germany, 6Christian-
Albrechts-University, Institute of Pathology, Kiel, Germany, 7Technische Universität
München, Institute of Pathology, München, Germany, 8Universitätsklinikum
Mannheim, Department of Medicine II, Mannheim, Germany
Testing for HER2-status (human epidermal growth factor receptor 2) in breast and
gastric cancers is mandatory for the treatment with trastuzumab. We hypothesized that
imaging mass spectrometry (IMS) of breast cancers may be useful for generating a
classifier that may determine HER2-status in other cancer entities irrespective of
primary tumor site.
A total of 107 breast (n=48) and gastric (n=59) cryo tissue samples were analyzed by
IMS (HER2 was present in 29 cases). The obtained proteomic profiles were used to
create HER2 prediction models using different classification algorithms.
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A breast cancer proteome derived classifier, with HER2 present in 15 cases, correctly
predicted HER2-status in gastric cancers with a sensitivity of 65% and a specificity of
92%. In order to create a universal classifier for HER2-status, breast and non-breast
cancer samples were combined, which increased sensitivity to 78%, specificity was
88% respectively.
Our proof of principle study provides evidence that HER2-status can be identified on a
proteomic level across different cancer types suggesting that HER2 overexpression
may constitute a unique molecular event independent of the tumor site. Furthermore,
these results indicate that IMS may be useful for the determination of potential
drugable targets, as it offers a quicker, cheaper and more objective analysis than the
standard HER-testing procedures immunohistochemistry and fluorescence in situ
hybridization.
Differential expression of stem cell related genes in neoadjuvant treated gastric cancer. Bauer L.
1, Langer R.
1, Becker K.
1, Ott K.
2, Novotny A.
3, Hapfelmeier A.
4, Höfler H.
1,5,
Keller G.1
1Institut für Pathologie der TU München, München, Germany,
2Cirurgische Klinik der
Universität Heidelberg, Heidelberg, Germany, 3Cirurgische Klinik der TU München,
München, Germany, 4Institut für Medizinische Statistik und Epidemiologie der TU
München, München, Germany, 5Institut für Pathologie, Helmholtz Zentrum, München,
Germany
Neoadjuvant treatment of gastric cancer offers the opportunity to investigate residual
tumor cells after chemotherapy (CTX). According to the cancer stem cell (CSC)
hypothesis, there might be an enrichment of cells expressing CSC related genes within
the residual tumor. We analyzed the expression of CSC related genes in residual
gastric tumors for an association with overall survival and compared corresponding
pretherapeutic biopsies and resected tumors for CTX associated expression changes.
An initial screening of 44 genes selected according to their relevance for
differentiation and development or as putative CSC markers was performed. Resected
specimens from 63 gastric cancer patients treated with neoadjuvant platinum/5-FU
Abstracts of Poster Presentation
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based CTX that demonstrated a partial response based on histopathological tumor
regression (i.e. 10-50% residual tumor cells) were included. mRNA was isolated from
macrodissected FFPE tissues and gene expression was quantified by real time PCR
using TaqMan®
low density arrays. Data were analyzed for correlation with overall
survival and clinicopathological parameters. Selected genes (n=12) were compared
between corresponding biopsies and resected specimens from patients with partial
(n=22) and minimal/no tumor regression (i.e. >50% residual tumor cells, n=22).
Genes involved in Wnt and notch signaling pathways, such as GSK3B, β-catenin and
Notch2, were among the genes demonstrating a prominent association with overall
survival (p=.006, p=.043 and p=.072, respectively) in the initial screening.
Comparison between biopsies and resected specimens revealed an increase of Notch2
and LGR5 expression in tumors with partial response (p=.002 and .017) and of
POU5F1 in both partial and minimal/non responding tumors (p=.028 and .002).
Notch1 expression was significantly decreased in minimal/non responding tumors
(p=.001) and DNMT1 expression was significantly down regulated in both groups
(p=.009 and .002).
In tumors with partial regression after neoadjuvant CTX, the expression of genes
involved in CSC associated signaling pathways, such as the Wnt and notch pathway,
showed a prognostic significance, which may be used for risk stratification in this
patient group. The comparison of expression levels between corresponding biopsies
and resected specimens revealed gene expression alterations that might be a result of
enrichment of chemotherapy resistant residual tumor cells.
Cell mobility and metastatic spreading: a study on human breast
cancer cells using the nano-mechanical approach Tavano F.
1, Stanta G.
1, Cojoc D.
2, Pinato G.
2, Migliorini E.
1, D'Este E.
1, Bonin S.
1
1University of Trieste, Trieste, Italy,
2CNR-IOM, National Laboratory TASC, Trieste,
Italy
Background: The primary causes of death in cancer patients are local invasion and
metastasis. The common classification of cancers according to tumor stage and grade
is not sufficient to predict the clinical outcome. The mechanism that characterises
Abstracts of Poster Presentation
78
tumor progression from the primary to the metastatic sites is not completely
unravelled. Metastatization is accompanied by alterations of the cytoskeleton and
membrane structure leading to changes in their biomechanical properties 1. Recently
mechanical tests have demonstrated that tumor cells are softer than their normal
counterpart 2.
Methods: In this study we analyzed, by means of Optical Tweezers (OT) 3,4
and
atomic force microscopy (AFM), the mechanical properties of two different breast
carcinoma cell lines corresponding to different metastatic potential (MDA-MB231 and
MCF7). Then we compared this value with those obtained by the analysis of a breast
line, assembling the normal breast tissue, HBL-100 (mamma lactans). The data gained
by OT and AFM were compared with the expression of proteins obtained by Western
Blot (WB). As for mechanical data we also determined the Young's Modulus for the
Integrin-Fibronectin bound by AFM. The proteins we analyzed by WB were involved
in epithelial-mesenchimal transition (Vimentin), in cell adhesion (Integrinαvβ3,
Viculin and E-Cadherin) and in breast cancer differentiation (Cytokeratin 8).
Experimental data were also implemented by the confocal images of the Actin
cytoskeleton obtained with the Actin Cytoskeleton and Focal Adhesion Staining Kit
(Millipore).
Results: The rigidity of the membrane and the tether stiffness obtained by OT were
significantly different among the analysed breast cell lines (p=0.01 and p=0.02
respectively), displaying higher values for the not-cancerous HBL-100. The Young's
modulus also significantly differed among the breast cell lines (p=0.000). Regarding
the MCF-7, associated to a low metastatic potential, and the MDA-MB 231, poorly
differentiated with a high metastatic potential, they showed different tether stiffness
with a value four times lower for the MDA-MB231. The Actin cytoskeleton resulted
highly organized in HBL-100 and MCF7 in comparison to MDA-MB231. The
mechanical data were confirmed by WB analyses, which demonstrated that the
molecules associated with poorly outcome in breast Ca, such as Vimentin, were
positive in MDA-MB231, but not in MCF7. On the other hand E-Cadherin was
positive in MCF-7 but not in MDA-MB231.
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Conclusions: Our results seems to confirm the hypotesis that metastasis prone cells
are softer than less aggressive cancer cells, and support the use of OT for these
measurements for its sub-pN force resolution and because cells are manipulated
without damage.
References:
1 Suresh S. Acta biomaterialia 2007; 3: 413-38.
2 Suresh S. Nature nanotechnology 2007; 2: 748-9.
3 Tavano F, Bonin S, Pinato G et al. Int. J. Optomechatronics In Press.
4 Schmitz J, Benoit M, Gottschalk KE. Biophysical J 2008; 95: 1448-59.
The HIV reverse transcriptase inhibitor efavirenz selectively kills
human cancer cells Brüning A.
1, Burger P.
1, Vogel M.
1, Kost B.
1, Burges A.
1, Friese K.
1
1University Hospital Munich, OB/GYN, Munich, Germany
Aims: Several HIV drugs have recently been described to exert anti-tumour effects.
We have investigated the effect of efavirenz on the cell survival of human cancer cells.
Methods: A variety of human cancer cell types, including breast cancer, ovarian
cancer, and leukemia cells were analysed by MTT assays, clonogenic assays and
FACScan analysis for the occurrence of cell cycle arrest and apoptosis. Immunoblot
analyses were performed to investigate the effect of efavirenz on intracellular
signalling pathways.
Results: Efavirenz selectively induces cell death in human cancer cells, but not in non-
malignant human epithelial cells or fibroblasts. Efavirenz-induced cell death was
associated with the induction of apoptosis, as revealed by caspase activation and p53
phosphorylation. In breast cancer cells, the cytotoxic effect of efavirenz could be
enhanced by tamoxifen.
Conclusions: Efavirenz, although a non-nucleosidic reverse transcriptase inhibitor,
selectively induces apoptosis in human cancer cells with little effects on
untransformed human cells, and could thus be of high interest for cancer treatment
purposes.
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80
Non-specific binding of antibodies in immunoassays: fakes and
facts Buchwalow I.
1, Samoilova V.
1, Boecker W.
1, Gergs U.
2, Mohamed S.A.
3, Tiemann
M.1
1Institut für Hämatopathologie, Hamburg, Germany,
2Martin-Luther-Universität
Halle-Wittenberg, Institut für Pharmakologie und Toxikologie, Halle (Saale),
Germany, 3Universitätsklinikum, Schleswig-Holstein, Klinik für Herzchirurgie,
Lübeck, Germany
The current protocols for blocking background staining in immunohistochemistry are
based on conflicting reports. Background staining is thought to occur as a result of
either non-specific antibody (Ab) binding to endogenous Fc receptors (FcRs) or a
combination of ionic and hydrophobic interactions. This concept has been mentioned
in all publications regarding immunohistochemistry since its inception half a century
ago, but we have been unable to find the original source of the idea. This prompted us
to explore whether commercially available Abs have a propensity for random non-
specific binding in the immunolabelling of routinely fixed cell and tissue samples.
In this study, cell and tissue samples were processed according to routine protocols
either with or without a blocking step (goat serum or BSA). In view of the fact that
FcRs are expressed primarily on monocytes, macrophages, B cells, dendritic cells,
neutrophils and platelets, we paid special attention to cell and tissue samples where
these cells are abundant, specifically bone marrow, spleen, tonsils and blood cell
smears. Surprisingly, no Abs in samples processed without a blocking step showed any
propensity for non-specific binding leading to background staining, implying that
endogenous FcRs do not retain their ability to bind the Fc portion of Abs after standard
fixation. Likewise, we did not find any non-specific Ab binding ascribable to either
ionic or hydrophobic interactions. After performing immunostaining using
fluorophore-conjugated Abs, we also found that the omission of the protein blocking
step did not lead to non-specific background staining in single or multiple fluorescence
immunolabelling with the use of either fluorophore-conjugated Ab or streptavidin.
This was a clinically oriented study focused on human patient tissue samples.
However, many researchers perform immunohistochemistry on tissues from
Abstracts of Poster Presentation
81
experimental animals, particularly rodent tissues. Similar to immunostaining of human
cell and tissue samples, omission of the protein blocking step did not lead to unwanted
background staining in histological samples from experimental and farmed animals.
In contrast to the commonly accepted view, we found that the protein blocking step
traditionally used in immunohistochemistry is unnecessary in the immunostaining of
routinely fixed cell and tissue samples taken both from human patients and from
nonhuman species. Omission of the traditional protein blocking step may save
substantial reagent costs and preparation time both in research and
immunohistopathology.
Signal amplification in immunoassays: heteropolymeric HRP
conjugates vs. polymer backbone conjugates Buchwalow I.
1, Samoilova V.
1, Boecker W.
1, Wolf E.
1, Tiemann M.
1
1Institut für Hämatopathologie, Hamburg, Germany
The peroxidase-labeled antibody (Ab) method, introduced in 1968, was the first
practical application of Abs to paraffin-embedded tissues for most clinical and
research studies (Nakane, 1968). During the past few decades, improvements in the
reagents and protocols used for immunohistopathology have led to increased
sensitivity of detection systems. A significant level of signal amplification was
achieved by the peroxidase-antiperoxidase complex system (PAP). This technique uses
large complexes which contain many detection agents bound together. The binding of
one of these complexes to each secondary antibody amplifies the signal manyfold. For
many years, the PAP procedure represented the most sensitive and hence most popular
techniques in many pathology laboratories. However, today these techniques are only
rarely used being substituted by modern more sensitive methods - heteropolymeric
HRP conjugates and polymer backbone conjugates.
In this study, we compared the level of signal amplification achieved with the use of
heteropolymeric AmpliStain™-HRP conjugate (SDT GmbH) and EnVision™+-HRP
backbone polymer conjugate (DAKO Corporation). Primary Abs were applied for 1h
at room temperature in series of several dilutions of twofold increments. Detection of
bound primary Abs with AmpliStain™ and EnVision™+ was performed pairwise on
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82
two immediately adjacent sections for each Ab dilution. Chromogen development after
application of the polymeric HRP conjugates was monitored under microscopic
control until reaching the optimal staining and stopped simultaneously for the entire
series of primary Ab dilutions.
Immunohistochemical staining with various anti-mouse and anti-rabbit primary Abs
revealed a significantly higher level of signal amplification with the use of AmpliStain
for detection of bound primary Abs. In order to quantify the immunostaining
efficiency of AmpliStain™ and EnVision™+, we run ELISA tests for specific Rabbit
and Mouse IgG Abs. Quantitative ELISA readout data show that, compared with
EnVision, anti-Mouse AmpliStain enables at least three times more sensitive detection
of mouse Abs, whereas anti-Rabbit AmpliStain is on the average ten times stronger
than anti-Rabbit EnVision.
Our results show that the AmpliStain™ HRP system is a more sensitive method that
allows higher dilutions of the primary Abs. Based on the German market pricing, price
per test of the EnVision™+ system is markedly higher than that of the AmpliStain™
system. Moreover, the aggregate cost per test with AmpliStain™ HRP can be further
reduced thanks to the much higher dilution of the primary Abs. Apart of allowing
cheaper immunohistochemical assays, higher dilutions of the primary Abs with
AmpliStain™ HRP system also enable obtaining more reliable results since higher Ab
dilutions prevent unwanted background staining.
The advantages of coupling high efficiency ion mobility separation with MALDI MS imaging of on-tissue digested proteins Claude E.
1, Langridge J.
1
1Waters Corporation, Manchester, United Kingdom
Aims: The main advantages of MALDI imaging mass spectrometry (MSI) introduced
in 1997 of by Caprioli et al are the unnecessecity to label the molecules (i.e. proteins)
to image them but also the ability to image multiple molecules within one MS
experiment. The disadvantages of this technique when analyzing proteins is the lack of
identification of the proteins of interest which help with the understanding of the
biological processes. Indeed to identify a protein by MSI, one needs to fragment the
Abstracts of Poster Presentation
83
protein to get the full or part sequence of the amino acid sequence that constitute the
protein due to the lack of collision energy but also to perform tandem mass
spectrometry, the precursor ion needs to be selected and it becomes problematic after a
certain m/z regardless of the mean of selection. By performing a form of enzymatic
digestion of the proteins (typically trypsin) directly from tissue section, one can have
access to its peptides which have a lower m/z and therefore are more suitable to
MS/MS identification. A means of increasing the separating power of a MALDI
imaging experiment is the use of high efficiency ion mobility separation (IMS),
coupled with time-of-flight mass spectrometry which offers a new dimension of
separation. Using this technique it is possible to separate different compound
classes.However in this study, high efficiency ion mobility separation (IMS) was used
for the analysis of tryptic digestion on-tissue sample as a mean of selectivity to
decongest the MS imaging data but also to differentiate isobaric species.
Methods: The sample under investigation was a 12 µm section of rat brain. The tissue
section was washed with ethanol and chloroform. Trypsin solution was sprayed onto
the tissue section using the SunCollect (SunChrom, Germany) and incubated for 5
hours at 37°. 5 mg/mL of CHCA mixed with aniline solution was subsequently
sprayed onto the tissue using the SunChrom. Data were acquired on a MALDI G2
HDMS system (Waters Corporation, Manchester, UK) operated in HDMS mode over
the m/z range of 700 to 3000 with a laser repetition rate of 1000 Hz. After acquisition
HDMS data were processed and visualised using High Definition Imaging (HDI)
software (Waters Corporation, Manchester, UK) where ion mobility dimension is fully
integrated in the software. Subsequently, MS/MS experiments were carried out on an
adjacent tissue section for identification of the tryptic peptides, directly from tissue.
Results: Here we show how ion mobility separation can be used to provide a
dimension of separation that can be used post ionisation and hence can be utilised in a
MALDI imaging experiment. Using the new HDI software, example of ions images
from tryptic peptides and lipids that have been separated by ion mobility will be
presented.
Furthermore, identification by MS/MS of tryptic peptides directly from tissue will be
demonstrated.
Abstracts of Poster Presentation
84
Combining lipidomics and proteomics by MALDI-MSI Crecelius A.C.
1,2, von Eggeling F.
3, Schubert U.S.
1,2
1Friedrich-Schiller-University Jena, Laboratory of Organic and Macromolecular
Chemistry (IOMC), Jena, Germany, 2Friedrich-Schiller-University Jena, Jena Center
for Soft Matter (JCSM), Jena, Germany, 3Friedrich-Schiller-University Jena, Institut
für Humangenetik und Anthropologie Universitätsklinikum Jena, Jena, Germany
Introduction: Matrix-assisted laser desorption/ionization mass spectrometric imaging
(MALDI-MSI) is a method that allows the investigation of the molecular content of
tissues within its morphological context.[1] Recently, the use of multiple matrices by
applying inkjet-printing on a single tissue surface has been presented.[2] The aim of
this contribution is to show its useful application by analyzing two different compound
classes, lipids and proteins, in a variety of different tissue organs. This developed
methodology is in particular interesting when precious tissue samples are employed.
Experimental part: Organs, such as liver, brain, and kidney were rapidly extracted
from a C57BL/6J mouse and snap frozen in liquid nitrogen and stored at -80 °C. For
the MALDI-MSI analysis 12 µm thick sections were cut in a microcryotome and thaw
mounted on ITO glass slides. Prior to MALDI-MSI analysis the sections were dried in
a vacuum desiccator for 10 minutes. For the analysis of lipids a-cyano-4-hydroxy
cinnamic acid and for proteins sinapinic acid were used. The matrices were applied
with a spatial resolution of 500 x 500 µm across the tissue section with an offset of
250 µm x 250 µm using an inkjet-printer. A volume of 3 drops per spot during each
pass and 12 passes of matrix application were applied. Mass spectra were acquired on
an Ultraflex III MALDI-TOF/TOF instrument (Bruker Daltonics) running in the
positive mode. A m/z range of 500-1,600 for lipids and of 2,000-23,000 for proteins
were analyzed, respectively. A total sum of 500 shots per spot was acquired in steps of
50 shots in a random pattern. MS acquisition and visualization was performed using
FlexImaging and FlexControl software (v. 2.0, Bruker Daltonics).
Results and discussion: The methodology was first tested on mouse liver tissue, since
this is the most homogeneously available tissue. As expected the distribution of lipids
as well as proteins was more or less homogeneously as determined by a series of
MALDI-MSI images. The next investigated tissue type was mouse brain. Since the
Abstracts of Poster Presentation
85
limit of resolution is currently 500 µm for the application of two matrices on a single
tissue slice, some anatomical features of the mouse brain are presented by only a few
pixels in the recorded MALDI-MSI images, however a lower resolution caused the
generation of a homogeneous film instead of discrete spots. Further improvements are
still ongoing. Finally, mouse kidney was analyzed and the distributions of lipid and
protein signals were compared. The developed methodology shows the simple
combination of two compound classes or areas (lipidomics and proteomics) in the
emerging technique MALDI-MSI.
References:
[1] B. Balluff, C. Schöne, H. Höfler, A. Walch, Histochem. Cell Biol. 2011, DOI
10.1007/s00418-011-0843x.
[2] J. T. Delaney, A. Urbanek, L. Wehder, J. Perelaer, A. C. Crecelius, F. von
Eggeling, U. S. Schubert, ACS Comb. Sci. 2011, 13, 218.
Polarization optical and histochemical characterization and
supramolecular structure of carbohydrate-protein fibrils Csoka L.
1, Makovitzky J.
2, Eitner A.
2, Jirikowski G.
2
1University of West Hungary, Institute of Wood and Paper Technology, Sopron,
Hungary, 2Friedrich Schiller University, Institute of Anatomy II, Jena, Germany
Topooptical staining reactions were used to investigate chemically and structurally the
carbohydrate-protein structures of bacterial cellulose, chitosan and alginic acid from
brown algae. Chitosan and alginic acid are associated with glucosaminoglycans
(GaGs), which appear as a continuous sheath enveloping the axial layer or periodically
distributed along the fibrils. The results obtained with polarization microscopy indicate
that reactive carbohydrates and proteoglycans contain chemical end-groups similar to
the ones found in human amyloid fibrils. The introduced staining sequences can be
used as a reliable method for the histochemistry with light and polarization microscopy
of GaGs.
Abstracts of Poster Presentation
86
Normalization in MALDI-TOF imaging of proteins Deininger S.O.
1, Cornett D.S.
2, Paape R.
1, Becker M.
1, Pineau C.
3, Rauser S.
4, Walch
A.4, Wolski E.
1
1Bruker Daltonik GmbH, Bremen, Germany,
2Bruker Daltonics, Billerica, United
States, 3INSERM U625, Proteomics Core Facility Biogenouest, Rennes, France,
4Helmholtz Zentrum München - Institute of Pathology, Munich, Germany
Introduction: In MALDI imaging normalization is often used to improve the quality
of the images, but there is still discussion in the field whether this procedure creates
artifacts and is therefore generally applicable. In protein imaging, the normalization to
total ion count (TIC) is often used, but other procedures such as Median or noise level
have also been investigated. In this work, we have used a kidney dataset to do a
quantitative comparison of the effects of different normalization procedures. While
this kidney dataset showed no artifacts due to normalization, we used a testis dataset
that did lead to artifacts after normalization to TIC. We investigated how potential
artifacts can be detected and avoided by correlation of different normalization
methods.
We also show data that stress the importance of normalization in the comparsion
across multiple datasets and how normalization can remove the effect of varying laser
intensites and instrument settings.
Results: On the kidney data, the normalization on TIC, median, noise, and vector
norm did not change the ratio of the average intensities in the different regions of the
kidney, which did indicate that there is no artifact introduced by normalization. The
variance of the selected signal in the individual regions was decreased after all
normalizations, indicating a positive effect of the normalization. The improvement in
the variance was stronger for the TIC and vector norm as opposed to the median or
noise level normalization. This could be confirmed by the respective images.
In the testis dataset, a peak with an unusually large area in some regions led to an
artifact in normalization to TIC and the vector norm, which could be evaluated by the
comparison with the non-normalized dataset. Normalization was necessary though for
the display of certain histological features. Here, the normalization to median and
noise level was able to improve the image with respect to histology without creating
Abstracts of Poster Presentation
87
artifacts. The biggest improvement was achieved by normalization to TIC with the
exclusion of the aberrant peak (TICx).
On another kidney dataset we performed a measurement with deliberately different
instrument settings, such as detector gain and laser energy. A principal component
analysis with and without normalization clearly shows how the effect of different
instrument settings is removed by the normalization. This observation is of crucial
importance for the analysis of clinical imaging data, where a study can be ongoing for
years.
Immunohistochemical investigations of keratin expression for a
characterization of epidermal stem cell niches in bovine fetal skin Ebach K.
1, Kenngott R.A.-M.
1, Sinowatz F.
1
1Institute of Anatomy, Histology and Embryology, Department of Veterinary Sciences,
LMU, Munich, Germany
Purpose: The pattern of keratin expression characterizes different cell types of
ectodermal origin. Especially cell populations with a potential to regenerate the
epidermis, hair follicles or glands show an expression pattern that is useable as a
marker for those cell types. During the bovine fetal skin development it is possible to
differentiate and observe changes in those cell niches by analysis of expression of
keratin 8, 15, 18 and 19.
Results: Results that give an impression of the diversity of epidermal cells are for
example the strong expression of keratin 8 and 15 which is confined to the outermost
cell layer of the hair bulge, whereas keratin 18 and 19 can be detected in inner and
outer cell layers of this compartment. An interesting finding is the expression of
keratin 18 around the bulge region, which appears strongly positive in its main part
and negative in its upper part. The keratin 18 negative area of the upper hair bulge
shows a strong staining with the Periodic Acid Schiff reaction (PAS), whereas the
major part of the hair bulge is PAS-negative.
Conclusions: These findings support the observations of other authors that these two
areas of stem cells can be distinguished by their characteristic expression pattern of
keratin 15, nestin and Lgr6 in murine and human tissue. The upper part of this stem
Abstracts of Poster Presentation
88
cell niche serves as a reservoir for pluripotent stem cells with the potential to
differentiate also into the neuronal cell lineages, whereas the larger lower part contains
multipotent cells that serve as source for all epidermal cell types. The comparison of
markers in human, murine and bovine skin provides a closer inspection of those
important cell niches and provides a basis for further studies dealing with the
regeneration of epidermal skin compartments.
Topo-optical follow up of phenothiazine induced charge transfer reactions in the yeast cell wall Tigyi Z.
1, Emody L.
1, Molnar J.
2, Jozsi M.
3, Makovitzky J.
4
1University of Pecs, Department of Medical Microbiology and Immunology, Pecs,
Hungary, 2University of Szeged, Department of Medical Microbiology and
Immunobiology, Szeged, Hungary, 3Hans Knöll Institute, Leibniz Institute for Natural
Product Research and Infection Biology, Jena, Germany, 4University of Heidelberg,
Department of Neuropathology, Heidelberg, Germany
In this paper phenothiazine induced charge transfer reactions are studied on various
yeasts, i.e. Candida species, Cryptococcus neoformans and Saccharomyces cerevisiae.
Pre-treatment of the fungal smears with any of eleven phenothiazine compounds
resulted in a weak linear positive birefringence with respect to the cell surface even
without subsequent addition of dyes. Completing the CT reaction by staining the
preparations with Eosin, Erythrosin or Rose Bengal, respectively, did not change the
character of birefringence but strongly increased its intensity. Further polarization
optical analysis revealed that the phenothiazine and xanthen dye complexes are bound
in parallel orientation to the fungal cell wall in a sterically oriented manner. Pre-
treatment with 1% aqueous periodic acid abolished the charge-transfer reactions while
pre-treatment with lipid solvents did not affect them. These results suggest that
oxidation of the hydroxyl (OH) groups in the sugars to aldehyde groups could inhibit
either their interaction with the phenothiazine compound or the complex formation of
the xanthen dye with the fungal cell surface component. Methylation of the different
fungal cell wall sugars entirely ceased the charge transfer reactions. The individual
fungal species required different incubation times of methylation to abolish their
Abstracts of Poster Presentation
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charge transfer reactions, i.e. C. krusei, C. tropicalis 6 hours, C. glabrata 12 hours and
C. albicans 18-24 hours, respectively. These results suggest that methylation of the OH
groups in the sugar compounds could alter the interactions with phenothiazine
molecules, and consequently their complex formation with eosin molecules in fungal
cell surface structures. However, treatment with periodic acid-bisuphite after the
methylation reaction reconstituted the chlorpromazine-eosin CT reaction resulting in
an intensive deep green birefringence. Taking together our results point to a pivotal
role of yeast cell wall sugar OH groups in the phenotiazine induced topo-optical
phenomena. Enzymatic reactions and extraction procedures are planned to disclose the
fine molecular mechanisms of these charge transfer interactions.
MALDI imaging of endogenous compounds directly in human skin-
tissue sections Enthaler B.
1,2, Pruns J.
1, Wessel S.
1, Rapp C.
1, Fischer M.
2, Wittern K.-P.
1
Institute(s):
1Beiersdorf AG, Hamburg, Germany,
2University of Hamburg, Hamburg School of
Food Science, Hamburg, Germany
Since introduction of matrix-assisted laser desorption/ionization imaging mass
spectrometry (MALDI-IMS) by Caprioli et al. in 1997, there has been an increasing
number of publications in the last few years. However, only a few groups have dealt
with skin tissue and subsequent MALDI-IMS measurements up to now. The
localization of endogenous and exogenous compounds directly in tissue sections,
including the investigation of biological processes within tissue, is a challenging task
in skin research.
In this manuscript, we report the possibilities of MALDI-IMS in localization of
endogenous compounds directly in human skin-tissue sections. In general, handling
and properties of skin tissue differ from that of other organs, e.g. the kidney.
According to the practical procedure one main difficulty is caused by the low adhesion
of skin-tissue sections to commercially available indium-tin-oxide-coated (ITO) glass
slides, resulting in detachment of tissue sections while matrix coating or hematoxylin
and eosin (HE) staining. In this work, corona-discharge treatment was used to modify
Abstracts of Poster Presentation
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the glass-slide surface. As a result, adhesion of skin-tissue sections to ITO-coated glass
slides was improved. Surface modifications were investigated by electron
spectroscopy for chemical analysis (ESCA). This represents an improved sample
preparation procedure for MALDI-IMS measurements of skin-tissue sections (1).
Furthermore, we describe the development of an approach that performs MALDI-IMS
on skin-tissue sections followed by removal of the matrix by washing steps and
subsequent histological staining. Corresponding to the size of some specific skin areas,
e.g. sweat glands, the use of serial sections is not advisable, because of their non-
identical character. It cannot be assumed that specific skin areas are present in both
serial sections or rather located at the identical area. Hence, it is advisable to use one
single skin-tissue section.
For localization of endogenous skin compounds by MALDI-IMS, different matrices
were applied. Using 9-aminoacridine and the negative-ion mode, the distribution of
primary metabolites like ATP within tissue sections were acquired. The assignment to
specific skin areas was enabled trough co-registering of MALDI results and the
corresponding HE-staining image. The compounds were identified by post-source
decay.
Additionally, the poster employs the possibilities of MALDI-IMS in mapping proteins
and tryptic peptides directly in skin-tissue sections.
(1) Enthaler et al. (2011), Improved sample preparation for MALDI-IMS and mapping
of endogenous and exogenous compounds directly in skin-tissue sections, Analytical
and Bioanalytical Chemistry (submitted)
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91
The intramitochondrial bacterium Midichloria: novel tools for its
detection Mariconti M.
1, Epis S.
1,2, Sacchi L.
3, Biggiogera M.
3, Sassera D.
1, Bandi C.
1,
Bazzocchi C.1
1University of Milan, Milano, Italy,
2University of Camerino, Camerino, Italy,
3University of Pavia, Pavia, Italy
Midichloria mitochondrii is a fascinating intramitochondrial bacterium symbiont of
Ixodes ricinus, an arthropod of medical and veterinary interest. Midichloria is present
in several cell types (luminal cells, funicular cells, and oocytes) of the reproductive
tissues of I. ricinus females. Electron microscopy shows that these bacteria multiply
inside the tick mitochondria, leading to the consumption of the mitochondrial matrix.
Current data indicate 100% prevalence in female I. ricinus and approximately 50% in
males. Available information suggests that Midichloria bacteria circulate among ticks,
likely through the infection of their vertebrate hosts. It is thus important to develop
tools for the specific detection and staining of these bacteria. Recently, the sequencing
of the Midichloria genome showed the presence of 26 genes coding for proteins of the
flagellar apparatus. This would be the first evidence for the presence of flagellar genes
in a bacterium of the order Rickettsiales. We thus decided to produce in recombinant
form the flagellar protein FLID of Midichloria, to be used as an antigen for serological
analysis, and for the production of polyclonal antibodies, with the aim of developing a
tool for the staining of these bacteria and their flagellum. Ticks ovaries were fixed and
examined by transmission electron microscopy (TEM), indirect immunofluorescence
assay and immunogold staining. Preliminary results indicate that anti-FLID antibodies
react with structures inside the Midichloria cells, but also at their surface and in the
surrounding cytoplasm and mitochondria of infected cells. This staining pattern appear
specific, since no staining was observed in oocytes of a closely related ticks, not
infected by Midichloria. The staining of Midichloria and its cellular structures might
have important implication in the study of tick symbiosis and tick-borne diseases, and
to investigate the possibility that these bacteria infect humans and animals.
Abstracts of Poster Presentation
92
Desorption Electrospray Ionization (DESI) imaging of tumor
samples - using mass spectrometry to support classical histology Gerbig S.
1, Golf O.
1, Schaefer K.C.
1, Balog J.
2, Takáts Z.
1
1Justus-Liebig-University, Analytical Chemistry, Gießen, Germany,
2Medimass Ltd.,
Budapest, Hungary
Aims: Identification of tissue samples based on their specific phospholipid profile
using desorption electrospray ionization mass spectrometry
Methods: Desorption Electrospray Ionization (DESI) is a mass spectrometric
ionization method described in 2004. DESI is suitable for the analysis of various types
of molecules and it is capable for imaging mass spectrometric investigation of intact
tissue samples. Tissue samples are sectioned using a cryotome and analyzed in their
native state without further sample preparation. The phospholipid profile of tissue
samples was investigated by scanning the surface with DESI. For the identification of
the detected ion signals, high resolution and tandem mass spectrometry was used.
Results: DESI imaging experiments included investigation of different healthy and
cancerous tissue samples. For comparison of the detected structures with histological
findings, H&E stained sections of the samples were produced and tissue areas on the
sections were assigned by pathologists.
Summarized spectra of the different tissue areas visible in the ion images were
extracted from raw MS data and stored in an SQL-type database. Data was processed
using multivariate statistical analysis scheme, comprising Principal Component
Analysis (PCA) and Linear Discriminant Analysis (LDA) which classify spectra
according to their similarities and differences. The results showed that the spectra of
similar tissues are grouped together, while distinct separation of data points was
observed for different histological types.
Construction of a spectral library using reference samples enables the mass-
spectrometry based identification of unknown samples. This identification of unknown
samples was performed in particular for every single pixel, resulting in an artificial
histological map of the tissue section visualizing tissue structures of 100-200 µm. This
extends the potential of automated tissue recognition and leads to a mass-spectrometric
histology-like approach.
Abstracts of Poster Presentation
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Conclusions: DESI imaging allows the mass spectrometric characterization of tissue
samples based on the phospholipid profile. Clinical applications of this approach
include the rapid identification of tissues during surgical interventions. Using
electrosurgical tools coupled to mass spectrometric analysis (e.g. REIMS), spectra are
collected in-situ. Real-time analysis of these spectra based on the spectral database can
provide assignment of tissue in-vivo and in case of tumor resection indicate whether
the surgeons keep sufficient margin or already dissect tumor-infiltrated tissue.
Ribonucleoprotein-containing foci increase in size upon cell-cycle exit in cell nuclei from patients affected by myotonic dystrophy
type 2 Giagnacovo M.
1, Malatesta M.
2, Cardani R.
3,4, Meola G.
5, Pellicciari C.
1
1University of Pavia, Department of Animal Biology, Pavia, Italy,
2University of
Verona, Department of Neurological, Neuropsycological, Morphological and Motor
Sciences, Verona, Italy, 3University of Milan, Department of Molecular Biology and
Biotechnology, Milan, Italy, 4Centre for the Study of Neuromoscular Diseases - CNM,
Milan, Italy, 5University of Milan, Department of Neurology, IRCCS Policlinico San
Donato, Milan, Italy
Myotonic dystrophies (DM) are genetically-based neuromuscular disorder with
multisystemic traits among which muscle hyperexcitability (myotonia), muscular
dystrophy with increased number of clumped or centrally located nuclei in skeletal
muscle fibres, dilated cardiomyopathy and cardiac conduction defects (1)
. Two DM
forms exist, called DM1-Steinert's disease (OMIM 160900) and DM2 (OMIM
602688). DM1 is more severe, and depends on the expansion of a (CTG)n
trinucleotide sequence in the 3' untranslated region of the Dystrophia Myotonic Protein
Kinase gene (OMIM 605377). DM2 displays a milder clinical phenotype and is caused
by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc
Finger Protein (ZNF)-9 gene (OMIM 116955). DM pathogenesis depends on the
accumulation of the expanded RNAs in the nucleus resulting in the ectopic
sequestration of several RNA-binding proteins (CUGBP1, MBLN1) and some
ribonucleoprotein (RNP) splicing factors (2)
into peculiar intranuclear foci. The
Abstracts of Poster Presentation
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functional deregulation of these protein factors results in the general alteration of the
mRNA pathways in the cells of different tissues (3)
. The cytochemical detection of
expanded RNAs or MBNL1 in the nuclear foci is an accepted diagnostic marker for
DMs (4)
. It is apparent that in DM patients the most significant pathological features
are found in the skeletal muscle, heart and the central nervous system where non-
cycling cell populations (i.e., myofibres, cardiomyocytes or neurons) are mainly
found; on the contrary, cells from self-renewing tissues (such as skin fibroblasts or
layering epithelial cells) appear much less affected.
In this investigation, we aimed to elucidate whether in DM tissues the exit of cells
from the cell cycle may lead to the increase in size of intranuclear foci due to the
progressive sequestration of protein factors (namely MBNL1); to do this, we
investigated by immunocytochemical and morphometric techniques the fate of
MBNL1-containing foci in proliferating cells during the cell cycle, and in non-cycling
cells. Cultured skin fibroblasts from DM2 patients were chosen as a model cell system.
We found that nuclear MBNL1-containing foci do not associate with chromosomes at
mitosis, and remain in the cytoplasm at cytodieresis, being disassembled in early G1
and re-formed in the nucleus, at each cell cycle. After cells had spontaneously stopped
dividing in senescing fibroblast cultures, the nuclear foci were observed to increase in
number and size. Interestingly (and consistently), morphometric measurements on
sections of muscle biopsies taken from the same DM2 patients at different ages
demonstrated that in myonuclei MBNL1-containing foci become larger with
increasing patient's age.
References:
1 - Meola G and Moxley RT 3rd
, J Neurol 251:1173-82, 2004
2 - Malatesta M et al., Histochem Cell Biol 135, 419-25 , 2011
3 - Schoser B and Timchenko L, Curr Genomics 11:77-90, 2010
4 - Cardani R et al., Eur J Histochem 50:177-82, 2006
Abstracts of Poster Presentation
95
Renal ischemia-reperfusion injury causes intercalated cell-specific
disruption of occludin in the collecting duct Lee S.-Y.
1, Shin J.-A.
1, Kwon H.M.
2, Weiner I.D.
3, Han K.-H.
1
1Ewha Womans University School of Medicine, Department of Anatomy, Seoul, Korea,
Republic of, 2University of Maryland School of Medicine, Division of Nephrology,
Baltimore, United States, 3University of Florida College of Medicine, Division of
Nephrology, Gainesville, United States
Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose
of this study was to examine the effects of ischemia-reperfusion (IR) injury on
expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat
kidney. IR injury was induced by clamping both renal pedicles for 30 minutes and
animals were sacrificed at 6 hours after the reperfusion. IR injury decreased blood
bicarbonate level but did not persistently alter pH, Na+, K
+, or Cl
−. The control kidneys
showed strong occludin immunoreactivity in the tight junctions of the thick ascending
limb, distal convoluted tubule, and collecting duct. Occludin staining was moderate in
the thin limbs of the loop of Henle, and was not detected in the proximal tubule,
glomerulus, and blood vessels. ZO-1 was expressed in the same sites as occludin, and
in addition was also expressed in the proximal tubule, glomerulus, and vascular
endothelial cells. In the IR injury kidneys, many collecting duct cells, as well as
proximal tubule cells, in the outer medulla were damaged. In the collecting duct,
intercalated cells lost their polarity and occludin was localized diffusely in the
cytoplasm after IR injury, whereas principal cells were unaffected. The ZO-1 patterns
showed abnormal cellular distribution in both the proximal tubule and collecting duct.
IR injury did not detectably alter the occludin and ZO-1 distribution in the thick
ascending limb and collecting duct principal cells. The abundance of occludin protein
expression was not changed with IR injury. In summary, renal IR injury causes
cellular damages not only in the proximal tubule but also in the collecting duct and
that the disruption of tight junctions, particularly occludin, is cell-specific to
intercalated cells in the collecting duct. The collecting duct is composed of two
histologically different cell types, the principal cells and intercalated cells. The
principal cells reabsorb water, whereas the intercalated cells are involved in acid-base
Abstracts of Poster Presentation
96
homeostasis. The results may have significant value in understanding clinical
manifestations of renal ischemic injury such as metabolic acidosis.
Key words: Collecting duct, ischemia/reperfusion injury, kidney, occludin, tight
junction
This work was supported by National Research Foundation of Korea (2009-0073733,
2011-0016068).
E-cadherin mutations contribute to gastric carcinogenesis by modulating multiple EGFR-dependent downstream signalling
pathways Heindl S.
1, Malinowsky K.
1, Wolff C.
1, Luber B.
1
1TU München, Pathology, München, Germany
Mutations in the gene encoding the cell adhesion molecule E-cadherin and
overexpression of the epidermal growth factor receptor (EGFR) represent fundamental
genetic alterations associated with diffuse-type gastric cancer. Mutations, e.g. in frame
deletion of exon 8 (del 8), frequently affect putative extracellular Ca2+
-binding sites of
E-cadherin, thereby impairing its functionality. Several studies have shown a
bidirectional crosstalk between E-cadherin and EGFR. Functional wild-type (wt) E-
cadherin inhibits ligand induced EGFR activation, but mutations lead to a loss of its
suppressive function, resulting in increased EGFR activation and recruitment of
downstream signalling components. The phenotype of cells harboring such mutations
is characterized by increased proliferation and elevated migratory and invasive
potential.
Our project aims at studying the impact of somatic mutations of E-cadherin on EGFR-
mediated signalling pathways, in order to identify signalling molecules of these
pathways as molecular targets for selective therapies in gastric cancer.
Cell lines expressing wt and mutant E-cadherin (del 8) were EGF-stimulated and
cellular lysates were analyzed by commercial proteome profiler antibody array (R&D
Systems). The array allowed detection of the phosphorylation status of 48 signalling
molecules and facilitated identification of differential activation. We found differential
response to EGF stimulation of proteins involved in DNA damage and repair. Whereas
Abstracts of Poster Presentation
97
activity of tumorsuppressors p53 and CHK2 increased with EGF-stimulation for
different time spans in cells expressing wt E-cadherin, it remained low or even
decreased in cells with mutations of E-cadherin. As upon DNA damage both proteins
are needed for induction for DNA repair and initiation of cell cycle arrest or cellular
apoptosis, a loss of function of these molecules can lead to uncontrolled cell cycle
progression and increased proliferation promoting tumor progression. Furthermore,
Src-family kinases (SFKs) like Src, Yes, Fgr, Lck and Lyn exhibited similar activation
profiles in response to EGF-stimulation as described above. As SFKs are key players
in multiple cellular processes like cell growth, differentiation, cellular adhesion,
migration and invasion, the impact of their differential activation on the cellular
phenotype is subject of ongoing experiments. To determine the role of the identified
targets in gastric carcinogenesis and the mechanisms of crosstalk between E-cadherin
and EGFR a protein microarray platform was established, allowing analysis of
multiple samples under the same experimental conditions. This approach will reveal
new insights in EGFR-mediated signalling pathways contributing to gastric
carcinogenesis and complete our data describing how tumor associated mutations of E-
cadherin affect cellular signalling.
Immunohistochemical detection and non-invasive U-SPECT
imaging reveal distinct tumor tissue distribution patterns of anti-EGFr antibody zalutumumab in a tumor xenograft model Houtkamp M.A.
1, Branderhorst W.
2, Blezer E.L.A.
2, Witteveen H.
1, Gerritsen A.F.
1,
Parren P.W.H.I.1, van Dongen G.A.M.S.
3, Beekman F.J.
4, Bleeker W.K.
1
1Genmab, Utrecht, Netherlands,
2University Medical Center Utrecht, Image Sciences
Institute, Utrecht, Netherlands, 3VU University Medical Center, Nuclear Medicine &
PET Research, Amsterdam, Netherlands, 4MILabs B.V., Utrecht, Netherlands
Therapeutic antibodies (Abs) for cancer treatment must penetrate solid tumors and
bind to a tumor-specific target in order to be effective. To gain further insight in these
aspects for zalutumumab, a human anti-EGFr monoclonal Ab (mAb), we have studied
an A431 xenograft model with high EGFr expression and intratumoral necrotic areas.
111In-labeled zalutumumab was administered at a non-saturating dose and total body
Abstracts of Poster Presentation
98
111In distribution was determined in vivo at different time points using ultra-high-
resolution focused pinhole SPECT imaging. After 48h, the A431 tumor was removed
for ex vivo SPECT imaging and immunohistochemical (IHC) analysis of zalutumumab
biodistribution and EGFr (target) expression . Consecutive whole tissue IHC images
were microscopically captured and co-registered to the SPECT images at equidistant
reference points. With IHC, the in vivo administered zalutumumab showed a diffuse
distribution throughout the A431 tumor, which appeared to be exclusively localized
within areas of high EGFr expressing tumor cells, but was absent in other tumor cells
with lower EGFr expression. Interestingly, ex vivo SPECT imaging after 48h showed
overlapping 111
In tumor distribution within all EGFr target expressing tumor cells but
also showed hotspots just outside of the tumor area. Both IHC and SPECT imaging
were negative for intratumoral necrotic areas. An explanation for the observed
differences between IHC and U-SPECT could be that IHC mainly detects intact tumor
cell-associated zalutumumab, whereas 111
In imaging also detects accumulated 111
In
derived from internalized and degraded zalutumumab. The hotspots might then be
indicative of uptake of 111
In-labeled tumor cell debris by tissue macrophages which
appear in clusters adjacent to the tumor. We conclude that SPECT imaging is a
suitable technique for non-invasive assessment of Ab penetration into the tumor,
which provides insight in target localization. When evaluating U-SPECT images it
should be kept in mind that part of the signal may be related to residual 111
In derived
from antibody uptake and subsequent degradation.
Immune laser capture microdissection of macrophages in engineered dental pulp tissues Kaneko T.
1, Yamanaka Y.
1, Yoshiba K.
1, Okiji T.
1
1Niigata University Graduate School of Medical and Dental Sciences, Division of
Cariology, Operative Dentistry and Endodontics, Niigata, Japan
We have recently developed a method of engineering dental pulp tissues from stem
cells from human exfoliated deciduous teeth, by culturing these cells in a poly-L-lactic
acid scaffold placed in the canal space of human tooth slices for 14 days. In the
engineered tissue, macrophages may play some roles in absorbing the scaffold by their
Abstracts of Poster Presentation
99
phagocytic ability or maintaining the tissue homeostasis by acting as antigen
presenting cells. To examine the roles of macrophages in the engineered dental
pulp tissue, we performed immune-laser capture microdissection (LCM) of CD68
(anti-human macrophages)-immunoreactive cells from formaldehyde-fixed (24 h),
formic acid-demineralized (7 days) and paraffin-embedded tissues, followed by a real-
time PCR analysis on the mRNA expression of class II MHC, which is highly
expressed in activated antigen presenting cells. Results demonstrated that class II
MHC mRNA expression levels were significantly higher in macrophages retrieved
from the area where most of the scaffold was absorbed, as compared with
macrophages in the area where most of the scaffold was still present. The results
suggested that macrophages show different activation levels in different area of
engineered tissues. The immune-LCM method presented here allows for the
quantitative analysis of gene expression in paraffin-embedded tissue sections from
demineralized specimens, suits for the analysis of relatively rare cell types within a
tissue, and thus may constitute a new approach for histochemistry of mineralized
tissues, Also, the immune-LCM method may open the door for the acquisition of new
data from archived specimens, and may improve our ability to perform differential
diagnosis of pathologies.
Autophagosome formation by quercetin Klappan A.
1, Mylonas I.
1, Friese K.
1, Brüning A.
1
1University Hospital Munich, OB/GYN, Munich, Germany
Aims: Many studies have shown that quercetin, a bioflavonoid, causes anti-cancer
effects. However, the mechanism behind this remained unknown. We have performed
cell biological studies to analyse the effect of quercetin on human cancer cells.
Methods: The occurrence of autophagy was shown in viable cells by using fluorescent
autophagy markers and acidotropic dyes. Proteasome activity was analysed by a
bioluminescence assay using synthetic substrates. Western blot analysis was
performed to investigate the mTOR signalling pathway.
Abstracts of Poster Presentation
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Results: Application of quercetin to epithelial human cancer cells induced strong
intracellular vacuolation that caused cell cycle arrest and apoptosis. These vacuoles
could be characterized as phagolysosomes. Immediately after application of quercetin,
inhibition of mTOR activity, an autophagy-controlling pathway, occurred in quercetin-
treated cancer cells, as revealed by reduced phosphorylation of 4E-BP1 and p70S6
kinase, all major mTOR substrates. Evaluation of cellular proteasome activity in
quercetin-treated cancer cells showed an effective and immediate inhibition of all three
enzymatic activities of the proteasome and, as a result, accumulation of
polyubiquitinated proteins and protein aggregates.
Conclusions: Proteasome inhibition by quercetin and triggering of massive autophagy
due to the accumulation of polyubiquitinated proteins can be seen as a major
contributor to quercetin-induced cancer cell death. Since proteasome inhibitors
represent a highly effective group of anti-cancer drugs, these results suggest potential
new applications for quercetin in cancer treatment.
Spatial segmentation of hyperspectral 3D volume data Kobarg J.H.
1, Alexandrov T.
1,2
1University of Bremen, Zentrum für Technomathematik, Bremen, Germany,
2Steinbeis
Innovation Center SCiLS (Scientific Computing in Life Sciences), Bremen, Germany
Segmentation of hyper-spectral imaging data using clustering requires special
algorithms, which consider spatial relations between the pixels. This strategy can
improve the clustering of noisy data, since neighbor pixels should usually be clustered
into one group. However, in the case of the spectral dimension p being large, cluster
algorithms already suffer from the curse of dimensionality and have high memory
needs as well as long runtimes.
We propose to incorporate neighboring pixels from a window of w×w pixels to define
a feature space of size npw², then apply a clustering method to the projected points.
The effect of improvement is controlled by weights depending on the spatial distance
between the pixels to be clustered. We propose a data-adaptive way to define weights
based on the similarity of pixels. Any vectorial clustering algorithm like k-means can
Abstracts of Poster Presentation
101
be directly applied to the projected points. In addition, we employ FastMap, to find a
Euclidean space of dimension nq corresponding to the projection.
The proposed algorithm is well suited for hyper-spectral imaging data as found in
imaging mass spectrometry where the number of pixels is relative high (n≈104).
Literature:
Alexandrov, T. and Kobarg, J.H. (2011). Efficient spatial segmentation of large
imaging mass spectrometry datasets with spatially aware clustering. Bioinformatics,
27, i230-i238.
Faloutsos, C. and Lin, K.-I. (1995). FastMap: a fast algorithm for indexing, data-
mining and visualization of traditional and multimedia datasets. ACM SIGMOD, May
1995, San Jose, CA, pp. 163--174.
Tomasi, C. and Manduchi, R. (1998). Bilateral filtering for gray and color images. Intl.
Conf. Computer Vision, Jan. 1998, Bombay, India, pp. 839--846.
pHPMA-based tissue embedding medium compatible with MALDI
mass spectrometry imaging experiments Strohalm M.
1, Strohalm J.
2, Kaftan F.
3,4, Krásný L.
1,5, Volný M.
1, Novák P.
1, Ulbrich
K.2, Havlíček V.
1
1Institute of Microbiology, Academy of Science of Czech Republic, Laboratory of
Molecular Structure Characterization, Prague, Czech Republic, 2Institute of
Macromolecular Chemistry, Academy of Science of Czech Republic, Prague, Czech
Republic, 3Institute of Organic Chemistry and Biochemistry, Academy of Science of
Czech Republic, Prague, Czech Republic, 4Charles University, Department of
Analytical Chemistry, Faculty of Science, Prague, Czech Republic, 5Institute of
Chemical Technology, Department of Biochemistry and Microbiology, Prague, Czech
Republic
Due to the ion suppression effect in MALDI ionization and number of background
peaks in the low-mass region, traditional tissue-sectioning embedding media are not
suitable for mass spectrometry imaging (MSI) experiments. Droplets of water, that
does not affect MALDI ionization, are often used to mount tissue on a sectioning
Abstracts of Poster Presentation
102
target. But, the ice block formed around the tissue does not provide a good support for
sectioning of fragile samples. In this work, we propose a novel embedding medium
based on the poly[N-(2-hydroxypropyl)methacrylamide], providing smooth cutting
surface and fully compatible with MSI experiments and conventional histological
haematoxylin-eosin staining.
Cordycepin induces double strand breaks and the DNA damage response in breast cancer cells Lee H.J.
1, Burger P.
1, Vogel M.
2, Brüning A.
1, Friese K.
1
1University Hospital Munich, OB/GYN, Munich, Germany,
2University Hospital
Munich, Munich, Germany
Aims: Cordycepin (3-deoxyadenosine) is a fungal metabolite known to exert anti-
inflammatory effects and anti-tumor activities. We have investigated the effect of
cordycepin on growth and cell survival of human breast cancer cells.
Methods: Cell survival and apoptosis of human breast cancer cells was analyzed by
the MTT assay and FACScan analysis. Activation of intracellular signalling pathways
was investigated by immunoblot and immunofluorescence analysis.
Results: Treatment of human breast cancer cells with cordycepin reduced cell
proliferation and clonal growth. Cordycepin treatment was associated with marked
morphological changes, resulting in a multi-nuclear and differentiation-like phenotype.
At the molecular level, a strong induction of the DNA damage response (DDR), which
included phosphorylation of ATM, ATR, and histone gammaH2AX, could be
observed. This indicates induction of DNA double strand breaks induced by
cordycepin in proliferating breast cancer cells, resulting either in apoptosis or
persistent cell cycle arrest.
Conclusions: The genotoxic effect of cordycepin on proliferating breast cancer cells
indicates a new mechanism of cordycepin-induced cancer cell death, and its
preferential activity against highly proliferative and undifferentiated breast cancer cells
indicates new perspectives for cordycepin in the treatment of advanced breast cancer.
Abstracts of Poster Presentation
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MALDI MSI for ovarian cancer biomarkers research: latest
developments of the technology for screening and tracking. Longuespée R.
1,2, Boyon C.
1,3, Kerdraon O.
4, Desmons A.
1, Vinatier D.
3, Fournier I.
1,
Day R.2, Salzet M.
1
1Laboratoire de Spectrométrie de Masse Biologique Fondamentale et
Appliquée/Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France,
2Institut de Pharmacologie de Sherbrooke/Université de Sherbrooke, Sherbrooke,
Canada, 3Service de Chirurgie Gynécologique/Hôpital Jeanne De Flandre/CHRU
Lille, Lille, France, 4Centre d'Anatomie et de Cytologie Pathologiques/CHRU Lille,
Lille, France
Ovarian cancer is the second major cause of gynecological death in Europe and USA.
Ovarian cancer has been studied at a proteomic level for biomarkers hunting with the
emergence of diverse techniques such as 2D electrophoresis and SELDI TOF MS. At
this time, CA125 is still the only biomarker used for the monitoring of the pathology.
Its sensitivity is 80% for stage III and stage IV cancers but only 30% for early stages
with high rates of false positives. Since its introduction in the early 90's, MALDI
Imaging Mass Spectrometry (IMS) has widely been used for biomarkers discovery in
many pathologies thanks to the possibility to discriminate the localization of
compounds in tissues of interest. Using this technology, we found the Cter part of
PA28, “Reg Alpha” present in the cancerous regions of ovarian biopsies. Recently, we
found this peptide in stage I epithelial cancer tissues, making it a potential marker for
wide screenings among women populations. Then, our recent developments in the
field of IMS allowed us to associate Histo Immuno Chemistry and IMS for the specific
tracking of this molecule in cancerous tissues. We indeed propose to incubate tissues
of interest with known anti-markers antibodies, then digest the surface of the tissues
with a solution of trypsin and finally perform the MALDI Image of the prepared
tissue, to reveal the presence of the tryptic peptides of the antibodies. It's now possible
to proceed to the quantitative detection of markers by directly analyzing the presence
of tryptic peptides of the antibodies targeting Reg Alpha. This procedure allows the
use of Immuno Histo Chemistry with the Mass Spectrometric devices as revealers
instead of a microscope, and would be strongly relevant for the tracking of cytotoxic
Abstracts of Poster Presentation
104
antibodies in tumors. These developments also pave the way for new pharmacologic
applications such as the monitoring of the effect of treatments of the pathology by
screening the evolution of the presence of the compound in malignant tissues.
Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-
fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the
Arbeitsgemeinschaft Internistische Onkologie (AIO) v Luber B.
1, Deplazes J.
1, Keller G.
1, Walch A.
2, Rauser S.
2, Langer R.
1, Höfler H.
1,
Fend F.3, Peschel C.
4, Lordick F.
5
1Technische Universität München, Pathology, München, Germany,
2Helmholtz
Zentrum München, Pathology, Neuherberg, Germany, 3Universität Tübingen,
Pathology, Tübingen, Germany, 4Technische Universität München, 3rd Medical
Department, München, Germany, 5Klinikum Braunschweig, 3rd Medical Department,
Braunschweig, Germany
Aims: The activity of the therapeutic antibody cetuximab against the epidermal
growth factor receptor (EGFR) and chemotherapy was assessed in first-line metastatic
gastric and gastro-esophageal junction cancer in a phase II study of the
Arbeitsgemeinschaft Internistische Onkologie (AIO) with objective tumor responses in
65% of patients and a low mutation frequency of KRAS (3%). The aim of the
correlative tumour tissue studies was to investigate the molecular mechanisms
underlying clinical response to treatment.
Methods: Patients were a subset from the phase II trial of cetuximab plus weekly
oxaliplatin, 5-fluorouracil and folinic acid (n=39). We performed FISH analysis,
immunohistochemistry and mutation analysis.
Results: We analysed the association of clinical outcome with (1) EGFR gene copy
number, (2) expression levels of EGFR, phosphorylated EGFR (pEGFR),
phosphorylated mitogen-activated protein kinase (pMAPK), phosphorylated Akt
(pAkt) and E-cadherin and (3) the mutation profile of selected exons of the E-cadherin
gene CDH1, KRAS and BRAF.
Abstracts of Poster Presentation
105
Our study showed a significant association between increased EGFR gene copy
number (≥4.0) and OS in gastric and OGJ cancer, indicating the possibility that
patients may be selected for treatment on a genetic basis. Furthermore, a significant
correlation was shown between activated EGFR and shorter TTP and ORR, but not
between activated EGFR and OS. No V600E BRAF mutations were identified. On the
other hand, an interesting trend between high E-cadherin expression levels and better
OS was observed and two missense mutations in exon 9 of the E-cadherin gene
(CDH1) (A408V and D402H) were detected.
Conclusions: Our data suggest that the EGFR gene copy status, activated EGFR and
E-cadherin are clinically important factors that may help to identify subgroups of
patients who are likely to benefit from EGFR-targeted therapy in combination with
chemotherapy.
The topo-optical investigation of Alzheimer Amyloid Makovitzky J.
1, Kovacs G.
1, Appel T.
1
1Niedersächsisches Internatsgymnasium, Bad Bederkesa, Germany
In 2010 we presented the examination of human amyloid fibrils with various topo-
optical staining reactions. We showed that different ex vivo amyloid fibrils (fibrils
isolated from post-mortem tissue) all contain lipids, chondroitin sulfate
glycosaminoglycans and sialic acid. The carbohydrates are accessible from outside of
the fibril, and highly ordered.
In the current study formalin-fixed paraffin-embedded brain samples of 10 Alzheimer
patients were investigated with topo-optical reactions. Neurofibrillary tangles, amyloid
plaques and vessels gave positiv staining reactions with Congo red, PSI, Pinacyanol,
Toluidine blue and Rivanol.
The amyloid deposits are resistent to potassium permanganate / trypsin and to
permanganate / pronase treatment. We found similar results after performic acid /
trypsin and performic acid / pronase treatment. The carbohydrate (ABT) and sialic
acid specific reactions were negative. We selectively demonstrated
glycosaminoglycans with the critical electrolyte concentration method (CEC). After
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106
GAG-specific enzymatic treatment the binding of Congo red to amyloid was
increased.
As a control, in vitro amyloid beta (1-40) and amyloid beta (1-42) fibrils are also
positiv with Congo red, PSI, Pinacyanol, Toluidine blue and Rivanol. In vitro fibrils
are negative with carbohydrate and sialic acid specific reactions and the critical
electrolyte concentration method (CEC).
Using topo-optical staining reactions we could differentiate between aggregated
protein, glycosaminoglycans, non-acidic carbohydrate and carbohydrates containing
sialic acid in Alzheimer tissue deposits. The staining reactions followed by microscopy
with polarised light provide strong analytical evidence for molecular components of
amyloid in a native (tissue) setting. Topo-optical reactions should be applied to other
kinds of amyloid tissue and studied in more detail.
Late gastrointestinal metastases of invasive lobular breast carcinoma mimicking Crohn´s disease Baranyay F.
1, Szabó J.
1, Makovitzky J.
2
1Kanizsai Dorottya Hospital, Pathology, Nagykanizsa, Hungary,
2University of
Heidelberg, Department of Neuropathology, Heidelberg, Germany
Introduction: Invasive lobular carcinoma - comprising approximatelly 10 percent of
breast cancers - is considered to be a histologically, molecular genetically distinct
entity metastatizing mainly the gastrointestinal tract. They manifest after 3-20 years
from the recognition of the primary tumor and they appear to be inflammatory disease
or a secundary tumor
Case report: The female patient with breast cancer died at the age of 53 years. 8 years
after tumor-free state upper abdominal spastic pain emerged irradiating into the back
with belt-like pattern. Radiologically Crohn´s disease was diagnosed. Ileum biopsy
was negative. Patient was treated ex juvantibus with methylprednisolon. In the
background of mechanic ileus the resection of the terminal ileum and partly the
ascended colon was surgically removed. The patient died in 3 weeks after operation.
Histology, immunohistochemistry: We compared the immunohistochemical reactivity
of the primary breast cancer, the axillary lymph glands, the terminal ileum, liver and
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107
spleen metastatic cells with ABH blood group specific lectins, antibodies to
cytokeratis, breast carcinoma antigen (BRCA), CA15-3, estrogen-progesteron
receptors, C-erbB-2, EMA, CEA, Ki 67 and ABH blood group antigens.
Autopsy findings: The whole stomach wall and the initial section of the duodenal wall
was diffusely thickened 4-5 mm in linitis plastica-like emergence. The lobular
structure of the liver was blured, firm palpation and diffusely with miliary reddish
nodules. The cut surface of the spleen appeare to be like a ham blured structure and
pale reddish colour.
Results: The sections of the terminal ileum showed intensive fibrotic, desmoplastic
reaction with lobular carcinoma cells infiltratring from the serosa to the mucosa in
"indian file" pattern.The carcinomatous cells in the primary tumor didn´t possess
estrogen receptors, however progesteron receptors could be detected in breast
carcinoma cells and in the metastatic cells of the axillary lymph glands. The
gastrointestinal tumor infiltrates, the cells of the liver and splenic metastases didn´t
react with anti-progesteron, nor with cerbB-2. All the applied antibodies were reacting
with cells of the primary tumor, the tumor cells of axillary lymph nodes, gastrintestinal
and hepato-lienar metastatic cells. The Ki-67 proliferation antigen detected with MIB-
1 labelled only 10% of metastatic carcinoma cells. The patient according to her red
blood cell phenotype belonged to AB blood group and the ABH specific lectins and
mabs reacted with both the primary carcinoma and the metastatic cells intensively.
Conclusion: Late invasive lobular carcinoma metastasis may mimic wide scale of
gastrointestinal disease or may simulate Crohn´s disease. They may cause linitis
plastica of the gastroduodenal wall and may bring about pseudo-cirrhosis and special
splenic metastasis. In our case all these listed gastrointestinal metastases occured in
one patient.
Abstracts of Poster Presentation
108
Role of macrophages in brown adipose tissue remodeling in
hyperinsulinemic rats Markelic M.
1, Velickovic K.
1, Golic I.
1, Otasevic V.
2, Stancic A.
2, Jankovic A.
2,
Vucetic M.2, Buzadzic B.
2, Korac B.
2, Korac A.
1
1University of Belgrade, Faculty of Biology, Belgrade, Serbia,
2University of Belgrade,
Institute for Biological Research 'Sinisa Stankovic', Belgrade, Serbia
There are two types of fat in mammals including humans - white (WAT) and brown
adipose tissue (BAT). WAT is an energy-storage, endocrine organ while BAT
functions as an energy-dissipating organ through adaptive thermogenesis. Previous
studies have suggested that macrophage (MA) functions and phenotypes vary
considerably in different fat depots. Evidence has accumulated indicating that obesity
is associated with a WAT chronic, low-grade inflammation (INF) with presence of
MAs proinflammatory phenotype, suggesting that INF may be a potential mechanism,
whereby obesity leads to insulin resistance (IR). Given the fact that little is known
about BAT-MA interactions, we investigated their possible role in BAT under
hyperinsulinaemia (HI) which is known to precede IR and represents one of the
metabolic syndrome symptoms.
The effects of different doses of insulin (0.4 IU or 4.0 IU/kg, i.p., 1 or 3 days) were
studied on Wistar rats. Saline-treated animals served as physiological controls. 3 hrs
after the last injection animals were sacrificed.
In BAT, MAs of all groups were mostly sparsely distributed. Generally they had a
round appearance, more alike to resident than to proinflammatory phenotype. In some
cases, massive MA infiltration around capillaries was visible, with an activated
phenotype (with secondary lysosomes, indicators of prominent phagocytic activity)
even in control animals. MAs were also present in the regions of erythrophagocytic
activity, which is common for BAs, still increased by insulin. In these regions, they
showed heme oxygenase 1 (HO-1) and HO-2 immunopositivity, indicating their
assistant role to BAs in hemoglobin degradation. Even monocytes inside of capillary
lumen near erythrophagocytic region were HO-1/HO-2 positive. MAs in other regions
showed no HOs positivity.
Abstracts of Poster Presentation
109
Although it is not clear what the responsible molecular mechanisms are, we have
shown that there are significant interactions between BAs and MAs that affect their
functions and probably differentiation. MAs in BAT of HI rats have no
proinflammatory phenotype, but rather serve in tissue remodeling. It is possible that
HOs expression in BAT MAs contributes to suppression of apoptosis, necrosis, INF
and oxidative stress, since it is known that CO and bilirubin suppress these processes
while the iron formed enhances the synthesis of the antioxidant ferritin. Also, HO
system has been shown to modulate both the metabolic and inflammatory systems
suppressing IR and INF. In this study, we have seen no signs of INF in BAT, and MAs
appeared quiescent, with exception of those in erytrophagocytic and apoptotic regions,
and some cases of their infiltrations around capillaries. Previously showed increased
angiogenic, adipogenic and hypertrophic effects of HI allow us to postulate MAs role
mainly in BAT remodeling, maintenance of insulin sensitivity and anti-inflammatory
state, even in condition of extreme HI.
Supported by Serbian Ministry for Education and Science, Grant 173055.
Selective Induction of Inhibin beta E by the ER stress reaction Matsingou C.
1, Brüning A.
1, Friese K.
1, Mylonas I.
1
1University Hospital Munich, OB/GYN, Munich, Germany
Aims: The endoplasmic reticulum (ER) is highly sensitive to changes in the pH value,
redox state, and calcium content. Especially in cancer cells, its ability to respond to
changes in homeostasis is of crucial importance for cancer cell survival under
conditions of hypoxia, interstitial acidification, and nutrient deprival. We have
investigated factors that could contribute to the survival of cancer cells under these
conditions.
Methods: ER stress was induced by various means, including the use of fungal
antibiotics, such as tunicamycin and calcimycin (A23187), and chemotherapeutics
such as nelfinavir and bortezomib. Induction of the ER stress reaction was verified by
XBP1 splicing analysis, and expression of ER stress target genes was monitored by
RT-PCR and immunofluorescence analysis.
Abstracts of Poster Presentation
110
Results: In addition to the induction of the canonical downstream targets of the ER
stress response, a strong induction of the peptide hormone Inhibin beta E could be
observed. Inhibin beta E expression was in direct association with the induction of the
ER stress response, independent of the inducing agent. Ectopic overexpression of
ATF4, a key transcription factor induced by the ER stress response, enhanced the
expression of Inhibin beta E. Other inhibin subunits remained unaffected by the ER
stress response.
Conclusions: The selective and strong induction of Inhibin beta E by the ER stress
reaction, mediated by activation of the transcription factor ATF4, provides a new link
of the ER stress response to the expression of inhibin beta E and its involvement in
cancer cell survival.
Proteomic markers for regional lymph node metastasis in primary
colon cancer tissues Meding S.
1, Balluff B.
1, Elsner M.
1, Schöne C.
1, Rauser S.
1, Nitsche U.
2, Maak M.
2,
Schäfer A.3, Hauck S.
3, Ueffing M.
3,4, Langer R.
5, Höfler H.
1,5, Friess H.
2, Rosenberg
R.2,6
, Walch A.1
1Helmholtz Zentrum München, Institute of Pathology, Neuherberg, Germany,
2Klinikum rechts der Isar, Technische Universität München, Department of Surgery,
München, Germany, 3Helmholtz Zentrum München, Research Unit Protein Science,
Neuherberg, Germany, 4Institute for Ophthalmic Research, Center for Ophthalmology,
University of Tübingen, Germany, 5Technische Universität München, Institute of
Pathology, München, Germany, 6Kantonsspital Baden, Klinik für Allgemein-, Viszeral-
und Gefässchirugie, Baden, Switzerland
Regional lymph node metastasis negatively affects prognosis in colon cancer patients.
Patients without metastases can often be fully cured by resection of the primary tumor.
For a priori determination of the metastatic potential of primary tumors, reliable
markers, especially proteomic ones, are still lacking. In this study, two complementary
proteomic methods, MALDI Imaging and label-free, quantitative proteomics, have
been employed in order to identify proteomic markers for regional lymph node
metastasis. A tissue cohort comprising of primary colon tumors which were either
Abstracts of Poster Presentation
111
without metastasis (UICC II, pN0, n = 21) or with lymph node metastasis (UICC III,
pN2, n = 33) was analyzed for proteomic marker identification. Validation by
immunohistochemistry was performed on an independent tissue cohort consisting of
primary colon tumors specimens (n = 168). MALDI Imaging yielded 10
discriminating m/z species and label-free, quantitative proteomics 28 proteins. Two
MALDI Imaging derived candidate proteins (FXYD3 and S100A11) and one coming
from label-free, quantitative proteomics (GSTM3) were validated on the independent
tissue cohort. FXYD3 (p = 0.0110), S100A11 (p = 0.0071) and GSTM3 (p = 0.0173)
had a significant correlation with lymph node metastasis. With our complementary
approaches it was possible to identify proteomic markers for regional lymph node
metastasis directly in primary colon cancer tissues.
S100-A10, Thioredoxin, and S100-A6 as biomarkers of papillary thyroid carcinoma with lymph node metastasis identified by
MALDI Imaging Nipp M.
1, Elsner M.
2, Balluff B.
2,3, Meding S.
2, Sarioglu H.
4, Ueffing M.
4, Rauser S.
2,
Unger K.5, Höfler H.
2,6, Walch A.
2, Zitzelsberger H.
1
1Helmholtz Zentrum München - Research Unit of Radiation Cytogenetics, Department
of Radiation Sciences, Neuherberg, Germany, 2Helmholtz Zentrum München - Institute
of Pathology, Neuherberg, Germany, 3Technische Universität München - Klinikum
rechts der Isar, Department of Medicine II, Munich, Germany, 4Helmholtz Zentrum
München - Core Facility Proteomics, Department of Protein Science, Neuherberg,
Germany, 5Imperial College London, Hammersmith Hospital, Human Cancer Studies
Group, Department of Surgery and Cancer, London, United Kingdom, 6Technische
Universität München - Institute of Pathology, Munich, Germany
In papillary thyroid carcinoma (PTC), metastasis is a feature of an aggressive tumor
phenotype. To identify protein biomarkers that distinguish patients with an aggressive
tumor behavior, proteomic signatures in metastatic and non-metastatic tumors were
investigated comparatively. In particular, matrix-assisted laser desorption/ionization
(MALDI) imaging mass spectrometry (IMS) was used to analyze primary tumor
samples. We investigated a tumor cohort of PTC (n=118) that were matched for age,
Abstracts of Poster Presentation
112
tumor stage and gender. Proteomic screening by MALDI-IMS was performed for a
discovery set (n=29). Proteins related to the discriminating mass peaks were identified
by 1D-gel-electrophoresis followed by mass spectrometry. The candidate proteins
were subsequently validated by immunohistochemistry (IHC) using a tissue
microarray for an independent PTC validation set (n = 89). In this study, we found 36
m/z (mass-to-charge-ratio) species that specifically distinguished metastatic from non-
metastatic tumors, among which m/z 11,608 was identified as thioredoxin, m/z 11,184
as S100-A10, and m/z 10,094 as S100-A6. Furthermore, using IHC on the validation
set, we showed that the overexpression of these three proteins was highly associated
with lymph node metastasis in PTC (p < 0.005). For functional analysis of the
metastasis specific proteins we performed an Ingenuity Pathway Analysis (IPA) and
discovered a strong relationship of all candidates with the TGF- β dependent EMT-
pathway. Our results demonstrated the potential application of the MALDI-IMS
proteomic approach in identifying protein markers of metastasis in PTC. The novel
protein markers identified in this study may be used for risk stratification regarding
metastatic potential in PTC.
The MALDI-imaging/ MULTI-ARRAY core facility: A new service
platform at the University of Bremen Oetjen J.
1,2, Maass P.
1,3, Maedler K.
2,4, Alexandrov T.
1,2,3
1University of Bremen, Center for Industrial Mathematics, Bremen, Germany,
2University of Bremen, MALDI-imaging lab, Bremen, Germany,
3Steinbeis Innovation
Center SCiLS (Scientific Computing in Life Sciences), Bremen, Germany, 4University
of Bremen, Islet Biology Laboratory, Centre for Biomolecular Interactions, Bremen,
Germany
The MALDI-imaging/ MULTI-ARRAY Core Facility at the University of Bremen
(http://www.maldi.uni-bremen.de) is a newly established institution providing
expertise and service in matrix assisted laser desorption/ ionization (MALDI)-imaging
mass spectrometry, also called MALDI-imaging, and MULTI-ARRAY® technology
combined with computational solutions for data analysis. MALDI-imaging is a
powerful tool for the analysis and visualization of (bio)molecules in their spatial
Abstracts of Poster Presentation
113
proximity directly from tissue sections, plant samples and bio- or polymer films.
Biomarkers can be detected in a molecular discovery process and validated by
exploitation of the MULTI-ARRAY® electrochemiluminescence-based platform for
high throughput analysis of proteins in serum-, cell and tissue lysates.
The MALDI-imaging/ MULTI-ARRAY Core Facility holds the Autoflex SpeedTM
MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany)
equipped with the smartbeamTM
-II laser technology providing a repetition rate of 1000
Hz for a maximum performance and guaranteed spatial resolution down to 50 µm for
linear and reflector analysis both in positive and negative modes. Our service model
envisages access to the technology for internal and external users by the
straightforward 'sample in - data out' approach. The MALDI-imaging workflow will
be executed by our specialists and encompasses sectioning, tissue and requirement-
specific sample processing, matrix application by ImagePrep® sample preparation
device (Bruker Daltonik), measurement, staining and data analysis in a user-tailored
fashion. Complimentary data analysis and evaluation can be achieved by specialized
and up-to-date developed computational methods provided by long-standing experts in
the analysis of MALDI-imaging data.
Here, we aim to introduce the MALDI-imaging/ MULTI-ARRAY Core Facility and
the service we are providing. We present the organization structure and the
instrumentation of the Core Facility. The power of the MALDI-Imaging technology
will be demonstrated exemplified by own executed tissue samples pointing out the
importance of data analysis methods for improved information extraction from
MALDI-imaging datasets using our own computational pipelines.
Abstracts of Poster Presentation
114
Glycohistochemical characterization of the avian inner perivitelline
layer Rodler D.
1, Sinowatz F.
1
1Ludwig-Maximilans-University, Department of Veterinary Sciences, Munich,
Germany
The avian inner perivitelline layer (IPVL), is a homologous structure to the
mammalian zona pellucida. Is is deposited between the granulosa cells and the huge
polylecithal oocyte during folliculogenesis. In our study, a panel of FITC-labelled
lectins was used to characterize and localize the oligosaccharide sequences of the
IPVL glycoproteins at different stages of follicular development in the ovary of two
bird species, the small quail (Coturnix japonica) and the huge ostrich (Struthio
camelus). Control reactions using competivite inhibition with the corresponding
sugars, deacetylation and sialidase digestion were also performed. Contrary to
mammals, where the topographical distribution of the investigated carbohydrate
residues is not uniform throughout the zona pellucida, indicating the regionalization of
oligosaccharide chains, we found a homogenous lectin staining of the comparatively
thin IPVL of birds. In the quail, variations in the presence and distribution of the
carbohydrate residues in the IPVL during different stages of follicular growth could be
demonstrated. The IPVL of previtelline follicles distinctly stained with WGA, sWGA
and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α−D-GalNac in the
glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-
D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc), VAA
(Gal), DBA (α-DGAlNAc (1-3)-GalNAc) and UEA1 (α-L-Fuc). With continuing
follicular growth of the oocyte and the follicle, this staining pattern changed. PNA-
and LCA-staining in the IPVL now became distinctly positive. The IPVL of the ostrich
(Struthio camelus) showed a much simpler staining pattern. During oocyte growth, its
IPVL which covers the huge oocyte was only distinctly labelled with WGA and
sWGA, whereas all other lectins are only weakly bound to the IPVL or were
completely negative. The differences in the lectin staining pattern between these
different avian species are shortly discussed.
Abstracts of Poster Presentation
115
Thyroid gland-commonness of hyperthyreodism and
hypothyreodism in the female and male population Salovska J.
1, biochemistry
1Institute of Biology, Faculty of Natural Sciences and Mathematics, Biology, skopje,
Macedonia, the Former Yugoslav Republic of
Introduction: The hormones of the thyroid gland are essential for normal growth and
development of the organism. In order to keep the metabolic processes in normal, the
thyroid hormones must be constantly secreted in the proper amount. The reduced or
excessive secretion of the thyroid hormones in the adult population causes serious
problems. The diseases which develop on the thyroid gland due to lack or excessive
secretion of the thyroid hormones are hyperthyreodism and hypothyreodism.
Hyperthyreodism appears as a result of hyperactivity of the thyroid gland, i.e.
excessive secretion of hormones in the gland , and their release in the bloodstream ,
and they appear during the maturity, and more in the woman population.
Hypothyreodism is defined as lack of biologically active hormones in the tissue or
incapability of the tissues to use the thyroid hormones, condition which is due to
subnormal concentration of thyroid hormones in the blood, it appears mostly due to
lack of iodine in the food.
Materials and methods: For the analysis of the serum at the patients, the apparatus
VIDAS had been used, which is automatic analysis for the Vidas system, which
enables for the human serum to be quantitatively measured, and the principle of
analysis combines the method of enzyme imuno-analysis and final fluorescent
detection, for the detection of the free FT3 and FT4, and for the detection of the human
thyrotropin in the human serum TSH, by using the ELFA technique (Enzyme Linked
Fluorescent Assay). The serum of 220 patients from Gostivar and the region has been
divided in 6 age groups: ≤ 20 years, 21-31 years, 31-40 years, 41-50 years, 51-60 years
and above 60 years.
Discussion: Out of 220 patients taken for analysis, 26 are male, while 194 are females
divided by age, and of all 100 % of the patients taken for analysis, 5.6 % are patients
with Hyperthyreodism, 10.4 % are patients with Hypothyreodism and 84 % of the
patients are in good health condition.
Abstracts of Poster Presentation
116
Through the aspect of sex, hypothyreodism is the most common among the female
population with 88 %, while males have 12 %, and as for the hyperthyreodism it is the
most common in the female population with 79 percent while the male has only 21 %.
According to the results from 250 patient, we can see that 79 % are female patients
with hyperthyreodism in the age of 41-50 years, while 11 % are males in the age of 51-
60.
On the other hand, 88 % of the female patients between the age 41-50 have
hypothyreodism, while males are 12 % in the age 41-60.
Conclusion
1) The most common population with hypothyreodism is the female with 88.46 %
2) The most common population with hyperthyreodism is the female with 78.58%
MALDI Imaging as a novel approach for the systematical study of
intratumoral heterogeneity Schöne C.
1, Rauser S.
1, Balluff B.
1, Elsner M.
1, Meding S.
1, Feuchtinger A.
1, Schmitt
M.2, Aubele M.
1, Walch A.
1
1Helmholtz Center Munich, Neuherberg, Germany,
2Klinikum rechts der Isar der TU
München, Munich, Germany
Aims: Tumors are complex. Cancer cells within a tumor may vary in their protein
expression and their therapy response. While this intratumoral heterogeneity is well
known, its systematic analysis has proven to be difficult so far. Here we want to
present MALDI Imaging as an innovative tool for the analysis of this phenomenon. Its
capability to perform label-free, multimodal proteomic measurements, while retaining
the morphoplogical information of the tissue, makes it very well suited to tackle this
problem.
Methods: A total of 40 invasive-ductal breast cancer samples were measured using
MALDI Imaging. Tumor specific proteomic profiles where then selected by virtual
microdissection. After selection hierarchical clustering was applied to these spectra, to
discern internal differences between the tumor cell proteomes. The resulting clusters
were then superimposed onto the histological staining of the respective tissue in order
to correlate them with the morphological information of the tumors.
Abstracts of Poster Presentation
117
Results: Using this approach we were able to identify intratumoral heterogeneity of
varying degrees in about 20% of the analyzed samples. In addition, we could perform
comparisons between the proteomes of the different cancer cell populations allowing
us to find masses with expression levels varying between these populations that might
be of special interest for identification.
Conclusion: We could show that MALDI Imaging is a method very well suited for the
study of intratumoral heterogeneity. It allows the combination of proteomic
measurements with morphological analysis. This could allow the systematical study of
intratumoral heterogeneity and the gain further insights in the clonal variation within
cancers.
Immunohistochemical localization of transit cells, putative stem
cells and stem cells in benign prostate hyperplasia (BPH) and adenocarcinoma of the prostate Sinowatz F.
1, Albanaw A.
2, Rodler D.
1, Kenngott R.
3
1Ludwig-Maximilians-University, Department of Veterinary Sciences, Munich,
Germany, 2Kuwait University, Department of Medical Laboratory Sciences, Faculty of
Allied Health, Kuwait, Kuwait, 3Ludwig-Maximilians-University, Department of
Veterinary Science, Munich, Germany
Understanding the processes leading to benign and malign prostate tumour is of great
importance for the assessment of disease progression and the choice of treatment.
Recent gene expression studies have demonstrated that processes that regulate the
development of the prostate gland are also relevant for prostatic tumours. The
transcription factor SOX9 has been shown to be essential for normal prostate
development. Oct3/4 also plays a role during embryogenesis of the human prostate. In
our investigation, we studied the expression of SOX9 and Oct3/4 in 20 cases of BPH
and 36 cases of prostatic carcinoma of different Gleason grades using
immunohistochemistry. Additionally the localization of keratin 14 (marker for
“putative stem cells) and keratin 18 (marker for “transit cells”) were investigated. Our
results show that in BPH and prostate cancer a nuclear localization of Oct3/4 and
SOX9 was found. Using keratin-immunophenotyping we could demonstrate a
Abstracts of Poster Presentation
118
significant number of transitional cells, both in BPH and malignant prostate tumours.
If these results could indicate an etiological relationship between the two diseases
appears possible, but certainly needs more extensive studies.
Cell adhesion molecules in melanocytic tumors Stoemmer P.
1, Torres-Galea P.
1
1Forschungslabor und Gemeinschaftspraxis Pathologie, Augsburg, Germany
Cell adhesion molecules are very important for the intercellular connections, the
resulting architecture and the biological behaviour of tumours in general and
particulary in melanocytic proliferations.They are the basis of the functional
communications with other epithelial and mesenchymal cells.
In contrast to other proteins, usually tested in malignant and not-so-malignant
melanocytic tumours such as S-100, HMB-45 or MALT-1, their expression is not a
feature of melanoma or naevus in general, but an affair of a peculiar cell in a specific
surrounding, and thus more a functional than a histogenic marker in the tumour.
Therefore we found great local differences of their expression in the various
melanocytic tumours.
Materials and methods: we analysed 10 formalin-fixed paraffin-embedded (FFPE)
tissue sections of each junctional, compound and dermal nevi, blue nevi and different
melanomas (SSM, LM and NMM and our sole case of nevoid fungating melanoma) in
respect of the expression and location of E-, N-, P-cadherin, alpha- and beta-catenin
and H-CAM/CD44v. We tested FFPE blocks with different antibodies in a qualitative
and semiquantitative evaluation of their expression in respect of the architectural
structure in the tumours.
Results and discussion: Cell adhesion molecules seem to be of great importance for
the connection of cells in their environment. They contribute to the architecture of a
tumour and by shifting from one type of CAM to another, the tumour changes not only
the morphological appearance, but also -probably and in concert with other factors- its
ability for infiltrative growth pattern and formation of metastases.
Abstracts of Poster Presentation
119
According to conventional ideas of nevi, intraepidermal nevomelanocytes may
proliferate, penetrate the basal lamina and invade the dermis; the interaction with this
microenvironment drives them to maturation and senscence. Melanocytes type A have
E-cadherin, beta-catenin and CD44v as main adhesion complex molecules; no N- and
P-cadherin. Type B-melanocytes lose their E-cadherin without expriming N-cadherin
and blue nevi have never E-cadherin, only N-cadherin in their type C melanocytes.
In malignant melanomas and their precursors there is a shift from E-cadherin to N- and
P-cadherin at first, in the in situ tumour (cells resembling type A melanocytes) there is
a coexpression of E-cadherin and N-cadherin; in massively invasive cells
(monocellular infiltration, invasionfront, spindle cells) E-cadherin is no more
exprimed but N- and P-cadherin; DC44v surrounds only the mesenchymal border of
tumor complexes. This may enable these tumour cells for the better invasive growth
and metastasis.
Nuclear lamina in organization of DNA replication. Strelkova O.S.
1, Dianova V.D.
1, Kurchashova S.Y.
1, Abramchuk S.S.
2, Alieva I.B.
1,3,
Kireev I.I.1,4
1A.N.Belozersky Inst. of Phys-Chem Biology, Moscow, Russian Federation,
2Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Moscow, Russian Federation, 3People's Friendship University of Russia, Department
of Histology, Сytology and Embryology, Moscow, Russian Federation, 4Moscow State
University, Dept. of cell biology and Histology, School of Biology, Moscow, Russian
Federation
Nuclear lamina is a main structural component of nuclear skeleton, which provides
mechanical rigidity of the cell nucleus. It also takes part in the chromosome
organization by linking special chromosome loci to nuclear lamina. Association of
chromosomes with nuclear lamina is correlated with transcription activity and
replication timing, and lamina might plays an important role in regulation of this
processes. On he other hand, tight connection of late-replicationg heterochromatin to
lamina may create additional topological constrains during replication. It can be
Abstracts of Poster Presentation
120
overcome by temporally dissociating replicating chromatin from lamina. We studied
correlation between spatial lamina organization and replication status of preipheral
heterochromatin using several expreimental models. First, we analysed distribution of
replicating chromatin loci relative to nuclear lamina at ultrastructural level in cell lines
expressing GFP-PCNA (Leonhard et al, 2000). Asynchronous cell culture was labeled
with mouse-anti GFP Abs, which were detected with Nanogold-coupled secondary
Abs and silver enhancement. Cells with replication pattern 3 were selected under
bright-field microscope and sectioned. We found that in many cases peripherally
located labeled replication sites moved away from nuclear lamina to a distance of 0.2-
0.3 mkm. Interestingly, this detachment was observed mainly when the label
concentrated on the distal part of replicating chromatin domain facing nuclear lamina.
Next, we studied intranuclear position of large artificial heterochromatic locus that
demonstartes orderly relocalization from its peripheral position in G1 to more central
location just before and during its replication (Li et al, 1998). Surprisingly, we found
that this locus never lost its association with nuclear lamina even during replication.
Rather, movement of the locus towards the central nuclear region was accompanied by
formation of deep intranuclear invaginations of nuclear envelope. These invaginations
always contained lamins A and B. Apparently, we failed to observe complete
dissociation of this locus from lamina during replication due to its complex replicative
organization, where individual replicative clusters fire asynchronously. Nevertheless,
our studies demonstrate dynamics of chromatin-nuclear envelope interactions and high
plasticity of nuclear lamina during replication.
This work was supported in part by RFBR (Grants No. 09-04-01352-а to I.I.K, 09-04-
00363-а to I.B.A.)
Abstracts of Poster Presentation
121
In-tissue lipid profiling by mass spectrometry imaging Strohalm M.
1, Volny M.
1, Faltyskova H.
1, Pol J.
1, Hulkova H.
2, Novak P.
1, Havlicek
V.1
1Institute of Microbiology, v.v.i., Laboratory of Molecular Structure Characterization,
Prague, Czech Republic, 2Institute of Inherited Metabolic Disorders, Charles
University, 1st Faculty of Medicine and Teaching Hospital, Prague, Czech Republic
There are many diseases known to be related to altered metabolism of different
species. To better understand physiology and biochemistry of the disease, information
about the spatial distribution of selected metabolites is often very important. In
addition to traditional histological techniques, recent development in mass
spectrometry has enabled a new approach called mass spectrometry imaging (MSI).
Using this method, metabolites can be identified and visualized based on the exact
mass, which allows us to monitor different species separately, whenever their masses
are not equal. Here, we provide an overview of a method for lipid identification in
MSI experiments. Matrix-assisted laser desorption/ionization (MALDI) in
combination with Fourier transform ion cyclotron resonance mass spectrometry
(FTICR MS) was utilized to determine the accurate mass and spatial distribution of
lipids in various tissue samples. Several software were involved in subsequent data
analyses. An average mass spectrum was generated for each region of interest using
in-house made software and analyzed by an open source program mMass
(www.mmass.org). This program enables fast and easy way to identify different lipids,
as well as the corresponding adducts, by comparison with publicly accessible Lipid
Maps database (www.lipidmaps.org). Detailed information about particular lipid can
be accessed from generated report, providing direct link to the Lipid Maps server.
Finally, identified lipids were visualized by FlexImaging software (Bruker Daltonics,
Germany) and combined with the results obtained by traditional histological staining.
Abstracts of Poster Presentation
122
Spatial proteomics: A new LC-MS/MS tissue imaging workflow
providing protein identities and their distribution in tissue Schürenberg M.
1, Lübbert C.
1, Becker M.
2, Paape R.
1, Suckau D.
1
1Bruker Daltonik GmbH, EAppM, Bremen, Germany,
2Bruker Daltonik GmbH,
MALDI Imaging Applications, Bremen, Germany
Background: MALDI-Imaging of undigested proteins in tissue sections has
established itself as a powerful new approach to biomarker discovery and
histopathological research in recent years. Digestion, on the other hand, increases the
sample complexity significantly and caused direct MS/MS identification from digested
tissue to fail providing reasonable protein coverage and more than a few identifications
of high abundant proteins. Here we introduce a novel proteomics technology that
combines the spatial information with the routine identification of proteins from tissue
sections without the need of MS/MS analysis from the tissue.
Methods: Highly resolved protein digests were generated by applying trypsin onto
two subsequent tissue sections by supersonic nebulization. One of the sections was
coated with DHB matrix and analyzed by MALDI imaging mass spectrometry at 50
µm spatial resolution yielding a list of 200-800 peptide signals per image.
Peptides were extracted from the other sections entire surface and submitted to
standard proteome analysis using LC-MALDI-TOF/TOF identification. The identified
peptide list is then matched to the peaklist from the image by newly developed
software tools. All matching peaks in the image can then be assigned to a protein and
the co-localization of 2 or more tryptic peptides typically confirms their protein
association.
Results: We analyzed rat brain and testis/epididymis using the new Spatial Proteomics
approach typically yielding peptide distributions at the 50-100 µm level. In both tissue
types approx. 80% of all peaks visible in the image matched to peptides identified in
LC-MS/MS. These peptides belong to ~100 different proteins. More than 20 proteins
showed co-localization of >2 IDed peptides. The developed methods and software
tools are available from the authors.
Conclusion: Simultaneous determination of protein distribution and identity was
achieved in a combined bottom-up imaging-LC-MALDI workflow for the first time
Abstracts of Poster Presentation
123
for ~100 proteins. In addition, the method has great potential for the MALDI imaging
analysis of FFPE tissue or membrane proteins. In this work we confirmed protein
identifications from previous top-down work on rat testis1 (e.g. b thymosins and Long
Chain fatty acid CoA ligase(19-48), at ~ 50 vs. 20 µm spatial resolution.
References:
1. Revisiting Rat Spermatogenesis with MALDI Imaging at 20-µm Resolution.
Lagarrigue M et al. MCP. 2011 Epub 2010 Dec 12.
MRI molecular imaging with targeted albumin-based nanoparticles: conceptual design strategies to create the Magic
Bullet Thurner G.C.
1, Wallnöfer E.A.
1, Rohr I.
2, Talasz H.
3, Kremser C.
1, Abdelmoez A.A.
2,4,
Klammsteiner N.2, Dietrich H.
5, Matuszczak B.
6, Jaschke W.
1, Debbage P.
2
1Medical University of Innsbruck, Clinic for Radiology, Innsbruck, Austria,
2Medical
University Innsbruck, Department of Anatomy, Histology & Embryology, Innsbruck,
Austria, 3Medical University Innsbruck, Sektion für Klinische Biochemie, Biozentrum,
Innsbruck, Austria, 4Assiut University, Department of Pharmaceutical Organic
Chemistry, Assiut, Egypt, 5Medical University Innsbruck, Central Animal Research
Facility, Innsbruck, Austria, 6University of Innsbruck, Institute of Pharmaceutical
Chemistry, Innsbruck, Austria
Introduction: Paul Ehrlich in 1900 [1] described the idea of a targeted vehicle
carrying a drug. His notion of a „Magic Bullet“ changed the whole concept of medical
treatment. Richard Feynmann in 1959 [2] challenged scientists to create
nanomachines. Since then Nanomedicine has promised improved drug delivery,
reduced dosages and side-effects: "personalized medicine". Why has Nanomedicine
not delivered on this promise? Two major challenges hinder realisation of this dream:
1. identification of suitable biomarkers; 2. poor availability of nanoparticles capable of
homing to biomarker molecules hiding behind intact tissue barriers. We review the
reasons for these difficulties and suggest some approaches to overcome them.
Materials and methods: Albumin-based nanoparticles bearing gadolinium were
developed and extensively characterised. The lectin LEA was attached to the particles
Abstracts of Poster Presentation
124
to target oligolactosamines. During MR Imaging of living rats both imaging and
quantitative approaches were applied. Coordinated with the MRI, chemical and
immunohistochemical analyses tracked components of the nanoparticles in
longitudinal time series after injection, 15 minutes to 6 weeks, obtaining large runs of
quantitative data.
Results: Our nanoparticles were ~30 nm diameter. They were pure, contained no
starting materials and had good imaging properties with relaxivities ~1 • 107 1/Ms.
They were stable in various in vitro testings though not in SDS-gel-electrophoresis. In
haemagglutination tests they agglutinated red blood cells; after intravenous injection
into living rats they gave high-resolution MR imaging of the vascular wall lasting >2
hours. The numbers of (lectin) targeting groups required for molecular imaging and of
gadolinium ions per nanoparticle necessary for high-resolution MRI were assayed in
relation to particle size and type of crosslinking.
Discussion: We determined critical parameters for MR Molecular Imaging by use of
nanoparticles. These particles require a second type of targeting group to migrate
across vascular walls and access subendothelial interstitial compartments, for
Molecular Imaging and Molecular Targeting of disease sites behind intact tissue
barriers. The concept of multiple targeting is new in Nanomedicine and represents the
hurdle that limits present-day techniques [3]. Assuming that quantitative aspects of
targeting will be similar for each of the multiple targeting groups, we already know
how to design nanoparticles for targeting both drugs and contrast agents to disease
lesions hiding behind blood-tissue barriers. Multiply-targeted nanoparticles are
Ehrlich´s "magic bullets".
Acknowledgements: Austrian Nano-Initiative (Project N201-NAN); Austrian
National Bank Jubilee Program (Projects 9273, 10844, 11574)
References:
1. Ehrlich, P. (1900) Proc R Soc Lond 66: 424 - 448.
2. Feynman RP. (1959) Miniaturization. Horace D. Gilbert, Ed. © Van Nostrand
Reinhold. New York
3. Debbage P., Thurner GC. (2010) Pharmaceuticals 3: 3371 - 3416
Abstracts of Poster Presentation
125
EGFR, HER2 and HER3 expression and dimerization in esophageal
cancer Timme S.
1, Fichter C.D.
1, Schoepflin A.
1, Bogatyreva L.
2, Hauschke D.
2, Tang L.
3,
Klimstra D.3, Opitz O.G.
4, Werner M.
1, Lassmann S.
1
1University medical Center Freiburg, Institute of Pathology, Freiburg, Germany,
2University medical Center Freiburg, Institute of Medical Biometry and Medical
Informatics, Freiburg, Germany, 3Memorial Sloan Kettering Cancer Center New York,
Dept. of Pathology, New York, United States, 4University medical Center Freiburg,
Tumorzentrum Ludwig Heilmeyer - Comprehensive Cancer Center, Freiburg,
Germany
Text:
Introduction: Receptor tyrosine kinases (EGFR, HER2, HER3) are therapeutic targets
in epithelial tumors, whose distinct expression and/or dimerization may affect
therapeutic responses. Here, we examined EGFR, HER2 and HER3 protein expression
and dimerization in esophageal squamous cell carcinoma (ESCC) and Barrett's
adenocarcinoma (BAC).
Materials+Methods: Serial tissue sections of 110 pre-treatment biopsies of ESCCs
and BACs from three centers were examined for EGFR/HER2/HER3 expression by
immunohistochemistry (IHC) according to routine diagnostics. HER2/HER3 double
immunofluorescence (dIF) and proximity ligation assay (PLA) was performed in
selected BAC biopsies. For evaluation of dIF and PLA, three-dimensional image
stacks were recorded on a fluorescence microscope. Statistical univariate analyses
were performed by SPSS v18.
Results: In tissue specimens, HER2 (18/61, 30%; p< 0.001) and HER3 (16/61, 26%;
p< 0.001) were significantly overexpressed in BACs. EGFR expression was found in
4/61 (6.5%) BACs and in 7/49 (14%) ESCC (p=0.088). EGFR (p=0.350), HER2
(p=0.224) and HER3 (p=0.194) expression was not associated with histologic grading.
By PLA, we found that HER2 and HER3 heterodimers do appear in invasive tumors
cells of BACs expressing high levels of HER2 and HER3. In contrast, a substantial
number of ESCCs and BACs „over“express only EGFR or HER2, suggesting presence
of EGFR homodimers in ESCCs particularly.
Abstracts of Poster Presentation
126
Conclusions: Together, this may contribute to differentially regulated signalling
events and effects on tumour progression in ESCCs and BACs, or subgroups thereof.
In view of current receptor tyrosine kinase targeted therapies, inhibition of EGFR
and/or HER2 may represent a therapeutic option for ESCC and BACs, respectively.
HER3 appears to be the predominant interaction partner of HER2 in BACs,
presumably affecting also therapeutic responses.
Support: Deutsche Forschungsgemeinschaft DFG CRC850 project C5, and in part
Mushett Family Foundation, Chester, NJ, US
Software for 3D MALDI imaging data: Visualization and 3D spatial
segmentation Trede D.
1, Schiffler S.
1, Becker M.
2, Wirtz S.
3, Strehlow J.
3, Kobarg J.H.
4, Aichler
M.5, Heldmann S.
6, Walch A.
5, Thiele H.
2, Maass P.
4, Alexandrov T.
4
1Steinbeis Innovation Center SCiLS (Scientific Computing in Life Sciences), Bremen,
Germany, 2Bruker Daltonik GmbH, Bremen, Germany,
3Fraunhofer MEVIS, Institute
for Medical Image Computing, Bremen, Germany, 4University of Bremen, Zentrum für
Technomathematik, Bremen, Germany, 5Helmholtz Center Munich, Institute of
Pathology, Munich, Germany, 6Fraunhofer MEVIS, Institute for Medical Image
Computing, Lübeck, Germany
In the last decade, matrix-assisted laser desorption/ionization (MALDI) imaging mass
spectrometry, also called MALDI-imaging, has proven its potential for proteomics
analysis of 2D samples and was successfully applied for histological analysis of thin
tissue slices. MALDI-imaging is used as a general analytic tool revealing the
functional proteomic structure of tissue slices, and as a discovery tool in detecting new
cancer biomarkers discriminating an annotated tumor region. Recently, a technological
breakthrough has been reported when a spatially 3D model corresponding to one m/z-
value was reconstructed from consecutive slices each measured with MALDI-imaging.
Certainly, 3D MALDI-imaging has immense potential for representing not only one
slice through the organ but showing the complete 3D model which can be virtually
sliced in any direction. However, the progress stopped after a proof-of-principle
demonstration, since 3D MALDI-imaging requires not only much measurement time
Abstracts of Poster Presentation
127
but a bunch of efficient and user-friendly computational algorithms specifically
developed for 3D MALDI-imaging.
Here, we present for the first time our recently developed software SCiLS Data
Explorer for import, construction, visualization, and analysis of 3D MALDI-imaging
data. It takes several 2D MALDI-imaging datasets as an input, each dataset
corresponding to a consecutive slice. Then, it creates a 3D cloud of measurement
points in the 3D Cartesian space with coordinates (x,y,z), with a spectrum assigned to
each point. We demonstrate how this huge dataset (approximately 500,000 points with
each spectrum of 10,000 m/z bins are considered) can be visualized given a specific
m/z-value, representing 3D distribution of a compound with this m/z-value. Moreover,
we illustrate how mass spectra pre-processing (baseline correction, normalization,
image denoising) improves the visualization.
Spatial segmentation of 2D MALDI-imaging data has been introduced several years
ago and is currently a well-accepted computational method in the MALDI-imaging
field. It splits a sample into regions of similar chemical composition, clustering the
spectra, and representing the clustering results with a spatial segmentation map. One
color in this map represents a region with similar mass spectra. A spatial segmentation
map can be explored and interpreted from the mass spectrometry viewpoint providing
a unique approach to fast and sensitive analysis of complex MALDI-imaging data.
In this poster, we present for the first time the 3D spatial segmentation of a real-life 3D
MALDI-imaging data. The spatial segmentation tool is of high importance for any
analysis of 3D data, because a manual annotation in 3D space can hardly be done. Our
software makes 3D histological analysis possible by automatic selection of 3D regions
of interest which can be then manually assigned to histological tissue classes.
The project is funded by the Bremen Economic Development foundation (WFB).
Abstracts of Poster Presentation
128
Zonal distribution of CD105+/CD166+ cells in growing rat
humerus proximal epiphyseal cartilage Ustunel I.
1, Ozbey O.
1, Acar N.
1
1Akdeniz University, School of Medicine, Histology and Embryology, Antalya, Turkey
Objectives: The coexpression of cell surface receptors, CD105 and CD166, are
characteristic of mesenchymal stem cells in cartilage. However, there is limited data
regarding their immunolocalization in the cartilage of growing rat epiphyseal cartilage.
The purpose of this study was to determine the presence of CD105+/CD166+ cells in
the proximal epiphyseal cartilage of developing rat humerus and specify their zonal
distribution with age.
Materials and methods: The tissues of rat humerus were taken on embryonic day 15
(E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and
we detected the immunolocalization of CD105+/CD166+ cells.
Results: Our results showed that CD105+/CD166+ cells were scattered in early stages
of development in humerus epiphysis. We observed that CD105+/CD166+ cells were
only in the hypertrophic zone on day E15, predominantly in the resting and
hypertrophic zones of the epiphysis cartilage on E19, whereas we observed
CD105+/CD166+ cells only in hypertrophic zone on PN10 and PN20.
Conclusion: We observed CD105+/CD166+ cells predominantly in the resting zone
and hypertrophic cells of the epiphyseal cartilage. These results may show the
presence of stem cell-like cells in the epiphyseal cartilage and they may have an
important role in vitality, cell-matrix interaction, migration, angiogenesis, growth and
differentiation of epiphyseal cartilage.
Key words: Cartilage, CD105, CD166, Differentiation
Abstracts of Poster Presentation
129
Is CRIP1 a target gene of HER2? Weber E.
1, Rauser S.
2, Mylonas I.
1, Walch A.
2, Brüning A.
1
1University Hospital Munich, OB-Gyn, Munich, Germany,
2Helmholtz Zentrum
München, Institute of Pathology, Neuherberg, Germany
Aims: It has recently been observed that HER2-overexpressing breast cancer tissues
display elevated levels of CRIP1 (cysteine-rich intestinal protein 1). CRIP1 is known
to modulate cytokine expression and might thus play a role in cancer progression,
activated by HER2. We have investigated the relation between CRIP1 and HER2 at
the cellular level.
Methods: Protein expression was analyzed by FACScan and immunohistochemical
analysis. Human CRIP1 was cloned from its cDNA and transferred into a mammalian
expression vector for functional analysis.
Results: Although some HER2-overexpressing breast cancer cells revealed elevated
CRIP1 levels, no association between HER2 expression and CRIP1 expression could
be observed when a larger number of breast cancer cells was tested (n = 8). Further,
neither heregulin nor lapatinib modified CRIP1 expression in breast cancer cells.
However, CRIP1 expression varied strongly among the breast cancer cells tested,
indicating a possible role for CRIP1 in cancer progression. Overexpression of
exogenous V5-tagged CRIP1 in breast cancer cells revealed Golgi localization and
secretion of CRIP1, indicating either a paracrine or autocrine function in cancer
tissues.
Conclusions: CRIP1 expression is not associated with HER2 expression, but appears
to play an individual role in breast cancer progression.
Abstracts of Poster Presentation
130
Class I HDACs in the developing heart and limb of chicken embryos Aichinger C.
1, Engelmaier C.
1, Murko C.
1, Schöfer C.
1, Weipoltshammer K.
1
1Medical University of Vienna, Dept. for Cell and Developmental Biology, Vienna,
Austria
Epigenetic mechanisms are important regulators of cellular and developmental
differentiation. It has been shown that histone deacetylases are relevant factors in
epigenetic regulation exerting their influence by modification of histones on the
compaction state and thus accessibility of chromatin. Class I HDACs were previously
thought to be ubiquitously expressed. However, genetic deletion of class I HDACs in
mice led to malformations in different organ systems including the heart, central
nervous system, liver and lung (Haberland et al. 2009). We mapped the class I HDAC
expression in mice and chicken embryos (Murko et al. 2010) and found a distinct
spatio-temporal expression pattern. In the chicken limb bud strong class I HDAC
expression was seen, whereas the expression in the heart was surprisingly low
concerning the fact that HDAC k.o. mice displayed severe cardiac malformations.
Therefore we decided to study the effects of class I HDAC down regulation in these
organs in the chicken model system which allows the controlled spatio-temporal
application of HDAC-inhibiting drugs by in ovo manipulation. We either implanted
beads soaked with trichostatin A (TSA) or directly injected TSA into wing buds and
into the pericardial cavity. Subsequently the embryos were reincubated and evaluated
according to the following criteria: external anatomy (whole mount staining),
morphometry (serial sections), proliferation and apoptosis (in situ assays).
After TSA application a mild retardation of limb development was observed. When
the beads were placed in the site of the presumptive zeugopodium region of limb buds
a significant reduction in tibia size at later stages could be demonstrated by
morphometric analysis.
In the heart, we observed dramatic phenotypic effects mostly showing an aberrant ratio
of atrial and ventricular muscular mass. Here the preferential abnormality was atrial
hypertrophy. In both cases, we did not observe major changes in cell proliferation rate
and rate of apoptosis after TSA treatment.
Abstracts of Poster Presentation
131
Taken together we could show that class I HDACs play important roles in the
development of chicken embryos. In case of limb development our results suggest an
effect of HDACs in skeletal development possibly by regulation of the chondrocyte
differentiation pathway. In the heart our data indicate an important role of HDACs in
chicken myocyte differentiation.
References:
Haberland M, Montgomery RL, Olson EN (2009) Nat Rev Genet 10:32-42
Murko C, Lagger S, Steiner M, Seiser C, Schoefer C, Pusch O (2010) Int J Dev Biol
54:1527-1537
The first two authors contributed equally.
MALDI-Imaging of frozen prostate tissue micro arrays Wurlitzer M.
1, Koop C.K.
2, Becker M.
3, Thiele H.
4, Maaß P.
4, Minner S.
1, Stahl P.
1,
Schlüter H.5, Sauter G.1
1University Medical Center Hamburg-Eppendorf, Inst. f. Pathology, Hamburg,
Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany,
3Bruker Daltonik GmbH, Bremen, Germany,
4Universität Bremen, Zentrum für
Technomathematik, Bremen, Germany, 5University Medical Center Hamburg-
Eppendorf, Inst. f. Clinical Chemistry, Hamburg, Germany
In cancer research, the elucidation of new diagnostic or prognostic markers for the
detection of malignant cells in biopsies and tissue sections is an important aim. Mass
Spectral Imaging (MSI) yields information about the spatial distribution of a huge
number of biomolecules in a single experiment and therefore is a helpful tool for
biomarker discovery. MSI is especially interesting for the analysis of tissue
microarrays (TMAs), since a large number of patient samples can be analysed rapidly
under identical analytical conditions [1]. A single TMA section allows the analyses of
several dozen different tumors and control tissues at once. Formalin-fixed paraffin-
embedded (FFPE) TMAs are widely available, but complicate MSI analysis because of
the formalin induced protein crosslinking. In addition, biomarkers found in FFPE
tissue samples will likely prove difficult to translate into assays based on fresh
Abstracts of Poster Presentation
132
biobsies. Therefore, in this study we investigated the analysis of TMAs from fresh-
frozen tissue by MALDI Imaging, focusing on peptides and proteins. To perform a
heterogeneity analysis we constructed a TMA consisting of 34 samples from different
areas of one prostate cancer biopsy. In addition, seven samples from associated lymph
node metastases were included in the TMA. For MALDI Imaging analysis, the TMA
sections were coated with sinapinic acid matrix deposition using an ImagePrep device
(Bruker Daltonik GmbH). MALDI Imaging analysis of the TMAs was performed
using an Autoflex Speed MALDI-TOF system (Bruker Daltonik GmbH). The mass
spectrometric data was processed and interpreted with flexImaging and ClinProTools
software (Bruker Daltonik GmbH). 136 signals ranging from m/z 2000 to m/z 20000
were detected in the TMA. We could elucidate one specific cancer associated signal
which was present in only a few primary tumor samples but in several lymph node
metastases. In conclusion we were able to show that MALDI Imaging from frozen
TMAs is a promising approach for searching for markers of (prostate) cancer.
Reference:
[1] Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S,
Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-
throughput molecular profiling of tumor specimens, Nature Medicine 1998, 4:844-847.
General Information
133
General Information
Organizer
Society for Histochemistry
Congress Venues
Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe
Klinikum der Universität München Maistraße 11
80337 München
Helmholtz Zentrum München
Ingolstädter Landstr 1
85764 Neuherberg
Congress Language
The official congress language is English.
Registration Desk and Congress Office
You will find the registration desk on E0.94C / Stair 6. Please follow the signage on-
site.
Opening hours: Wednesday, 12 October, 2011: 14:00 – 18:30
Thursday, 13 October, 2011: 07:30 – 18:00
Friday, 14 October, 2011: 08:00 – 13:30
Saturday, 15 October, 2011: 08:30 – 16:00
Name Badges
Every registered participant will receive a name badge upon registration. It is
necessary to wear this to gain access to the scientific program and coffee / lunch
breaks.
Coffee / Lunch
Coffee and lunch are included in the registration fee. They will be served of the wing
ends of the congress area.
General Information
134
Mobile Phones
Participants are kindly requested to keep their mobile phones in the off position in the
meeting rooms, as well as during the poster session.
Media Check
Speakers will find the media check in the Main Lecture Hall. Presenters are requested
to provide their talk on an USB stick in advance (at least 2 hours before their talk or
the evening before).
Awards
The Poster Award winners are determined by a scientific committee and will be
announced at the closing plenary session.
The Young Histochemist Travel Awards will be announced at the oping plenary
session on Wednesday and they have the honour to present the contents of the
submitted abstracts at the Free topics communication session on Saturday.
Instructions for Speakers
To ensure the congress runs smoothly and fairly for all participants please don’t allow
your presentation to run overtime. The chairs are instructed to strictly monitor the time
and to interrupt the presentation if the speaking time is exceeded.
The only multimedia technology accepted is Microsoft PowerPoint. The operating
system is Microsoft Windows XP. No support is provided for overhead or slide
projectors. The conference room is equipped with laptop and data projector. Using
your own laptop is not recommended.
Smoking Policy
The symposium is a non-smoking event. Participants are kindly requested to refrain
from smoking in the congress venue, including the exhibition area.
General Information
135
Please notice the obligatory additional registration for the following workshops:
Industry Workshop (Carl Zeiss NTS GmbH):
Introduction to Energy Filtering TEM" - Live practical session on the microscope
Guided Tour at Helmholtz Zentrum München
Industry Hands-On Workshop (Intas Science Imaging & Huron Technologies):
Laser Confocal Slide Scanning System
Scan your small or big Tissue section with the unique
TissueScope4000 - Laser Confocal Slide Scanning System.
Slide Dimensions: 25x75mm up to 150x200mm
Immunhistochemistry or Fluorescence probes can be scanned in that workshop.
Access to Venues
136
Access to Venues
Frauenklinik Maistraße
Access to Venues
137
Helmholtz Zentrum München
Introduction to the Helmholtz Zentrum München and Meeting Point
(Lecture room 052 / Building 57) – (green)
Guided Tour at the Helmholtz Zentrum München
- GMC: German Mouse Clinic – (orange)
- GAC: Genom Analysis Center – (pink)
- AMSD: Research Unit Medical Radiation Physics and Diagnostics – (pink)
Industry Workshop presented by Carl Zeiss NTS GmbH (Building 37) –
(red)
Access to Venues
138
Copyright Open Street Map
OpenStreetMap is open data, licensed under the Creative Commons Attribution-ShareAlike 2.0 licence (CC-BY-SA).
Frauenklinik
Ratskeller
Social Events 139
Social Events
Opening Plenary and Welcome Reception
Date Wednesday, October 12, 2011
Time Welcome Drink 17:30; Get Together 19:45
Location Frauenklinik Maistraße 11
Symposium Banquet
Date Friday, October 14, 2011
Time 19:00
Location Ratskeller, Marienplatz, located at the Rathaus (“City Hall”)
We would like to invite all participants to
attend the Symposium Banquet. Join us and
all the other delegates from around the
world on this evening for networking within
the Histochemistry Community.
A Welcome Drink at 17:30 will be offered
to delegates and registered accompanying
persons at the exhibition area.
A Get Together directly following to the
Robert Feulgen Lecture will be offered.
All participants are invited to attend the
Welcome Drink and the Get Together of
the 53rd Symposium of the Society for
Histochemistry 2011.
Exhibitors and Sponsors
140
Exhibitors and Sponsors
Gold Sponsor
Comprehensive Index of Exhibitors and Sponsors
AB SCIEX Deutschland GmbH Booth 5
Aperio Booth 8
BioLabs --
Bruker Daltonik GmbH Booth 3
Caliper Life Sciences GmbH Booth 4
DCS Innovative Diagnostik- Systeme
Booth 1
GSG-Analytical --
Exhibitors and Sponsors
141
Hamamatsu Photonics Deutschland
GmbH Booth 10
Intas Science Imaging Instruments --
W.Reichert-LABTEC Booth 2
Leica Microsystems Booth 5
Nikon GmbH Booth 1a
Olympus Deutschland GmbH -
Mikroskopie Booth 6
Springer Booth 7
Carl Zeiss NTS GmbH --
Map of Exhibition Area
142
Notes