SEVENTH FRAMEWORK PROGRAMME THEME [ Health-2007-A]

HEALTH – 2007 -1.2-1: Development of a hybrid imaging system

Grant agreement for: Collaborative projects (Small or medium-scale focused research projects

with maximum EC contribution of € 6,000,000/project). Annex I - “Description of Work” Project acronym: FMT-XCT Project full title:

Hybrid Fluorescence Molecular Tomography (FMT) – X-ray Computed Tomography (XCT) method and system

Grant agreement no.: 201792

Date of preparation of Annex I (latest version): March 01, 2011

Date of approval of Annex I by Commission: (September 30, 2011)

List of beneficiaries

Beneficiary No.

Beneficiary’s organization name Short name

Country

Date enter project

Date exit project

1.(Coordinator) Helmholtz Zentrum Muenchen, German Research Center for Environmental Health

HMGU Germany 1 48

2. Commissariat a L’Energie Atomique CEA France 1 48 3. Foundation for Research and Technology Hellas FORTH Greece 1 48 4. University College London UCL U.K. 1 48 5. Fudacion para la Investigacion Biomedica del

Hospital Gregorio Marañon FIHGM Spain 1 36

6. 7. 8.

Universität Zürich Ct Imaging Universidad Carlos III de Madrid

UZH VAMP UC3M

Switzerland Germany Spain

1 1

37

48 48 48

FMT-XCT 201792

2

Table of contents:

PART A A1. Budget breakdown and project summary

A.1 Overall budget breakdown for the project 3 A.2 Project summary 4 A.3 List of beneficiaries 5

PART B

B1. Concept and objectives, progress beyond state-of-the-art, S/T methodology and work plan B.1.1 Concept and project objective(s) 6 B.1.2 Progress beyond the state of the art 7 B.1.3 S/T methodology and associated work plan 7

B.1.3.1 Overall strategy and general description 7 B.1.3.2 Timing of work packages and their components 8 B.1.3.3 Work package list /overview 9 B.1.3.4 Deliverables list 10 B.1.3.5 Work package descriptions 13 B.1.3.6 Efforts for the full duration of the project 37 B.1.3.7 List of milestones and planning of reviews 38

B2. Implementation

B.2.1 Management structure and procedures 39 B.2.2 Beneficiaries 41 B.2.3 Consortium as a whole 45 Sub-contracting 47

B.2.4 Resources to be committed 47

B3. Potential impact B.3.1 Strategic impact 50

B.3.2 Plan for the use and dissemination of foreground 51 B4. Ethical issues if applicable 53 B5. Consideration of gender aspects 57

FMT-XCT 201792

PART A

A.1 Overall budget breakdown for the project A3.2: What it costs Project Number 1 201792 Project Acronym 2 FMTXCT

One Form per Project

Particip

ant number in this

project 3

Participant

short name

Estimated eligible costs (whole duration of the project) Total

receipts

RequestedEC

contribution RTD/Innova

tion (A)

Demon stration

(B)

Management (C)

Other (D)

TOTAL A+B+C+D

1 HMGU 1,188,020.00 0.00 299,750.00 103,337.00 1,591,107.00 0.00 1,294,102.00

2 CEA 1,552,714.00 0.00 14,942.00 32,912.00 1,600,568.00 0.00 1,212,389.00

3 FORTH 622,600.00 0.00 21,600.00 00 644,200.00 0.00 488,550.00

4 UCL 528,000.00 0.00 6,400.00 7,200.00 541,600.00 0.00 409,600.00

5 FIHGM 467,470.08 0.00 0.00 0.00 467,470.08 0.00 350.602,56

6 UZH 460,800.00 0.00 5,600.00 14,400.00 480,800.00 0.00 365,600.00

7 Vamp 332,000.00 0.00 6,000.00 7,000.00 347,000.00 0.00 263,500.00

8 UC3M 156,529.92 0.00 3,200.00 7,200.00 166,929.92 0.00 127.797,44

TOTAL 5,310,134.00 0.00 357,492.00 172,049.00 5,839,675.00 0.00 4,512,141.00

3

FMT-XCT 201792

4

A.2 Project summary

FMT-XCT 201792

 

 

A.3 List of beneficiaries

5

Beneficiary No.

Beneficiary’s organization name Short name

Country

Date enter project

Date exit project

1.(Coordinator) Helmholtz Zentrum Muenchen, German Research Center for Environmental Health

HMGU Germany 1 48

2. Commissariat a L’Energie Atomique CEA France 1 48 3. Foundation for Research and Technology Hellas FORTH Greece 1 48 4. University College London UCL U.K. 1 48 5. Fudacion para la Investigacion Biomedica del

Hospital Gregorio Marañon FIHGM Spain 1 36

6. 7. 8.

Universität Zürich Ct Imaging Universidad Carlos III de Madrid

UZH VAMP UC3M

SwitzerlandGermany Spain

1 1

37

48 48 48

FMT-XCT 201792

6

PART B

B1. Concept and objectives, progress beyond state-of-the-art,

B.1.1 Concept and project objective(s) This proposal offers to develop quantitative Fluorescence Molecular Tomography (FMT) – X-ray

Computed Tomography (XCT) system, by appropriately advancing optical tomography and X-ray CT imaging methods so that they can be seamlessly integrated into a hybrid imaging system and method. As showcased in this proposal, this approach offers a highly synergistic multi-modality system that will perform in a superior manner than any of constituents if they were to be considered as two stand-alone modalities by:

1. Providing hybrid FMT-XCT images by superimposing fluorescence images of functional genomics,

proteomics and physiological responses on X-ray anatomical images to optimally visualize the spatial distribution of various biomarkers.

2. Improving the imaging accuracy and quantification of the optical tomography problem and the

performance of the hybrid system by utilizing the anatomical information provided by XCT into the FMT inversion problem.

The overall goal of this proposal is to develop quantitative hybrid FMT-XCT technology, engineer

the optimal theory and inversion approaches for achieving a highly performing and synergistic system and perform pre-clinical imaging with a view towards clinical translation and therapeutic intervention. Major focus is given to preclinical imaging of breast cancer, as the most probable entry point of this approach to the clinic. The general aims of this proposal are:

Aim 1: To research optimal methods for yielding improved XCT contrast and deliver a corresponding

XCT design and prototype (Wp2). Optimal methods will be defined as the methods that provide better contrast to noise ratio of target biological soft tissues, i.e. lung vs heart vs adipose tissue.

Aim 2: To research and validate new inversion schemes that offer optimal FMT stand-alone imaging performance in free-space FMT implementations (Wp3). Optimal performance is defined as the one that more closely represents the underlying structures of known objects being utilized as gold-standards exactly for testing inversion algorithms.

Aim 3: To research and deliver optimal methods for utilizing a-priori information into the FMT inversion code for developing the best FMT performance ever developed (Wp4). Similarly to Aim2, optimal performance is defined as the one that more closely represents the underlying structures of known objects being utilized as gold-standards exactly for testing inversion algorithms.

Aim 4: To build a highly performing quantitative FMT-XCT prototype (Wp5). Success will be measured by means of a fully functional system able to acquire fluorescence and X-ray CT images of equal or better quality compared to its stand-alone counterparts.

Aim 5: To apply FMT-XCT in-vivo in pre-clinical imaging of mouse models of breast cancer (Wp6). Success would be defined by means of accurately reconstructing the underlying fluorescence bio-distribution as confirmed histologicaly.

Aim 6: To research the quantitative ability of FMT-XCT in visualizing treatment in-vivo (Wp7). Success will be defined by means of verifying in-vivo performance against well established post-mortem (in-vitro) laboratory tests obtained at key selected points.

Aim 7: Compare FMT-XCT and PET-XCT to quantitatively assess the sensitivity and overall image fidelity achieved by FMT-XCT, using the PET-XCT as a gold standard (Wp8). Success will be

FMT-XCT 201792

7

Figure 1. Schematic of the work-packages involved in the proposal

measured by reporting quantitative metrics on image quality for FMT-XCT and PET-XCT obtained from the same animal model.

B.1.2 Progress beyond the state of the art This proposal offers to develop a unique FMT-XCT hybrid system, the likes of which exist nowhere. This

is a major starting point of this proposal. Besides the relatively straightforward integration of two different modalities, this proposal offers to significantly advance each of the systems components, well beyond the current state of the art in order to further advance technological European competencies and provide a truly high performing system that utilizes the strengths of XCT and FMT while significantly reducing their weaknesses by the hybrid approach. It is proposed to: 1. Improve XCT contrast differentiation while retaining reasonable dose exposure by dual energy, multi-

resolution approaches. 2. Advance stand-alone FMT by utilizing novel free-space non-contact approaches operating in the

360-degree projection implementation, offering the best yet performance of stand alone FMT. 3. Advance the knowledge on optimal implementation of anatomical information into the FMT problem

by (a) improving XCT contrast in Wp2 to offer advanced segmentation in Wp4 and highly adept annotation of XCT structures with optical attenuation properties in Wp5 and by (b) researching the use of priors that offer no-strong anatomy-function relations as is appropriate for fluorescence bio-distribution (Wp4). This advancement can only be achieved herein due to the proposed construction of the first XCT-FMT prototype worldwide.

4. Deliver the best performing optical imaging method by means of incorporating XCT information. 5. Deliver a highly evolved hybrid XCT-FMT to complement XCT with functional and molecular contrast

and complement FMT with anatomical contrast and the ability to improve its performance by means of XCT-based attenuation correction.

6. Advance the knowledge into the sensitivity and quantitative ability of the method in resolving functional and molecular signatures in-vivo.

While there is no direct basis to compare progress against, since a truly unique system is being developed, comparisons to stand-alone modalities and to PET imaging, as described in the previous paragraph offer direct evaluation metrics by which to monitor work progress.

B.1.3 S/T methodology and associated work plan

B.1.3.1 Overall strategy and general

description The work is split into 9 work

packages (Wp) as shown in Fig.1. Work package 1 regards the management and co-ordination of the project by partner 1 while Wp 9 considers dissemination and training activities. Each partner has been carefully selected as he brings a unique aspect into this development, while utilizing expertise from other partners. WP2-WP4 build therefore unique XCT and FMT technology and know-how that is then integrated into

FMT-XCT 201792

8

one system prototype in WP5. WP6 and WP7 research appropriate imaging strategies for in-vivo imaging, and perform pre-clinical imaging while WP8 compares the FMT-XCT system with a previously developed PET-XCT system to test the hypotheses in this proposal. The effort varies in different work-packages based on the corresponding complexity of each package as summarized in Table 1.3d.

B.1.3.2 Timing of work packages and their components

The timing chart is given according to the objectives (tasks) described in each work-package

 

 

FMT-XCT 201792

B.1.3.3 Work package list /overview Work package list

Work

package No

Work package title

Type of activity

Lead

participant No

Person- months

Start

month

End

month

WP1 Management MGT 1 50 1 48 WP 2 XCTdevelopment RTD 2-Leti 77.7 1 24 WP 3 FMT forward problem RTD 3 54 1 36 WP4 FMT inversion with priors RTD 4 60 1 48 WP5 FMT-XCT integration RTD 1 131.7 1 48 WP6 Breast cancer imaging RTD 2-Lime 80 1 48 WP7 Treatment imaging RTD 6 68 1 48 WP8 FMT-XCT vs. PET-XCT RTD 5/8 70.6 1 48 WP9 Training & Dissemination OTHER 1 16 12 48

TOTAL 608

9

 

FMT-XCT 201792

B.1.3.4: Deliverables List

List of Deliverables – to be submitted for review to EC1

Del. no.2

Deliverable name

WP no.

Lead bene-ficiary

Estimated indicative person-months

Nature3

Dissemi-nation level 4

Delivery date 5 (month)

1.1 Consortium agreement 1 1 2 R RE 1

1.2 Minutes of the kick off consortium meeting

1 1 2 R RE 1

1.3 Project website and updates

1 1 15 O PU,RE 12,24,36

1.4 Program of the consortium meetings

1 1 15 R RE 12,24,36

1.5 Half time report 1 1 6 R RE 24

1.6 Program of the closing meeting

1 1 6 R RE 48

1.7 Final report 1 1 4 R RE 48

2.1 XCT design 2 2 6 R PP 1

2.2 Dual energy prototype 2 2 30 P PU 15

2.3 Dual energy processing software

2 2 15 O RE 15

2.4 Preliminary technical specification

2 2 6 R PU 18

2.5 Scattered energy measurements

2 2 8.7 R PU 21

2.6 Comparison of contrast enhancement strategies

2 2 9 R PU 24

2.7 Final technical specification for XCT system

2 2 3 R RE 24

3.1 Direct inversion algorithm 3 3 12 O PU 9

3.2 Experimental trainingset measurements

3 3 3 O PU 12

3.3 Quantitative evaluation of direct inversion with FMT

3 3 6 R PU 18

1 In a project which uses ‘Classified information’ as background or which produces this as foreground the template for the deliverables list in Annex 7 has to be used

2 Deliverable numbers in order of delivery dates: D1 – Dn 3 Please indicate the nature of the deliverable using one of the following codes: R = Report, P = Prototype, D =

Demonstrator, O = Other 4 Please indicate the dissemination level using one of the following codes:

PU = Public PP = Restricted to other programme participants (including the Commission Services) RE = Restricted to a group specified by the consortium (including the Commission Services) CO = Confidential, only for members of the consortium (including the Commission Services)

5 Month in which the deliverables will be available. Month 1 marking the start date of the project, and all delivery dates being relative to this start date.

10

FMT-XCT 201792

data

3.4 Multi spectral algorithm 3 3 12 O RE 21

3.5 User friendly software environment

3 3 9 O RE 24

3.6 Quantitative evaluation of direct inversion with hybrid data

3 1 12 R PU 32

4.1 Inversion algorithms 4 4 24 O PU 9,15

4.2 Quantification of algorithmic performance

4 4 6 R PU 21

4.3 Inversion algorithm for estimating optical attenuation

4 4 12 O PU 24

4.4 Feature extraction algorithm

4 1 6 R PU 24

4.5 Quantification of algorithmic performance with experimental data

4 4 6 R PU 32

4.6 Userfriendly software 4 4 6 O RE 36

4.7 Optimal inversion using data and image compression

4 4 0 R RE 44

4.8 Assessement of combined reconstruction/classification

4 4 0 R RE 46

5.1 360 degree FMT prototype 5 1 24 P PU 12

5.2 Optimal settings 5 1 6 O RE 15

5.3 Gantry development 5 7 15 P PU 24

5.4 FMT-XCT prototype 5 1 36 P PU 27

5.5 Training datasets 5 1 8.7 O RE 28

5.6 Multi-spectral capacity 5 3 12 O PU 30

5.7 Compute and assign optical attenuation values

5 1 12 O RE 32

5.8 Functional specification of optimal acquisition and operational parameters

5 1 9 R RE 32

5.9 Functional user-friendly operational software

5 1 9 O RE 36

6.1 To develop molecular probes

6 2 30 O PU 9, 18

6.2 Prepare mammary tumor animal models.

6 1 9 O RE 18

6.3 Breed and make available PyMT animal models

6 2 9 O RE 24

6.4 Develop U87 animal models

6 2 9 O RE 27

6.5 Study FMT alone vs. FMT-XCT in resolving tumors

6 1 9 R PU 36

6.6 Study the imaging performance in cancer in animal organs

6 1 12 R PU 40

6.7 Study the overall imaging performance

6 2 2 R PU 42

7.1 Preparation of HIF 7 6 12 O PU 9

11

FMT-XCT 201792

transfected breast cancer cells

7.2 FMT of HIF induction in breast tumors.

7 6 10 R PU 15

7.3

FMT-XCT vs. Histological correlates in MDA-MB-231 tumors.

7 1 10 R PU 32

7.4 FMT-XCT vs. Histological correlates in PyMt tumors.

7 2 12 R PU 36

7.5 FMT-XCT to resolve differential treatment levels in tumors invivo.

7 6 12 R PU 40

7.6 Assessment of antiangiogenic therapy withFMT-XCT

7 6 12 R PU 46

8.1 Construct imaging phantoms.

8 5 12 O PU 12

8.2

Phantom characterization with PET, FMT and XCT and appropriate probes

8 5 20 O PU 24

8.3 Coregister and compare FMT-XCT and PET-XCT.

8 8 28.6 O PU 42

8.4 Analyze and report on XCT-PET and XCT-FMT performance.

8 8 10 R PU 48

9.1 Dissemination implementation document

9 1 2 R RE 36

9.2 Training curriculum and implementation document

9 1 2 R RE 24

9.3 Public promotion leaflet 9 1 3 O PU 36

9.4

Meeting program and book of abstracts for the meeting atmonth 42.

9 1 3 R PU 42

9.5 Summary of achievement at Workshop at month 42

9 1 2 R PU 42

9.6 Report on technologytransfer and IP.

9 1 2 R RE 48

9.7 Report on training and PhD theses achieved

9 1 2 R PU 48

TOTAL 608

                       

 

                         

                         

12

FMT-XCT 201792

13

B.1.3.5 Work package descriptions

Work Package 1 Work package number 1 Start date or starting event: 1

Work package title Management

Activity Type21 MGT

Participant number 1 2 3 4 5 6 7

Person-months per participant:

42 2 2 1 1 1 1

Objectives: WP1 serves as a horizontal workpackage that enables: 1.1. Management of the interaction of the co-ordinator, the Executive Committee and the Advisory Committee in

order to design, monitor and optimise the experiments. 1.2 Management of the pre-existing and new intellectual property (IP) and know-how. 1.3. Maintenance of the Consortium Agreement. 1.4. Regular meetings and reports on the scientific and financial progress, ethical and welfare issues.

Description of work (possibly broken down into tasks), and role of participants This work-package contains all activities to ensure coordination of the consortium, reporting to the EU, enabling appropriate information flow between participants and the EU.

Management of the consortium will require active interaction of the co-ordinator, the Executive Committee (EC) and the Advisory Committee (AC). The co-ordinator in this proposal will be helped by a project manager/public relations person, hired exclusively for management purposes. Partners will also devote time effort for management activity, when participating in executive tasks and in relation to the generation of the deliverables listed herein. The project manager will be further responsible for communicating Executive Committee decisions and directions and actively work towards group networking, executive committee meeting preparation and gathering the necessary research and financial information for the deliverables and reports. The roadmap of WP1 is presented in the Gantt chart. Besides the general management, this WP will ensure the ethical and animal welfare management of the project under direct supervision of the co-ordinator. An ethical expert will be invited to the kick-off and annual meetings to provide advice for the project and to assist in the preparation of the final report including broader ‘science and society’ implications of the results.

Meetings: Regular meetings will be held to present, monitor and evaluate progress and to make decisions for future steps. Work-package (WP) meetings will be organised a month prior to Executive Committee (EC) meetings, thus the WP meeting results will be efficiently translated into consortium-level decisions. A kick-off consortium meeting will be held at the beginning. WP and EC meetings will be held every year (mo 12, 24, 36). Key WP participants may be invited to present on all EC meetings. If need arises, EC video conferencing meetings will be held at mo 6, 18, 30, 42 or ad-hoc. Half-time report preparation at month 24. Progress monitoring, financial corrections and redistribution of WP will be performed at EC meetings at month 24. New scientific and industrial partner evaluation will be also performed in EC meetings. Final report and consortium meeting program will be provided at month 48. All consortium meetings will contain a dedicated EC meeting for reaching executive decisions.

Reports: Internal WP reports and a summarized consortium report at month 12, and 36 to monitor financial and research progress. Full reports to the EC at month 24 (half time report) and 48 (final report). Tasks:

Task 1.1. Organisation of consortium meetings, at months 0, 12, 24, 36 and 48. Task 1.2. Organization of Executive Committee conferences as needed. Task 1.3. Preparation of the half-time report on the scientific progress and financial status. Task 1.4. Preparation of the final report on the scientific results, new IP and finances. .

FMT-XCT 201792

14

Deliverables (brief description and month of delivery) 1.1 Consortium agreement (mo.1) 1.2 Minutes of the kick off consortium meeting (mo. 1) 1.3 Project web site (mo 1) 1.4 Program of the consortium meetings (mo 12, 24, 36). 1.5 Half-time report for the EC (mo. 24) 1.6 Program of the closing meeting (mo.48). 1.7 Final report for the EC (mo 48).

FMT-XCT 201792

15

Work package 2 Work package number 2 Start date or starting event: 1

Work package title XCT development

Activity Type21 RTD

Participant number 2 5 7 6 1

Person-months per participant:

51.7 18 6 1 1

Objectives: 2.1 To design a micro X-ray CT system appropriate for small animal imaging 2.2 To develop the XCT system and implement a dual energy X-ray CT system. 2.3 To optimize delivered dose using a multi-resolution CT approach. 2.4 To research and minimize possible interference of X-rays with optical components. 2.5 To research the contrast between organs and tissues achieved by the dual energy method 2.6 To research the contrast between organs and tissues achieved by contrast agents. 2.7 To provide an optimal XCT design to be incorporated with the free-space FMT system in WP5.

Description of work Several designs and commercially available systems exist for X-ray CT of small animals. The need to

implement a separate work package on X-ray CT development stems from the diverse needs of the hybrid approach to produce an XCT design that is not only appropriate for small animal imaging but also:

1) provides adequate accommodation of the optical components, 2) eliminates X-ray interference with optical components 3) offers improved contrast between organs as is important for the optimal utilization of X-ray CT

information as priors in the FMT inversion procedure as explained and performed in WP4. Overall the integration of XCT experts and XCT development in this proposal allows high flexibility in changing specifications when issues of interference between optics and XCT components arise or functional specifications need to be changed. It creates a functional team that can respond to both XCT and FMT challenges and yield an optimal system and method.

2.1 X-ray CT design. While the partners have already discussed appropriate XCT designs, the design

finalization will begin at the kick-off meeting, based on functional specifications directed by all partners in a special “design” meeting, which will be part of the kick-off meeting. Partners CEA-LETI, FIHGM and VAMP have extensive experience with the development of XCT small animal systems. Compatibility with optical components and small animal imaging considerations will be directed by the other partners and will be taken into account. The proposed system will employ a microfocus X-ray tube, and a solid state digital X-ray image sensor for the image acquisition; implementing cone-beam geometry. A computer will operate the X-ray source, an X-ray stopping shutter (to minimize X-ray dose per study) and the frame grabber to acquire the projections. Administered dose will be within the European and LETI guidelines for small animal XCT as applied to similar commercial micro-CT systems, ensured further by VAMP. The system will be tightly enclosed into a 4mm lead sheet chamber to minimize stay radiation. The radiation emitted outside the box will be measured upon construction and regularly thereafter during measurements to ensure the effectiveness of the shielding constructed. Inversion will be based a cone-beam reconstruction package available to FIHGM, implementing a modified Feldkamp-Davis-Kress reconstruction algorithm for circular orbit cone beam geometries [34]. To further improve on artifacts caused by X-ray scattering when using flat panel detectors a scatter reconstruction approach for Cone Beam CT, developed at CEA-LETI, will be employed [35] for small animal CT imaging and for the dual energy context in 2.2.

2.2 X-ray CT dual-energy development Dual energy approaches have the potential to improve the

contrast between main organs by dual energy decomposition in (ρ,z) components. This is necessary in this proposal for optimal use of X-ray priors in the FMT problem, as described in WP4, but can more generally also improve the diagnostic and utilization potential of XCT and is also clinically investigated. CEA-LETI has been involved in several projects of dual energy XCT decomposition of bone from soft tissue in clinical bone densitometry and VAMP is actively looking at the utility of these methods as well. CEA-LETI will develop a dual energy system prototype based on the design of 2.1, implemented for in the horizontal geometry (i.e. non-gantry approach) to accelerate the

FMT-XCT 201792

16

development, since the objective here is not the highest possible imaging performance but the comparison of different alternatives for recommending an appropriate design in the gantry-based system developed in Wp5. In this case the mouse is vertically rotated, after immobilized in a special holder that does not allow movement due to rotation. The consortium will then investigate also through motility between partners, whether this approach is useful for differentiating between soft-tissues, over contrast agents as per tasks 2.4 and 2.5.

The particular implementation will utilize a single source that is emitting at different energies by applying different voltages and appropriate filtration. For low energy, the source will be filtered with a material presenting a K-edge around the energy of interest and utilizing a voltage higher than the K-edge. For high energy the spectrum is hardened using higher voltage and filtering with material such as copper to suppress lower energies. For animal imaging it will be possible to utilize lower energies, compared to dual-energy clinical approaches. These energies will be selected to yield maximum contrast between the organs while ensuring appropriate signal to noise ratio. Prior experience indicates that appropriate energies would perhaps peak at 70KeV and 30KeV but the exact energies and source parameters will be optimised based on X-ray attenuation model software and will be adjusted and confirmed experimentally. Since strong overlap is expected between the two spectra the set of data obtained at the two energies will have significant cross-talk. Dual-energy image analysis is therefore based on a calibration process which allows to decompose each voxel over a basis of material. This calibration is performed offline (without patient or animal) and provides attenuation measurements for different thicknesses of combination of 2 materials: a low density and higher density material, for example lucite or hydroxyapatie mixed at different concentrations with a resin to mimic different soft tissues (and bone). The measured projections are then converted in equivalent basis materials thickness decomposition by interpolation of the calibration data. After reconstruction of these projections converted in basis materials, the X-ray attenuation of each reconstructed voxel can be described as the X-ray attenuation of a linear combination of the basis material. The density and the atomic number of each voxel can then be approximated by the density and the atomic number of the material combination corresponding to this voxel.

2.3 Optimization of delivered dose. In order to minimize the dose delivered to the animal in dual energy approaches, a multi-resolution CT approach will be considered. Since the FMT forward problem is solved at the resolution level of a mean free-transport path, i.e. ~0.5mm (or larger) for small animal imaging, X-ray 3D differentiation of soft tissues in the animal does not necessarily require the highest of XCT resolution. Therefore, dual-energy based tissue contrast will be based on a low resolution scan of the second energy source and subsequent data interpolation on the first set of acquisition. LETI has already progressed in developing approaches for incomplete data reconstruction based on the RM Lewitt approach. The CT acquisition is performed with a smooth breathing gating to limit the blurring, while for high resolution, VAMP has available high speed scans that can further improve imaging performance.

2.4 Minimization of X-ray interference. Through motility activity, VAMP and HMGU will visit CEA-LETI,

upon prototype construction and perform measurements of scattered X-ray energy in the various directions of the XCT prototype, when using phantoms and animals, in order to design appropriate X-ray shielding and identify optimal areas where shielded optical components can be placed. This information will confirm simulations performed by VAMP and by CEA-LETI and it is important in anticipation of the developments in WP5. The participation of HMGU and UZH will contribute to optimal consideration in terms of optical components and animal imaging.

2.5 Dual-energy contrast. Currently it is largely unknown whether dual energy approaches can yield

appropriate tissue and organ discrimination, as per methods in Task 2.2 and the need for image segmentation in WP4. It is also unknown which energies will yield optimal contrast and how this compares to the use of XCT iodine-based contrast agents. For this reason, after appropriate simulations and phantom calibrations performed by CEA-LETI in task 2.2 representative animal species (nude mice (n=5) and Balb/c mice (n=5); see also Section 4) provided by CEA-LIME will be imaged in order to obtain dual energy images of head, torso and abdomen. The resulting, processed images resulting from the dual energy acquisition (see 2.2), will be then analyzed and compared to the single energy image in terms of optimal contrast achieved. Quantitative image analysis will be performed by Prof. Englmeier at HMGU to determine whether there are contrast benefits in the dual wavelength approach for image segmentation.

2.6 Use of X-ray contrast agents. In parallel to the dual energy approach, FIHGM and CEA-LETI will

investigate through motility the contrast achieved by the use of iodine-based contrast agents. This will be achieved by imaging the animals at Task 2.5 also after the administration of XCT contrast agents, immediately after the

FMT-XCT 201792

17

imaging session in Task 2.5, while the animals are immobilized in the imaging setup, by performing a second single energy acquisition. All animals will be euthanized immediately after this second imaging session. The relative ability to differentiate between the heart, lung, muscle and other organs will be explicitly determined by characterizing the contrast achieved herein and as it relates to the corresponding results in Task 2.5. UZH will aid in identifying different structures as it relates to high quality MRI images of the same species, that will be also provided by UZH. Image analysis will be similarly performed by the group of Prof. Englmeier at HMGU - IBMI. The optimal method that maximizes organ delineation under comparable administered dose will be selected for WP5. Special considerations of defining optical parameters are described in WP4.

2.7 To provide an optimal XCT design An important milestone of this work package is to propose an optimal XCT design that will be implemented in a rotational, industrial-grade gantry in WP5 and an appropriate technology recommendation (single-energy, dual energy and contrast agent enhancement) for optimizing XCT imaging performance. The decision of an optimal design will be reached in the Executive Committee meeting in month 18 and formally presented in the Consortium meeting in month 24 and in the EU report.

Risk This proposal examines several alternatives to minimize risk. The competency of the partners to develop laboratory prototypes (CEA-LETI, FIHGM) and commercial systems guarantees that several of the practical implementations details will be addresses with multiple skills and capacities. High motility between partners and web-conferencing as described under management will further ensure the high information flow and timely execution of the proposed tasks.

Training The tasks offer significant training opportunities as they will develop unique knowledge in terms of

advanced XCT use and contrast differentiation This information will be made available in the first consortium meeting held for this purpose in Grenoble, and will include tour of the facilities and a demonstration of the system prototype to the consortium. Importantly through motility activity the FIHGM and VAMP will actively participate in the prototype development and measurements , whereas CEA-LIME will visit and participate in animal imaging.

Milestones This Wp is involved in a significant milestone, i.e the selection of an appropriate XCT

technology to use for integration in the FMT-XCT prototype. A preliminary recommendation and decision will be performed in month 18 by the executive committee after circulation of an internal report and results, and the technology and decision will be officially presented in the meeting of month 24.

Deliverables 2.1 An optimal design for micro X-ray CT system that can facilitate optics (mo.1). 2.2 A functional prototype for dual energy cone beam XCT (mo. 15) 2.3 Calibrated, dual energy processing software (mo.15) 2.4 Preliminary technical specification for SCT design to be implemented with the hybrid system (mo 18) 2.5 Measurements of scattered X-ray energy (mo 21) 2.6 Identification of optimal contrast-enhancing strategy mo 24) 2.7 Optimized system design summary, technical specification and imaging protocol for integration in the hybrid FMT-XCT in WP5 (mo.24).

FMT-XCT 201792

18

Work package 3 Work package number 3 Start date or starting event: 1

Work package title Theory for 360 degree FMT .

Activity Type21 RTD

Participant number 3 1 4

Person-months per participant:

36 12 6

Objectives: 3.1. To implement direct inversion based on boundary removal method for media with arbitrary boundaries. 3.2 To research optimal direct inversion approaches with simulations and experimental data. 3.3. To compare the direct inversion performance with conventional, previously developed FMT inversion methods. 3.4 To incorporate algorithms for multi-spectral imaging. 3.5 To develop user-friendly software for inversion of XCT–FMT data based on direct inversion approaches. 3.5 To invert training data from acquired from the FMT-XCT system for algorithmic finalization.

Description of work The proposed FMT-XCT system implies the development of an optical tomography method based on complete projection tomography over 360-degree angles. To accomplish high imaging performance it is important to move away from fiber-based systems or the use of matching fluids (as common in older generation systems) and utilize direct measurement of photons, at multiple projections, using CCD cameras. Theoretical approaches developed by partners HMGU-IBMI and FORTH-IESL now enable this new generation of systems to be implemented. 360-degree free-space systems utilize hardware similar by that used by XCT and so they can seamlessly be integrated in an XCT platform. They are further expected to deliver the best FMT performance compared to fiber-systems, fluid-systems or limited view angle systems (for example slab geometry systems) that have been developed in the past due to the superior data quality and data size acquired. It has been shown that high spatial sampling improves imaging performance and resolution [36] [37], (despite original notions that only a few measurements suffice for optical tomography). With this recent progress however comes a new challenge. Since 360-degree systems acquire a significant larger number of data (~108 measurements) compared to fiber based systems (102-103 measurements) or slab geometry systems (~104 measurements), the computational requirements are tremendously increased.

Task 3.1 Direct inversion: A key component in the success of the proposed hybrid system and a major

objective therefore behind this work-package is the implementation of direct inversion algorithms that retain

robustness while utilizing a large data-sets and achieving realistic reconstruction times (5-15 minutes). Direct

inversion obtains an expression for the fluorophore concentration N given the measurements U, in the form.

{ }UgN =)(r (3)

where g is the direct inversion formula. This approach does not involve matrix inversion and benefits from large

datasets since it is based in Fourier-Laplace decomposition [See work by John Schotland, for example [38, 39]]. The

reason these approaches have not been implemented before is mainly due to the fact that direct inversion formulas

have been found only for 1) simple geometries such as the infinite, slab or cylinder case and 2) for homogenous

media.

It is a major contribution of WP3 therefore, and a novelty in this proposal, that a particular inversion

scheme, termed “boundary removal”, developed recently by partner FORTH-IESL and HMGU-IBMI, will be

employed to allow the application of direct inversion methods with measurement obtained from complex boundaries

[38, 40]. This scheme is further combined with a certain dual-wavelength normalization method (see Ref [41] ),

which has been shown to perform accurately even when imaging at highly optically heterogeneous media.

Collectively, the approach is expected to yield the most accurate and highly performing inversion method developed

yet for FMT and combined with the superior data set collected with the FMT-XCT system developed herein.

More practical details of implementation follow: The forward problem utilized is a solution of the diffusion

equation based on the normalized Born approximation [41], which is independent of instrument gain factors and

yields accuracy even at high degree of background optical heterogeneity [42]. In the past, fast modeling of the

boundaries by the Kirchoff approximation Ref. [15, 19] or higher order approximations [18] and the propagation of

FMT-XCT 201792

19

photon signals from this surface to a CCD camera were employed due to their computational efficiency in slab

systems. However this scheme is now replaced by the boundary removal scheme. In parallel partner UCL (Simon

Arridge) has previously developed the main theoretical mainframe for modeling photon propagation in tissues in the

forward sense and will contribute here code developments associated with the forward modelling of photon

propagation in tissues. HMGU and FORTH have also previously developed methods (also under FP6) to measure

and introduce the mouse surface into the reconstruction code (see also fig.3,4) in order to calculate an accurate

forward model for optical tomography. While the above description generally referred to a single wavelength, multi-

spectral measurements can be also be described by Eq.1 and inverted under one inversion scheme for increased

accuracy.

In summary the key innovations in task 3.1 are:

1. Algorithmic developments in accounting for boundary effects using a recently developed boundary removal

method.

2. Time efficient reconstruction of photon propagation in tissues using direct inversion formulas operating in

optically heterogeneous media bounded by arbitrary free surfaces that can be used for highly-performing

stand-alone FMT as per Concept 1.1.a 1 (page 3).

3.2 Experimental optimization. Simulated data generated by UCL, based on anatomical maps provided by CEA_LETI will be generated using the TOAST software (i.e. a public-domain finite element simulation of photon propagation in tissues developed by UCL), In addition experimental data on phantoms and controlled animal experiments will be generated by FORTH on the stand-alone system already developed in Crete. Experimental data sets will be obtained on phantoms, build by FIGHM together with HMGU (see WP8) and animal models (see Section 4 for animal numbers) provided by partner UZH. These data sets will be utilized to test direct inversion approaches in practical settings that relate to the final FMT-XCT prototype. Finally, upon completion of the FMT-XCT prototype at HMGU, free-space FMT data will become also available as described in WP5, which will be explicitly analyzed by partner FORTH. The overall goal of task 3.2 is to find optimal inversion settings (optimal number of data, optimal regularization parameters, as relate to the signal to noise ration contained in the data). These tasks have been previously analytical addressed by singular value decomposition approaches and will be explicitly studied herein in relation to direct inversion performance.

3.3 Direct inversion vs. conventional FMT inversion. To quantitatively assess the developments in Task 3.2,

FORTH and UCL will study the performance of the direct inversion method in relation to :

a. Old generation linear FMT inversion code developed by partners HMGU-IBMI and FORTH-IESL

b. Finite element-based non-linear inversions, under the TOAST software, performed by partner UCL.

Both approaches 3.3 a,b have been extensively tested in the past and can serve as a benchmark for FMT performance (albeit the impractical inversion times). In particular 3.3.b is based on a two-step approach, where the background attenuation (absorption and scattering) is first reconstructed and used to calculate more accurate weights for the fluorescence problem. This method, available with partner UCL, will be considered as the gold standard in simulations and phantom measurements, even if more computationally intensive, in order to evaluate the performance of the direct inversion approach, by comparison of results between the two methods. Training data sets obtained from controlled phantoms and animal experiments post-mortem (see Wp5 and Wp8) will provide a data set library to test different methods. The overall method that will give the best imaging performance while maintaining practical inversion times will be then utilized for subsequent in-vivo data analysis. Inversion accuracy and performance will be evaluated in terms of imaging accuracy against the known manufactured optical phantoms (as per Task 3.2) and it terms of the resolution and sensitivity achieved. 3.4. Multi-spectral imaging. As evident in Wp6 and Wp7, the ability to perform imaging of multiple fluorochromes, simultaneously administered in-vivo can facilitate the study of complex pathways, or improve the imaging accuracy (see for example Task 6.3 in Wp6. FORTH has been developing multi-spectral approaches to 1) remove auto-fluorescence background in fluorescence measurements owing to tissue intrinsic fluorochromes and 2) the ability to

FMT-XCT 201792

20

accurately reconstruct multiple fluorochromes when the emission spectra are partly overlapping. This technique is based on the recording of tomographic data in multiple spectral regions with excitation light of different wavelengths and on the application of linear unmixing algorithms for targeting multiple fluorescent probes. These methods are available to FORTH as part of other funding (also FP6 funding) for imaging at the visible, but will be adapted herein in for imaging at the far-red and near-infrared (600nm – 900nm) to enable accurate imaging. We note that in the 600-900nm tissue auto-fluorescence is significantly reduced compared to the visible and these methods are expected to work even better than in the visible, after appropriate determination of the relative strengths of the near-infrared fluorochromes employed in commercially available probes (see table I) or in developments in Wp6. 3.5. Software development. Important in the technique dissemination and training activities, is the development of user-friendly software that can be accessible by users and not only developers. The software, developed by partner FORTH, will have easy to handle inputs and correspondingly straightforward visual outputs to simply guide a user through the inversion process and allow for at least some simple visualization tasks in order to easily view and quantify the reconstructed images. Risk and alternatives. The tasks of the work package follow highly inventive developments that present unknown development challenges as to their exact implementation. The team assembled however has major contributions in the developments of these methods and are very well skilled in programming computational methods for tomography. In addition accurate validation against established gold standards are contemplated to ensure that a highly performing inversion method will be developed. Furthermore the utilization of experimental data will enable the development of a practical method appropriate for in-vivo imaging. Training. This WP will enable training of consortium scientists in methods for FMT inversion methods by appropriate participation in the workshop at month 42, and interaction also through motility of the UZH and UCL groups and other group participation as well. Milestones. Important decision points in this work-package concern the development and final decision on an inversion algorithm with fundamental superior characteristics compared to the current state of the art. After validation with experimental data from phantoms and in-vivo, the partners involved will direct optimal algorithms, implementation settings and a functional inversion code for stand-alone inversion.

Deliverables (brief description and month of delivery) 3.1. A direct inversion algorithm for fluorescence implementing boundary removal principles (mo. 9). 3.2. Experimental measurements to serve as a training set for algorithmic optimization (mo. 12) 3.3. Quantitative evaluation of direct inversion performance vs. established FMT inversion methods (mo.18) 3.4 Functional code of multi-spectral algorithm for simultaneously resolving multiple fluorochromes (mo. 21). 3.5. Software that implements these algorithms in a user-friendly environment (mo. 24). 3.6 Optimization and quantitative evaluation of direct inversion performance with hybrid XCT-FMT data (mo. 32)

FMT-XCT 201792

21

Work package 4 Work package number 4 Start date or starting event: 1

Work package title FMT inversion with image priors

Activity Type21 RTD

Participant number 4 1 3 2

Person-months per participant:

36 12 6 6

Objectives: 4.1 To develop FMT inversion utilizing XCT image priors without strong anatomy function correlations. 4.2 To incorporate XCT image segmentation into the FMT code. 4.3 To calculate spatially varying optical attenuation in tissues in-vivo. 4.4. To develop FMT inversion based on simultaneous XCT segmentation and classification. 4.5 To quantitatively examine optimal inversion methods based on experimental data. 4.6 To develop user-friendly software for inversion of XCT–FMT data based on a-priori inversion.

Description of work (possibly broken down into tasks), and role of participants Utilizing XCT information for improving the FMT imaging performance is a key development herein and a

cornerstone towards a highly synergistic hybrid system. Many of the partners are very experienced in the use of image priors in inversion problems but also identify that the use of image priors may lead to biased solutions. Therefore the consortium focuses here on developing methods, beyond the current state of the art, that improve the accuracy of the FMT problem without biasing the solution. We will explore three inversion methods:

1. the use of anatomical priors without strong anatomy-function correlation. 2. the use of prior segmentation and classification techniques to identify tissue classes 3. the use of the methods developed in Wp3 (without priors) to obtained truly unbiased solutions for

comparisons (ANY investigation with priors herein implies also the of the unbiased solutions in Wp3 as means of internal reference).

In order to build more accurate forward problems, and to make the Bayesian framework feasible, it is necessary to estimate optical parameters. While this is experimentally achieved in WP5, many of the methodological components are enabled herein as explicitly described. Task 4.1 Inversion based on priors. The FMT inversion method (as also described in Wp3) can be written as the minimization of a cost function that in the generic sense can be written as:

2( ) || || ( )C x y Wx Q x= − +

� � � �

(1)

where y�

is the vector of measurements for different source detector pairs (in this case fluorescence over emission

measurements) , x�

is a vector of unknown fluorescence concentrations in different voxel elements in the medium

(after discretization) and W is the weight matrix, which maps the space of unknowns to the space of measurements.

The function ( )Q x�

is a penalty function which in conventional implementations is given under the Tikhonov

regulation i.e. 2

( )Q x xλ=� �

. For a simplistic use with image priors (in this case XCT priors but also potentially MR

or ultrasound priors), ( )Q x�

can be re-written as

2

( )

21 ( )

( )( ) 1 exp

2

Mi k i

i k i

x aQ x λ

σ=

−= − −

∑

�

(2)

where M is the total number of voxels used for the reconstruction, ( )k ia is the average expected optical property of

each organ or structure k seen on segmented X-ray CT images. ( )k iσ is the standard deviation associated with

each of the average optical property assigned. ( )k i is an index indicating which organs or structure each voxel i

belongs to. In essence we can define limits of fluorescence expected from different organs and mark our confidence

at these values using the appropriate standard deviation. One of the drawbacks of the above method is the

assumption that there is a simple relation between the pixel or region values defined in the auxiliary modality and

those in the optical image. This is by no means certain to be true, and if known is likely to be subject to considerable

estimation errors. For this reason Partner UCL considers herein three alternative methods for incorporation of priors

FMT-XCT 201792

22

developed with Partner FORTH.

Incommensurate Cross-Modality Priors It is therefore a particular contribution and novelty in this proposal that partner UCL will therefore develop methods for using XCT based priors when there is no simple relationship between the numerical values in the optical reconstruction and the anatomical modality, as is the case between fluorescence contrast and XCT density. We term this an incommensurate prior. Our approach will be to treat the mapping between values in one modality and the other as a set of hidden variables which must be estimated together with the reconstructed image. The effect of this will be to estimate a reconstructed image which is most likely the prior image in an information theoretic sense. The use of information theoretic metrics as a constraint in image reconstruction has been employed in only relatively few cases [chen2005a] and never to our knowledge in a nonlinear problem.

Hierarchical Bayesian framework. Hierarchical Bayesian methods, investigated by UCL and FORTH make no assumptions on strong anatomy-function correlations. This approach is also particularly appropriate for fluorescence imaging, since the anatomical images provide limited information regarding the underlying fluorescence activity. The method derives a “probability map” of the expected fluorescence activity for different tissue types, i.e. each organ/tissue is assigned a probability between 0 and 1 indicating the likelihood of fluorescence emission in that region. These maps are then used i) to impose discontinuities and ii) to constrain estimates with physiologically meaningful values in the fluorescence images. Then a hierarchical Bayesian approach will be used to incorporate potential inaccuracies in the prior information such as mismatch between the anatomical and optical edges and true optical coefficients and average coefficients available in the literature.

Object based reconstruction. UCL will represent shape implicitly using the level-set method. Here a function of one higher dimension is used whose zero set defines the boundaries of objects. The advantage of the level set method is that the topology of the segmented structures does not need to be known. In pilot work, we have demonstrated that direct reconstruction of the level set function is tractable using two simultaneous level-set functions to reconstruct absorption and scattering independently. We will extend this work to deal with multiple disjoint objects, using multiple level set functions following.

Task 4.2 XCT segmentation. FIHGM, CEA and HMGU will investigate methods for XCT segmentation based on the organ differentiation methods described in Wp2. In particular dual energy images obtained by CEA will be examined in parallel to iodine-enhanced images as described in Wp2 by FIHGM and HMGU. Dual energy images have the potential to enhance the contrast between organs. Blood-pooling iodine-based agents reveal images of vascularization, i.e. a bio-distribution of blood, which is the major absorber of light in the wavelengths of interest herein. Both methods therefore attain significant advantages in improving differentiation of structures on the XCT images with the goal of providing improved a-priori information to the FMT inversion.

Image analysis will be based on commercially available software and also segmentation tools developed in HMGU for micro-CT image segmentation. In particular Prof. Englemeier (HMGU) has developed methods based on active contours, which allow robust segmentation in images. The creation of the contour is not only based on adaptation to image features like edges, but also on the preservation of the contour smoothness. By assigning energies to the grade of adaptation and smoothness and then minimizing the total energy yields finding of optimal contours. A global optimal contour can be found by dynamic programming leading to automatic segmantation. This method has been so far applied to the more straightforward segmentation of the bone, (see Fig.Wp4-1) but it will be applied herein to contrast enhanced XCT images from Wp2 to identify more complex structures.

Fig Wp4-1: Automatic segmentation of the femur and the knee joint and labelling of the different bone structures (two left images) and cortex thickness measurement (two rightmost images) using active contours. Similar performance is expected on segmenting contrast-enhanced XCT images from Wp2 for accurate use as image priors.

Task 4.3 Determination of optical properties. Utilization of XCT images as a-priori information requires assumptions on the underlying optical property distribution based on an anatomical map or assumptions on the regularization of the hierarchical Bayesian methods. Therefore the use of XCT a-priori information, derived in Task 4.2 can be first used to yield a map of optical property variation in-vivo. This information can be used to derive an optical property atlas to be used with all animals of a species, or on a per animal case as described in Wp5. Here, UCL together with FORTH will develop an algorithmic inversion, based on the mainframe described in Task 4.1 to

FMT-XCT 201792

23

reconstruct optical properties (not fluorescence). This is a straightforward application of Eq.1, where the vector y is not a normalized fluorescence measurement but a measurement obtained just by illuminating a mouse by using light at a wavelength of interest. Partner 4 has been a major leader and inventor of these methods and this is a low risk step, resulting in accurate assignment of optical properties in the various structures seen in XCT. These XCT-based optical maps can then be used to contract more accurate forward problems of photon propagation, to improve the prior approaches considered in Task 4.1 or to directly resolve tissue intrinsic bio-markers such as haemoglobin, which is of interest to partner UZH and FORTH and is examined in Wp7.

Task 4.4 : Combining Reconstruction with Segmentation and Classification. Alternatively, UCL and FORTH will develop methods that combine an estimate of the optical properties of the tissue and segmentation techniques under a single inversion step. The hypothesis is that since the final segmentation or classification is of lower dimension than the full reconstruction, the estimation problem may be better posed.

Pixel based classification into tissue classes. We assume there are a fixed number of tissue classes present. We will develop a method to directly estimate the statistics of the optical properties of each image class, and to classify each voxel in the reconstructed image to one or the other. We will develop an iterative method based on the EM algorithm that alternately (i) estimates the posterior distribution for tissue class at each pixel and (ii) re-estimates the tissue class statistics (mean and variance).

Global classification into tissue classes. We will incorporate tissue classification into a Markov Random Field (MRF) based framework. This introduces a local smoothness constraint into the classifications that favours few continuous regions. Inference in the MRF will be performed using graph cuts (the push-relabel algorithm) or tree re-weighted message passing [kolmogorov2004]. An approximation to the posterior distribution of the pixels around the MAP solution will be used at each stage.

Task 4.5 Experimental validation. UCL will utilize simulated data in basic algorithmic development in Task 4.1 To further optimize and confirm these methods, experiments on animals will be performed in-vivo as per Wp5 – Wp7 to obtain realistic optical and X-ray heterogeneity and attenuation and controlled anatomical and fluorescence contrast. Optical attenuation and fluorescence values will be performed ex-vivo immediately following in-vivo imaging after surgically removing organs after appropriate suturing to minimize bleeding, i.e. loss of the major absorber haemoglobin by HMGU. This data set will be used as the gold standard in confirming results in Wp4. In addition to fluorescence, attenuation measurements performed at 670nm, 750nm and 780nm and at different body parts will be used to determine the attenuation of lung, liver, heart, bone, brain, intestine and “other tissues” as identified on co-registered CT.

Task 4.6 Software. Similarly to WP3, important in the technique dissemination and training activities, is the development of user-friendly software that can be accessible by users and not only developers. The software will have easy to handle inputs, XCT segmentation tools, and correspondingly straightforward visual outputs to simply guide a user through the inversion process and allow for at least some simple visualization tasks in order to easily view and quantify the reconstructed images.

A significant milestone herein is the selection of the most appropriate inversion methods using priors, from the alternatives described in Task 4.1 and 4.4 as well as the selection of optimal inversion approaches for optical property reconstruction.

Role of participants and training. UCL will perform algorithmic developments needed for inversion with priors. FORTH will share forward models from Wp3 and CEA-LETI will provide partners with XCT data sets (single energy, dual energy and contrast enhanced) from Wp2. FIHGH and HMGU will independently examine the XCT method that achieves optimal contrast and HMGU will develop segmentation methods for enabling the use of XCT structures as priors. UCL together with FORTH will develop methods for optical attenuation determination in data-sets obtained from FORTH and HMGU from animal models provided by UZH. Data exchange and active participation in this multi-disciplinary development from multiple partners fosters cross-disciplinary training further facilitated in consortium meetings, demonstration and workshop in month 42.

Deliverables 4.1. Inversion algorithms for improving reconstruction (mo. 9, 15) 4.2 Quantification of algorithmic performance with simulated data (mo. 21). 4.3. Inversion algorithm to determine the optical attenuation of different organs/tissues (mo.24) 4.4. An automatic feature extraction algorithm (mo.24). 4.5. Quantification of algorithmic performance with experimental data (mo.32). 4.6 User-friendly software that implements these algorithms in a user-friendly environment (mo.36).

FMT-XCT 201792

24

Figure WP5.1: Simplified drawing of

proposed FMT-XCT system using non-

contact measurements and 3600 geometry.

Work package 5 Work package number 5 Start date or starting event: 1

Work package title FMT-XCT integration

Activity Type21 RTD

Participant number 1 2 7 3 5 4 6

Person-months per participant:

96 9.7 9 6 6 3 2

Objectives: 5.1 To develop a fully functional multi-spectral XCT–FMT prototype and minimize XCT and FMT interference. 5.2 To integrate algorithmic developments from Wp2, Wp3 and Wp4 and control software operating the hardware components. 5.3 To acquire training data sets and optimize imaging settings. 5.4 To provide optical attenuation maps corresponding to the collected XCT images.

Description of work To achieve superior imaging performance the implementation of a fundamentally novel scanner based on

1) complete projection (3600) tomography, 2) non-contact approaches (no fiber-tissue coupling) and 3) free-space photon propagation (no matching fluids) is necessary. We will further integrate this system with X-ray CT to achieve concurrent high-resolution anatomical imaging. Although a commercial X-ray CT system could be employed, developing the hardware around the optical system maintains high FMT performance, optimal tissue contrast, high flexibility in X-ray utilization and cost efficiency, without compromising X-ray imaging performance, ,as explained in WP2. Several hardware components concerning the illumination, detection, minimization of optical and X-ray interference, dynamic range etc need to be explicitly researched herein, as briefly described in task 5.1. Task 5.1 FMT-XCT prototype Fig.Wp5.1 depicts a simplified drawing of the proposed system. A simplified prototype on a rotational motor stage will be developed in the first 12 months to serve as a pre-platform for the FMT-XCT integration, in order to optimize optical imaging parameters in the vertical geometry. Experience build by Partner 1 and 3 with horizontal free-space prototypes will be invaluable in this development. An industrial grade gantry will be provided by partner VAMP in month 24. VAMP will modify the gantry according to the design considerations and concerns from WP2, as it relates to component arrangement and special shielding for protecting the optical components from scattered X-rays. The gantry will be provided enclosed in a lead compartment for safe operation. The exact XCT components will be provided by VAMP in cooperation CEA-LETI according to the specifications derived in WP2. Partner HMGU will build the optical prototype using a laser light from a tunable CW Ti:Saph laser (690nm-870nm, 10-20 mW delivered) complemented by a few laser diodes for covering the complete far-red/NIR spectrum will serve as the light source for multi-spectral imaging. The laser light, after appropriate attenuation is directed through a 1x2 optical switch (not shown) to 1) an illumination branch that expands the laser light on the animal surface for front-illumination imaging and 2) to a fiber that is placed on the other side of the camera to implement the equivalent of fan beam geometry for FMT. The light beam is focused on the animal surface using a low numerical aperture lens so that the beam is always in focus during rotation. The animal is placed onto a quarter cylinder transparent glass window AR-coated which is mounted on a linear motorized stage for translation along z. The bed is mounted on both edges (not shown) to ensure accurate, reproducible and vibration-less movement. Signals propagated through the animal are collected via a highly sensitive CCD camera (and appropriate filters mounted on a rotating wheel) placed on the opposite side of the transillumination light spot. More viewing angles using mirrors rotating with the gantry or multiple cameras can be covered, however we have found in preliminary SVA analysis studies that this geometry is preferred for imaging by yielding an adequate data set while maintaining optimal

FMT-XCT 201792

25

dynamic range requirements and data set size. The camera and laser beam can be rotated around the animal using a high performance rotational rotor stage capable of high loads. While optical surface readings are feasible and can be easily implemented with the addition of a photo-luminescent plate, we expect to use the surface information extracted from the CT measurement for the FMT reconstructions. Practical considerations

Dynamic range We propose to implement a programmable, computer controlled attenuation which can automatically adjust the illumination light utilized in each measurement so that all projections are acquired under ideal illumination (i.e. best possible signal to noise ratio). This can be achieved using automated correction based on fast camera feedback measurements, before the actual measurement takes place.

Mouse positioning. Two alternatives for mouse placement will be considered. The first will use an anti-reflection coated glass quarter-cylinder plate mounted on the center of the gantry as shown on Fig.Wp5.1. The illumination beams will be perpendicularly incident on the glass window. We have previously utilized beam glass interfaces and we anticipate seamless operation. We will nevertheless test and correct (by changing alignment or with data post-processing) for photon distortions or reflections using controlled phantom measurements. Alternatively we will consider a bed of a few translucent thin strings for mouse placement. Gas anaethesia will be provided directly on the placement bed for mouse immobilization.

Acquisition parameters and field of view. X-ray and FMT acquisitions will be sequentially acquired due to the different acquisition practices required. The maximum field of view acquired at a single rotation is ~4cm for both systems. For larger fields of view, the animal will be translated and imaged. The proposed design is very flexible in imaging different body parts, in particular body vs. head, as compared with previously developed contact systems. Different optics (lenses or zoom parameters) may be required for head vs. torso due to the different dimensions. A typical FMT scan consists of linearly scanning the source along the animal at pre-set positions for each projection (1-3sec/source, 10-20 source positions and 12-18 rotations; i.e acquisitions of 2-18 min/wavelength, typical 5-10 min). An X-ray CT may require 10 or 20 steps scanning at 0.8-1.5sec per projection (~0.2s acquisition and 0.6-1.3s moving time) yielding <3-9 minutes per scan.

Animal movement. We will explicitly study the effects of mouse movement on imaging performance and will consider triggering, especially for thoracic X-ray CT imaging as also described in Wp2.

Illumination domains. CW has demonstrated implementation robustness in the past and offers favourable signal to noise characteristics. Under different funding Partner 1 has investigated time-resolved methods for the NIR. This technology (a Ti:Saph fempto-second laser and ultra-fast time-gated camera) will be available in IBMI and may be used as an alternative for accurately retrieving absorption and scattering or lifetime or to improve resolution. However to our experience, CW technology suffices for the goals of this study and enables the development of a more robust system compared to time-resolved of intensity-modulated methods.

FMT and XCT interference Issues of interference are important and will be explicitly studied by the consortium. Possible interference of optical components with scattered X-ray will be studied in Wp2. Scattered radiation will be also determined in the gantry system by VAMP, before optical components are added and any corrections and design considerations will be addressed by VAMP and FIGHM. Suspected stray radiation will be appropriately further shielded so that it eliminates radiation from hitting the CCS camera. By using appropriate lenses we will image only through a narrow opening in lead surface surrounding the camera. Alternative designs include the use of a mirror to image the subject through a completely otherwise concealed camera with no direct path to X-rays, or the use of sequentially imaging where X-ray is on while the optics are covered by appropriate movable shielding components. These decisions will be reached after experimental measurements, during executive committee meetings, ad-hoc if needed. Similarly, appropriate covering of hardware components in the gantry with totally absorbing black rough layers of matte black paint, and appropriate placement of surface towards angles not directly hitting the lens/camera will be designed to minimize reflections in the FMT-XCT chamber.

Metrics of assembly success At the end of instrument assembly a functional FMT/CT imager will be constructed with the following specifications: a) less than 15 minutes typical combined examinations per mouse (unless very high resolution imaging is performed) b) <100 micron resolution for the X-ray system and <millimeter resolution for the FMT system and c) <200 micron accuracy for the registration between the different images obtained. Furthermore the necessary software for component control and image registration will be implemented. Mock measurements simulating an actual experiment will be performed to test the ability of the software to control the different components and collect the necessary signals and images required within specifications. More extensive tests of

FMT-XCT 201792

26

performance related to imaging performance will be carried out as part of Task 5.3. Task 5.2: Integration of algorithms from Wp2 and Wp4. In order to facilitate high utility and dissemination of the project the need to incorporate hardware control software and algorithmic developments and software developed in Wp2 and Wp4 under one functional code, further bringing together developments in Wp5, such as the assignment of optical properties on XCT images in Task 5.4. This development will be achieved with close collaboration between HMGU, UCL, FORTH and CEA-LETI, each dynamically contributing into a common code accessible and tested by the other partners. Task 5.3 To acquire training data sets and optimize imaging settings. The functional prototype will provide measurements of phantoms provided by partner FIHGM (developed in Wp8) and animal models (n=10 animals; see also section 4) contributed by CEA-LIME to be utilized in the developments in Wp3 and Wp4. Importantly these first imaging sessions are required in order to optimize imaging parameters first with phantoms mimicking animals and then in-vivo, for example exposure and acquisition times, dynamic range/attenuation settings, minimization of reflections etc. Data sets acquired with optimal settings will be then communicated to partners FORTH and UCL, which will then use these data as training sets of the corresponding algorithms developed. Task 5.4 Optical property determination. Utilization of XCT images as a-priori information requires assumptions on the underlying optical property distribution based on an anatomical map as described in Wp4. Devising “mouse atlases” with average per organ optical properties assigned to different organs as seen on XCT images is a simple way to provide approximate maps that can be used in Wp4. However, herein we propose to develop the ability perform optical measurements on a per mouse basis, so that significantly more accurate maps can be produced. To achieve this optical measurements at wavelengths of interest will be performed at HMGU first in phantoms and then in animals in-vivo (same animals as in Task 5.3), together with corresponding XCT imaging. Then UCL will utilize the algorithms developed using priors, to “fit” or determine the optical properties (more precisely the attenuation) of different structures that appear on XCT so that experimentally based optical attenuation can be composed. While this information can be also use to compose maps of average optical properties, it can be directly also utilize to describe photon propagation in tissues or in the Bayesian approaches described in Wp4.

An alternative method that will be investigated is the use of contrast enhanced XCT images for correlating with areas of attenuation. In particular, macromolecular iodine-based agents enhance vascular areas. Since haemoglobin is the main light absorber in-vivo, certain correlations will be investigated between contrast enhanced XCT and optical images to determine optimal methodology to accurately relate XCT images with the underlying optical attenuation. These imaging sessions will be performed under guidance of partner FIHGM.

Training. Significant training activities will be performed under this work package. All partners will visit the Munich facilities for performing animal imaging or enabling software integration. In this process they will be exposed to hybrid XCT-FMT imaging and diverse aspects of in-vivo imaging and of theoretical developments. In addition, cross-training on the use of software and hardware and in animal imaging will be also facilitated in the workshop held in month 42. Milestones Technological decisions on the selection of optimal technology will be performed throughout the development. The most significant decision points are the decision on an optimal and final free-Space FMT implementation and shilding issues; all decision finalized by the executive committee.

Deliverables (brief description and month of delivery) 5.1 360-degree free-space FMT prototype developed on stepper rotational motor (mo 12) 5.2 Optimal illumination, positioning and dynamic range settings (mo 15) 5.3 Development of industrial grade gantry for the hybrid system (mo 24) 5.4 Functional FMT-XCT prototype (mo 27) 5.5 Data-sets to be used as training sets for algorithmic developments (mo 28). 5.6 Add multi-spectral capacity (mo 30) 5.7 Compute and assign optical attenuation values on XCT structures (mo 32) 5.8 Functional specification of optimal acquisition and operational parameters (mo 32) 5.9 Functional user-friendly operational software (mo 36).

FMT-XCT 201792

27

Work package 6 Work package number 6 Start date or starting event: 1

Work package title Cancer imaging with focus on breast cancer

Activity Type21 RTD

Participant number 2 1 3 4 6 5

Person-months per participant:

51 12 6 6 4 1

Objectives: 6.1 To provide key fluorescence probes and quantify the sensitivity and contrast achieved in the animal models

developed and as a function of tumor growth. 6.2 To develop animal models of breast cancer for studying FMT-XCT performance. 6.3 To develop animal models of other cancers for studying FMT-XCT performance. 6.4 To perform in-vivo imaging of key animal models of cancer and correlate the findings with standard laboratory

tests and growth measures 6.5 To predict clinical utility

Description of work (possibly broken down into tasks), and role of participants While phantoms are extensively utilized in virtually every work-package, the use of animal models of cancer is essential in order to develop FMT-XCT for its intended application, i.e in-vivo imaging. The overall goal of this work package is therefore to provide appropriate animal models, fluorescence probes and validation tools in order to :

1. Quantitatively examine FMT performance to visualize disease processes in-vivo and 2. To predict clinical utility in breast cancer imaging applications

A primary application of FMT-XCT is as a highly disseminated pre-clinical imaging tool for basic discovery, drug discovery and pre-clinical imaging as explained in the main text of Section 1. However it has been further shown in multiple studies that optical imaging can be applied in several pre-clinical models of human disease and to some clinical applications as well, for example in resolving breast cancer. The FMT-XCT system developed has the ability to i) offer significantly improve imaging performance and quantification over current state of the art optical imaging and ii) to offer higher detection sensitivity and specificity of detection over previous attempts due to the use of fluorescent reporters. In addition it can enable personalized treatment follow up as elaborated in Wp7. By the same token it can significantly enhance the diagnostic and overall clinical potential of stand-alone XCT. The proposed hybrid system is ideally suited to study the bio-distribution of molecular fluorescent probes and can be employed to quantitatively investigate and identify appropriate fluorescent probe candidates for clinical breast cancer imaging. Task 6.1 Animal models of breast cancer Animal models will be established or bred at CEA-LIME, shipped and housed at HMGU and made available for in-vivo FMT-XCT imaging. The rationale for working with several cancer models is based on (i) the necessity to assess FMT-XCT in different tumor localization sites, and sizes and (ii) the fact that different patterns of gene expression in cancers dictate different strategies for diagnosis and treatment strategies. Emphasis is placed on mammary gland tumors, due to the relevance in clinical propagation as follows a) mammary tumor xenograftsThe following cell lines will be implanted subcutaneously in mice: - MDAMB-231 human breast adenocarcinoma cells over-expressing MT4-MMP, a metalloproteinase which does not

affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated lung metastases in RAG-1 immunodeficient mice 8 weeks post-injection (CEA-LIME)

- primary cell cultures from human tumors that either express or do not express erb-B2, the target of trastuzumab (Herceptin®), a monoclonal antibody used in breast cancer chemotherapy; these cells are obtained by CEA-LIME through the Paris canceropole research collaboration.

- b) mammary tumor transgenic mice models - CEA-LIME breeds a transgenic strain of mice expressing the polyoma middle T oncoprotein (PyMT), under the control

of the mouse mammary tumor virus long terminal repeat (MMTV LTR). These mice develop breast cancer with four distinct stages of tumor progression, from premalignant to malignant stages. Tumoral development is often, at late

FMT-XCT 201792

28

stages, associate with lung metastases. This model presents many similarities with breast cancer progression encountered in womenthat can be quantitatively documented by PET imaging.

Task 6.2 Animal models of lung and brain cancer The consortium will investigate other tumors, developing at different sites, to assess FMT-XCT performance with imaging at diverse locations and underlying optical heterogeneity as a general accurate pre-clinical imaging tool that can be generally used for basic discovery and drug discovery applications. For this we will further develop a) lung tumor models - lung metastases from MDAMB-231 human breast adenocarcinoma cells over-expressing MT4-MMP (CEA-LIME) - lung metastases from PyMT transgenic mice (CEA-LIME) d) brain tumor models The mouse head represents a unique problem due to the smaller dimensions and more complex bone (cranial) structures and air-cavities involved. Guided by X-ray CT, we would be able to accurately study the relative performance of stand-alone vs. a-priori FMT. In this case CEA-LIME will image brain tumor progression in nude mouse bearing orthotopic intracerebral implants of U87 human glioma cells through motility activity and training activity at the HMGU site.

Task 6.3 Fluorescent probes This proposal does not aim in developing new fluorescence probes. However, successful completion, demonstration, training and dissemination of the proposed technology rely on the availability of fluorescent reporters to the consortium. To accomplish this we have taken certain steps to not only rely on commercially available fluorescent probes (see table I) but enable the availability of highly performing fluorescent probes due to own developments:

Increased accuracy through multi-wavelength labelling strategy based on commercially available probes: Previously reported cathepsin-sensitive or MMP-sensitive activatable fluorescent probes [26] are commercially available (see Table I, Section1) and will be utilized to image protease expression levels of cathepsin in tumors. To independently assess the bio-distribution of the activatable probes we will also administer here a probe, that consists of the same poly-lysine backbone used for the activatable probes (CaB–Cy-5.5, MMP 2/9–AF750), but conjugated to only 1 or 2 Cy7 fluorochromes per backbone so that no quenching occurs (Angio-810; see also Table I). The Angio-probe retains the bio-distribution characteristics of the activatable probe but continuously reports on permeability and probe delivery. Using this triple-wavelength strategy it is then possible to independently monitor Cathepsins, MMP’s and probe bio-distribution to reference Cathepsin and MMP reconstructed patterns to the actual amount of probe delivered to obtain accurate protease expression levels. This is because, for example, low signal in a single wavelength does not clearly indicate if the target concentration is low or if there was limited probe delivery. Multi-labelling can independently asses target concentration and probe delivery and therefore build a more precise picture of the underlying molecular and functional activity. This approach will be used in many in-vivo imaging protocols by the partners.

Non-commercially available probes To further enhance the impact of the present proposal, the partners will have

access to new classes of cancer targeting probes that are endowed with enhanced affinity, specificity, and favorable pharmacokinetic properties. This is achieved through CEA-LIME developments on using macromolecules for cancer, i.e. proteins / antibodies and oligonucleotides / aptamers, and establishing diverse labelling methodologies, developed largely inside the EU network EMIL and the EU Integrated project Cancer Degradome.

a) Trastuzumab, ( Herceptin®) is a monoclonal antibody that CEA-LIME will label with AlexaFluor® 680 for use with the human erb-B2 in various animal models. It will be used here to test the capacity of FMT-XCT to quantitatively image expression patterns of human erb-B2 in human mammary cancer cells as per models in Task 6.1.

b) Aptamers: Aptamers are favourable targeting molecules as they do not appear to trigger an immune response, (in contrast to antibodies), they attain smaller size (8–15 kDa) to promote better tissue penetration and they can be cost-effectively synthesized with excellent reproducibility and easier incorporation of chemical modifications, conferring plasmatic resistance to their degradation or improved pharmacokinetic. CEA-LIME has developed a new method to select aptamers against whole living cells, a new avenue with three major advantages 1) direct selection without prior purification of the targets; 2) conservation of membrane proteins in their native conformation similar to the in vivo conditions and 3) identification of (new) targets for a specific phenotype. This is a new way for drug design against cell surface molecules that represent 70% of all known drug targets. Using our differential whole living cells SELEX technology we were able to identify several aptamers. In the present project, we will focus on

- aptamers against metastatic forms of mammary cancer. Differential whole cell-SELEX can select aptamers specific for highly lung metastatic cells (versus low lung metastatic cells)

FMT-XCT 201792

29

- Metalloproteases overexpressed in breast metastasis (MT4-MMP). One of these aptamers is particularly attractive for imaging because it is internalized by the target cells and therefore likely to accumulate over time in metastatic tissue (Elina ZUEVA, Frédéric DUCONGE & Bertrand TAVITIAN, unpublished data).

We plan to optimize the bio-distribution of these aptamers in vivo and resolved them using XCT-FMT. Possible bio-distribution effects when labelling with fluorochromes will be examined in Wp8.

Task 6.4 In-vivo imaging and correlation. In vivo imaging of animal models will be performed originally with the CEA-LETI for XCT validation and in FORTH for stand-alone FMT validation using commercially available probes. Upon development of the FMT-XCT prototype in-vivo imaging activity will move to HMGU through motility and training activities. All partners are expected to participate in highly synergistic imaging sessions with physical presence or with communication and analysis/reconstructions of measurements that combine all expertise to obtain a highly performing imaging method. Stand-alone FMT, XCT and FMT-XCT will be all analyzed and compared to each-other. Animal imaging protocols will be according to institutional guidelines for animal safety and will follow the plan of the Table below. Generally two targeting strategies will be used, in all animal models, to study performance with varying types of cancer. Multi-wavelength imaging allows assessing multiple probes (for example aptamer and RGD-peptide based probes) or cathepsin, MMP’s and probe delivery/permeability probes. In this way multiple-probes can be evaluated on the same animal, overall reducing animal numbers utilized. The animal numbers will be adjusted to obtain statistically meaningful observations, given the standard deviation observed in the sample, however we do not expect to use more than 15 animals per study group and probe. Analytical description and justification of animal numbers is given in section 4. Data will be analyzed by FORTH ( MDA-231, PyMT data; direct inversion), UCL (MDA-231, PYMT data; a-priori inversion) and HMGU (all else) and the other partners through training activity.

Animals will receive a series of correlative studies for validation by CEA-LIME. After imaging, animals will be sacrificed and lesions and organs of interest will be removed. Each animal group will be divided in two sub-groups each containing half of the animals examined. For one of the subgroups, the excised tissues and lesions will undergo reflectance fluorescence imaging immediately after excision, for validating the fluorescence signals in a local macroscopic level. For the other subgroup organs will be prepared for histological examinations, including fluorescence microscopy to directly assess fluorescence activity and appropriate immuno-histochemical protocols for correlating underlying expression levels, physiology and structure.

TABLE Wp6: Cell lines , probes and interrogations considered in Wp6 Cell Line Site Probes – em. wavelength Interrogation & Purpose Partner MDAMB-231 Mammary/ lung AptamerHM1–690, RGD-770 or

CaB-690, MMP-780, Angio-810 Detection limit Quantification of protease levels Accuracy in quantifying growth.

2,3,4

Human erb-B2 + /- Mammary Trastuzumab – AF680 (680) or CaB-690, MMP-770, Angio-810

Accuracy in quantifying receptor levels Accuracy in quantifying growth

1,2,5

PyMT Mammary/ lung VEGF-690,RGD-peptide-770 or CaB-690, MMP-770, Angio-810

Test angiogenic switch. Detection limit of early development and metastasis

2,6,3,4

U87 Brain AptamerU87–690, RGD-770 or CaB-690, MMP-770, Angio-810

Image quality in imaging in the brain. Detection limit, image metastasis

All

[ ‘or’ indicates that half the animal will be imaged with the top line combination and the other half with the bottom line combination]

Task 6.5 To predict clinical utility. Predicting clinical utility will be accomplished by partner UCL through utilising in-vivo performance metrics obtained from animal models (probe concentration, contrast, signal-to-noise ratio achieved), as provided by HMGU, to predict using the TOAST software the sensitivity that can be achieved clinically for different optical probes and cancers. These results will be reported in the final report and used to draw prediction on the clinical utility of the FMT-XCT system.

Deliverables (brief description and month of delivery) 6.1 To develop and characterize molecular probes (mo.9, 18) 6.2 Prepare and characterize mammary cancer animal models (mo.18) 6.3 Breed and making available PyMT animal models (mo.24) 6.4 Develop U87 animal models (mo.27) 6.5 Study and report the quantitative accuracy of FMT-alone and FMT-XCT in resolving tumors (mo. 36) 6.6 Study and report cancer detection performance in various organs (mo. 40) 6.7 Report the overall imaging performance. (mo.42)

FMT-XCT 201792

30

Figure WP7-1: (a) Fluorescence epi-illumination (planar) imaging of a

chemo-sensitive and a chemo-resistant LLC tumor, treated with

cyclophosphamide and imaged with FMT of Cy5.5-labeled annexin V

fluorescent probe. (b) Corresponding FMT slices reconstructed (color) and

superimposed on a photograph of the animal (grayscale). The slice at

z=1.3cm is the most superficial; at z=1.06cm the deeper from the surface

shown in (a). A threshold was applied to 40% of the maximum to allow

for simultaneous visualization of the tomographic results and the animal

photograph and correlate reconstructed results with animal position. (c,d)

Histological images of the two tumors (TUNEL method: ApopTag kit;

Intergen, Purchase NY) confirmed the macroscopic findings. Overall FMT

demonstrated superior quantification accuracy compared to planar imaging

as shown in Proc. Natl. Acad. Sci. 101: 12294 (2004).

Work package 7

Work package number 7 Start date or starting event: 1

Work package title Imaging cancer therapy for enabling internention.

Activity Type21 RTD

Participant number 6 2 3 1 4 5 7

Person-months per participant:

36 9 8 10 2 1 2

Objectives: Task 7.1 To determine the quantification accuracy of the XCT-FMT system in imaging breast cancer response to

standard chemotherapeutic protocols as compared to histological gold-standards. Task 7.2 To measure the quantification accuracy of the XCT-FMT method to assess standard chemotherapy effects

vs. combinations of chemotherapy with targeted therapy. Task 7.3 To phenotypicaly characterize an animal model developed for imaging HIF-related pathways in-vivo using

conventional FMT and compare imaging findings with XCT-FMT. Task 7.4 To train scientists in imaging treatment effects in-vivo, with a view towards clinical translation.

Description of work (possibly broken down into tasks), and role of participants Significant research into the quantification capacity and overall in-vivo performance of FMT-XCT is delivered

as part of Wp6. However the XCT-FMT can further be used to enable new types of therapeutic intervention, by offering a highly capable platform of studying complex interactions related to treatment protocols and new drugs but also as it can lead to truly personalized clinical medicine, especially in breast cancer healthcare. This is because the technique could enable real-time monitoring of treatment response in a patient. While the exact practice and logistics of clinical application needs to be determined (for example one hybrid FMT-XCT session followed by multiple FMT sessions during treatment follow up), the clinical feasibility of the approach has been already demonstrated by Partner 4 together with the Yodh group (see Ref.33). In this role hybrid FMT could be used for monitoring personalized treatment and as a non-invasive tool for deciding the continuation or change of a therapeutic regime for each patient.

FMT-XCT is ideally suited to study multiplexed information (structural-functional-molecular) to allow the correlation of molecular imaging data with already established structural/functional imaging readouts with superior imaging performance of any of the methods alone. The particular hybrid system proposal offers a highly quantitative approach which is well suited for accurate observations of biological pathways and treatment response. Fig.WP7.1 shows an imaging example from assessing drug efficacy in animals implanted with chemotherapy-sensitive and chemotherapy-resistant Lewis Lung carcinoma (LLC) tumors in the left and right mammary pads respectively using stand-alone FMT. Tumors were grown for seven days and were subsequently treated with two injections of cyclophosphamide (200mg/kg i.p.) 24 hours apart. In-vivo targeting of apoptosis was achieved by intravenously administering a Cy5.5 dye labeled annexin V probe two hours prior to imaging. This probe had been confirmed to selectively target apoptotic

FMT-XCT 201792

31

cells [43, 44]. This proposal considers more elaborate approaches where an inactive (non-binding) probe labeled with a fluorochrome at a different wavelength is co-injected to independently assess probe delivery (permeability) and more accurately resolve actual target present, normalized to the amount of prove delivered. Overall, it was further observed in this study that FMT yielded better correlation to histological findings than fluorescence reflectance (epi-illumination) imaging, because it can account for changes in the optical properties of tumors and reports accurately on fluorescence concentration (within 20% of accuracy) even when the tumor absorption varies by 3x compared to the background measurements [2]. These findings have been independently confirmed by implanting plastic tubes of known fluorochrome concentrations in animals and measuring the effects of increased absorption in the plastic tube on FMT reconstructed intensity [45]. Herein, the use of FMT-XCT is expected to further improve the imaging performance in superficial and deep-seated tumors as well. Task 7.1: Imaging chemotherapy This task will interrogate the accuracy of the XCT-FMT system in monitoring and quantifying treatment response in a metastatic pre-clinical models of breast cancer MDA-MB-231 (adenocarcinoma) and one spontaneous breast mammary model PyMT spontaneous (as described in Wp6; developed and provided by CEA). FMT-XCT performance will be studied against standard measures of treatment progression (tumor size) and associated tumor biomarkers, for example cell survival as confirmed by histology and immuno-histochemistry. Correspondingly, successful fluorescent biomarkers employed herein can lead the way for quantitative, individualized assessment of treatment efficacy and dose response in animal models of breast cancer, with potential clinical propagation. Commercially available fluorescent probes targeting VEGF (Cy5.5; em: 690nm) and matrix-metallo-proteinases (MMP-AF750; em 770nm), as described in Wp6 and an apoptosis marker (annexin-Cy7; 800-830nm), used in Fig.Wp7.1 will be utilized as in-vivo biomarkers for evaluating vascularization, cell survival and cell death respectively.

Mice (n=12 / tumor type & probe combination; see also Section 4) will undergo doxorubicin+cyclophosphamide followed by docetaxel, at clinically relevant intervals and dose, adjusted to the mouse weight. 3 animals per cancer group will not receive treatment and serve as controls. Animals will be imaged each week by HMGU with FMT-XCT for the course of the treatment, but no more than 5 weeks. Each FMT session will collect two data sets, one at the emission wavelength and one at the excitation using appropriate filters. The animals will be kept anaesthetized with isoflurane gas anesthesia provided into the mouse bed. Animals will undergo imaging sessions every five days (maximum of three imaging sessions per animal) to monitor tumor growth and will be then euthanized (15 days after implantation). Selected animals will be euthanized at earlier time points than 5 weeks so that histological correlation data (see next paragraph) are also collected for earlier points of tumor growth.

Similarly to Wp6, animals will be sacrificed and lesions and organs of interest will be removed. Each animal group will be divided in two sub-groups each containing half of the animals examined. For one of the subgroups, the excised tissues and lesions will undergo reflectance fluorescence imaging immediately after excision, for validating the fluorescence signals in a local macroscopic level. For the other subgroup organs will be immersed in liquid nitrogen for histological examinations, including fluorescence microscopy to directly assess fluorescence activity and appropriate immuno-histochemical protocols for correlating underlying expression levels, physiology and structure. FMT-XCT results will be validated in terms of imaging accuracy against the location and size retrieved from X-ray CT by UZH, FORTH, UCL and in terms of molecular contrast against the laboratory tests in HMGU. In particular, we will plot correlation graphs investigating the agreement of location and size (volume) seen between FMT and X-ray CT during tumor treatment. Similarly, we will examine the correlation of the FMT values reconstructed for the tumors imaged against the fluorescence measurements on excised tumors or the corresponding fluorescence microscopy and immuno-histochemistry assessment of the targeted protease. In addition blood-vessel count (CD 31 staining) and fluorescence microscopy will be plotted against reconstructed probe bio-distribution. Results will be finally summarized with standard statistical measures (mean, standard deviation and statistical significance of results). Visualization will be in terms of observing FMT, CT and fused slices or via three-dimensional renderings as seen in Fig.2. Task 7.2: Quantitative Imaging of treatment effect. To characterize the quantification capacity of the system proposed, as it pertains to changes of disease response to treatment, the imaging protocol and histological validation of Task 7.1 will be repeated with adding differential imaging of treatment of standard chemotherapy (as in Task 7.1) together with targeted therapy (Trastuzumab or Bevacizumab) on the Human erb-B2 animal model described in Wp6, provided by CEA. The same correlation and data analysis and reporting will be followed as in Task 7.1.

FMT-XCT 201792

32

Task 7.3: characterizing HIF-related pathways. Focus of this task is given in the characterization of a transgenic mouse model that initiates HIF expression as a response to hypoxia, developed by UZH (Mueggler, Rudin). Hypoxia arises in a variety of physiological and pathological conditions and it affects treatment response in various therapeutic protocols. A key player in the process is the hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and which drives the expression of a large number of downstream genes. The mouse model in development is based on a reporter construct that expresses a fusion protein of HIF with a fluorescent reporter protein (mCherry) under the control of the endogenous HIF promoter, with expression pattern and functionality analogous to HIF (Lehmann et al. in prep). Similarly readouts for the downstream events (e.g. angiogenesis) are under development. This task proposes the phenotypic characterization of the animal model with standard histological/immunohistochemistry methods and with FMT and FMT-XCT. This animal model will be used in combination with imaging assays for signals downstream of HIF such as molecular targets involved in angiogenesis, anaerobic glycolysis or vasodilation, developed by UZH to offer a platform for quantitatively researching XCT-FMT performance. These molecular signals will be complemented by classical physiological imaging readouts (with AngioSense, MMP-Sense; Table I) but also by directly resolving oxy- and deoxy- haemoglobin using absorption optical tomography at multiple wavelengths. This process is very similar to reconstructing fluorescence and will be investigated herein, as an additional biomarker of physiological response. Studies will be then carried out in breast cancer disease models: i) hypoxia and induction of angiogenesis in subcutaneous tumor xenografts. Mice will be shipped to FORTH and initial characterization will be performed with the stand-alone FMT system already developed, also operating in resolving oxy- and deoxy-hemoglobin based on reconstructed absorption maps in different wavelengths. The acquired knowledge will then be translated to the FMT-XCT system that will allow investigating some essential aspects of hypoxia induced signaling and effects of therapeutic interventions on HIF signaling and general outcome such as tumor regression/stabilization/progression (RECIST criteria) in relation to generic treatment in Task 7.1. Hence, the combination of structural readouts (CT) with functional (optical) and molecular readouts (optical) readouts will be essential. UZH will further follow this animal model with MRI in order to establish imaging trends between FMT-XCT and MRI.

TABLE Wp7: Cell lines, probes and treatment considered in Wp6 Cell Line Site Probes – emission wavelength Treatment Partner MDAMB-231 &

Mammary/ lung VEGF-690, MMP-780, Annexin-810 or AptamerHM1-690, RGD-770, Annexin-810

Doxorubicin & cyclophosphamide followed by docetaxel

2,6,3,1

HIF-transgenic MDAMB-231

Mammary/ lung Annexin-810, MMP-780 and Oxy/Deoxy Hb intrinsic signatures and mCherry

Doxorubicin & cyclophosphamide followed by docetaxel

6,3,1

Human erb-B2 + /- Mammary Trastuzumab–680,MMP-780,Annexin-810

Doxorubicin & cyclophosphamide with or without Trastuzumab followed by docetaxel

1,2,5

PyMT Mammary/ lung VEGF-690, MMP-780, Annexin-810 Doxorubicin & cyclophosphamide followed by docetaxel

All

[ ‘or’ indicates that half the animal will be imaged with the top line combination and the other half with the bottom line combination ]

Training : Training of imaging treatment responses will be performed by Partner UZH on site in the Munich facilities of Parther 1. Active involvement in data collection or data analysis will be expected for the PyMT mouse model study which will be further used to train all partners on imaging treatment response; in particular the use of probes and the FMT-XCT performance. All partners will be either present on site through motility or participate remotely with data analysis and evaluation.

Deliverables (brief description and month of delivery) 7.1 Preparation of HIF transfected breast cancer cells: month 9 7.2 Assessment and in-vivo FMT imaging of HIF induction in subcutaneously implanted breast tumors stably transfected with

HIF reporter gene: month 15. 7.3 Characterization of the quantification accuracy of FMT-XCT vs. histological correlates in assessing treatment of the MDA-

MB-231 at the primary site of development in the mammary and in metastatic sites in the lung. month 32 7.4 Characterization of the quantification accuracy of FMT-XCT vs. histological correlates in assessing treatment of the PyMt

spontaneous mouse model at the primary site of development in the mammary and in metastatic sites in the lung. Mo. 36 7.5 Characterization of the quantification accuracy of FMT-XCT to resolve differential treatment levels in the Human erb-B2

animal model in the presence or absence Trastumazab as it relates to histological validation. month 40 7.6 Report on assessment of antiangiogenic therapy effects on HIF induction and downstream readouts of HIF (induction of pro-

angiogenic factors) with FMT-XCT: month 46.

FMT-XCT 201792

33

Work package 8 Work package number 8 Start date or starting event: 1

Work package title FMT-XCT imaging accuracy vs. PET-XCT

Activity Type21 RTD

Participant number 5 2 3 4 1 7 6

Person-months per participant:

36 12.6 6 6 6 2 2

Objectives: 1. To develop hybrid FMT-PET-XCT phantoms. 2. To develop aptamers labeled with fluorescence and 18F for PET to image cancer of the same animal model

and offer exact co-registration for validation purposes. 3. To validate the FMT-XCT image accuracy using co-registered PET-XCT imaging and the dual-labeled

aptamer probe from Task 8.2.

Description of work To precisely validate the ideas and prove the hypotheses driving this proposal, i.e. that the proposed FMT-XCT system can result in imaging performance of the same or better utility to nuclear imaging methods, it is imperative that we accurately validate the developed FMT-XCT method with established nuclear imaging gold-standards. The partners herein have collectively designed validation protocols that will accurately determine system performance. Besides offering a necessary scientific step, this work-package is further important in ensuring the accurate demonstration of the technique for commercial translation and for training purposes.

We note and remind here that while performance metrics between FMT-XCT and PET-XCT or SPECT-XCT will be recorded and reported (for example sensitivity, quantification accuracy etc), the essence of this work-package is not the direct comparison of FMT-XCT with PET-XCT, but rather the validation of the utility of FMT-XCT, i.e. the overall usefulness of FMT-XCT in biomedical research and potential clinical propagation in key applications, using PET-XCT as the established method for assessing FMT-XCT imaging accuracy. This is because an FMT – PET comparison makes sense only in terms of a specific application. For example PET could prove more sensitive than FMT, especially when larger animals are considered, in terms of absolute amount of labeled molecules detected; however fluorescence imaging can compensate with high administered dose and quenching probe mechanisms to maximize contrast and achieve similar ability to image and characterize disease. Similarly, FMT accuracy may significantly benefit from its straightforward ability to image multiple events simultaneously using spectral differentiation, despite the exact quantification metrics found for a single label when using FMT, SPECT or PET. For example, multi-spectral FMT imaging of an active and a scrabbled (non-binding) probe can independently image “permeability” and a “specific target” so that it can reveal the actual amount of the target present vs. the amount of probe delivered, independently of the absolute quanitifcation achieved (even though FMT-XCT absolute quantification is also expected to be very accurate). This was described and studied in Wp6, Task 6.3. Task 8.1 Hybrid phantoms In order to yield accurate system validation and minimize the overall number of animals utilized in this proposal, hybrid phantoms will be developed. Spatially varying optical properties are induced by casting resin mixed with TiO2 particles and India Ink to attain some optical property around pre-cured resin blocks of known shape location and different concentration of TiO2 and India Ink to induce different optical properties by FORTH. At the same time, the phantom will contain easily accessible and washable openings that are easy to fill with contrast agents. Identical phantoms will be imaged by HMGU with the FMT-XCT system and FIHGM by the PET-XCT system and metrics of imaging accuracy, resolution and sensitivity achieved as a function of fluorescence probe or PET tracer concentration will be measured. Task 8.2 Dual-labeled probes. In order to perform an accurate validation between two imaging modalities it is important to develop hybrid imaging probes so that both modalities image exactly the same bio-distribution. Such probes are not commercially available. For this reason, CEA-LIME will employ the aptamer-HM1 probe from Wp6, and chemically modify it by dual optical and PET labelling. Dual labelling of the aptamers will be to label with 18F at the 3’ extremity and with fluorescence (Cy5.5) at the 5’extremity. The methods for each individual labelling are

FMT-XCT 201792

34

already established at CEA-LIME; their combination and the resulting yield will be tested in-vitro with cell cultures MDAMB-231 cells and in-vivo in mouse xenographs of the cell-line using a standard micro-PET system available at CEA-LIME. The adbantage of using this cell line is that both superficial and deep-seated activity can be recorded due to the MDAMB-231 lung metastasis. Therefore imaging will be performed at different stages of tumor growth to ensure that lung signals will also be present. The probes will be tested in terms of their reference pharmacokinetics of the stand-alone PET probe, and bio-distribution and the bio-distribution observed for the dual PET and fluorescence probe, on the same animals, administered with a few hours difference (see Section 4 for animal description). While differences in bio-distribution between a PET-labelled aptamer and a PET-optical labelled aptamer does not affect the purpose of this Wp8, which is solely in characterizing the FMT-XCT and PET-XCT imaging accuracy, any such differences will be noted and reported. Task 8.3 FMT-XCT imaging performance validation with PET-XCT imaging. In order to perform accurate correlation of FMT-XCT and PET-XCT data, following original PET_XCT of the dual labelled probes in Task 8.2 by CEA, MDAMB-231 animal xenographs (nude mice) will be imaged with FMT-XCT and PET-XCT. Imaging will be performed in (n=10) animals after injection of the probe developed at Task 8.2. PET-XCT will be achieved in the Technical University of Munich, Nuclear Medicine department on a Siemens Inveon PET-XCT system already available since summer 2007. The cost of these experiments and corresponding analysis will be covered under a subcontract to the Technical University of Munich as provided in the HMGU budget. The rationale for performing those imaging sessions in Munich (even though FIHGM also has a PET-XCT systems) is because both FMT-XCT and commercial Siemens PET-XCT system is in close proximity. Co-registration will be facilitated by utilizing a mouse constraint/positioning system that places the animal between two transparent plates, which apply mild compression to the animal and ensure that no movement occurs when the animal is translated between the PET-XCT system and the FMT-XCT system while under anaesthesia. Co-registration will be then enabled by aligning the surface obtained with XCT with the optical surface reconstructed optical method. The alignment is performed by aligning the two three-dimensional surfaces and will be facilitated by software alignment tools developed at FIHGH and TUM for multi-modality co-registration. Risk assessment This step will compare the imaging performance achieved by FMT-XCT and PET-XCT. We anticipate that depending on the signal-to-noise achieved in FMT and in PET, that the dose of the probe administered will be adjusted to ensure good detection sensitivity by two methods. If this is not possible due to ionizing radiation dose restrictions, then we foresee increasing only the fluorescence dose by co-injecting the same aptamer labelled only for fluorescence so that imaging is achieved under similar signal-to-noise measurements. This is practically also nominal, since in practice the fluorescence injected dose can be more freely adjusted compared to the isotope dose. The bio-distribution of the fluorescence only labelled aptamer is not expected to be significantly different than that of the fluorescence and PET tracer probe. Partner interaction and Training. This work package plays a pivotal role as it will allows further cross-disciplinary training of multiple scientists in multi-modality imaging and allow significant understanding between the performance and utility of different techniques. Partners will have access to these key experiments and HMGU, CEA, FORTH, UCL, FIHGM actively participate either on site or remotely through data analysis. Milestones: A significant milestone herein, that is similar to the ones in Wp6 and Wp7, although not explicitly written there is the selection of optimal inversion approaches for FMT and XCT utilization in order to reach the maximum imaging accuracy and imaging utility, as it compares to PET and to the underlying histological analysis.

Deliverables 8.1 Imaging phantoms that will be used for assessing FMT- XCT performance and comparisons to PET-XCT

(mo.12). 8.2 Phantom characterization with PET, FMT and XCT (mo 24) 8.3 Coregistered FMT-XCT and PET-XCT images from mouse imaging and quantitative analysis of relative

performance (mo. 42). 8.4 A report on metrics of XCT-PET and XCT-FMT relative performance (mo. 48) .

FMT-XCT 201792

35

Work Package 9

Work package number 9 Start date or starting event: 1

Work package title Training and dissemination

Activity Type21 OTHER

Participant number 1 2 3 4 5 6 7

Person-months per participant:

6 3 2 0.5 2 2 0.5

Objectives: 9.1. Training of scientists on FMT-XCT technology and underlying technologies 9.2. Dissemination of the results and progress within partners and to scientific, industrial and public sectors. 9.3. Technology transfer activity

Description of work (possibly broken down into tasks), and role of participants

Training: The proposal depends on exchange between participant members for two main reasons: 1) the high inter-disciplinary scientific knowledge that constitutes the different WP and 2) due to the locality constraints of prototype development and animal model developments. Motility of participants will enable the training of the consortium on XCT-FMT, animal models and targeting strategies for breast cancer and treatment imaging. CEA-LETI will house a training session on XCT on mo.12, Crete jointly with UCL on free-space FMT on mo. 24, CEA-Paris jointly by ETH on animal models/cancer imaging on mo. 32, and Munich together with Madrid on FMT-XCT technology and in-vivo animal imaging in mo.42. Formal teaching by WP leaders and hands-on imaging sessions will be organised to educate consortium participants and potentially other interested parties on in-vivo hybrid FMT-XCT imaging and the utility of this tool in biological research and drug discovery. Active participation in experiments and data analysis will go beyond the delivery of results but will actively incorporate partner participation. For example specific imaging of animal models in Wp6 and Wp7 have been described that will invite all participants to participate in the experiments and the corresponding correlative analysis and data analysis reconstruction.

The development of user friendly software further facilitates the ability to train multi-disciplinary scientists in imaging aspects. By the same token participation in XCT, fluorochrome and animal model developments will increase the information exhange and training of scientists in cross-disciplinary scientific areas.

Workshops & Dissemination. Organization of one workshop at month 42 at Munich, organised together

with the training workshop at month 42 described above. The workshop will contain one day of presentations with title “Hybrid XCT-FMT imaging: from mouse to human” with invited speakers from the consortium, the advisory committee and the scientific community at large and will be open to scientists and industry and a second day of hand-on imaging session for selected interested scientific and industrial representatives, in order to raise awareness of the technology and facilitate commercialization. The workshop will be announced and its outcome followed with press releases and possibly solicited news coverage. The Workshop is needed to 1) present the work to interested parties outside the consortium, and to 2) present the system prototype and perform imaging sessions to interested parties from the industrial sector. Selection of participants and industries solicited will be decided in consortium meeting at month 36.

Communication to the public. Through press releases, a web site and a public promotion leaflet public awareness will be raised for the activity and successes of the consortium.

Technology transfer and intellectual property issues will be explicitely handled by the executive and

advisory committees and the consortium agreement, with the help of IP experts as more analytically described under management. This is an important aspect of this proposal, developing a technology with high commercialization potential and awareness will be further increased through dissemination of achievement and results outside the consortium, in scientific meetings, industry roll-outs etc. and through the workshop in month 42.

FMT-XCT 201792

36

Tasks:

Task 1.1. Workshop with invited potential users and industry at month 42 (Munich). Task 1.2. Meeting entitled: “FMT-XCT from mouse to man” co-localized with the workshop at month 42 Task 1.3. Training workshops on months 12 (Grenoble), 24 (Herakleon), 32 (Paris) and 42 (Munich). Task 1.4. Dissemination activities and press releases after major publications Task 1.5 Solicitations before the workshop on month 42 of appropriate attendees and press releases of workshop

and meeting outcome. Task 1.6 Activities for use of results within 2 years after the end of the project.

Deliverables 9.1 Dissemination implementation document (mo. 36) 9.2 Training curriculum and implementation document (mo. 24) 9.3 Public promotion leaflet (mo 36) 9.4 Meeting program and book of abstracts for the meeting at month 42. (mo. 42) 9.5 Summary of achievement at Workshop at month 42 (mo 42) 9.6 Technology transfer document and report on patent applications (mo 48) 9.7 Report on training accomplished and PhD Thesis achieved (mo 48)

FMT-XCT 201792

37

B.1.3.6 Efforts for the full duration of the project

Participant no./short

name

WP1 WP2 WP3 WP4 WP5 WP6 WP7 WP8 WP9 Total person months

HMGU 42 1 12 12 96 12 10 6 6 197

CEA 2 51.7 0 6 9.7 51 9 12.6 3 145

FORTH 2 0 36 6 6 6 8 6 2 72

UCL 1 0.5 6 36 3 6 2 6 0.5 60.5

FIHGM 1 18 0 0 6 1 1 36 2 65

UZH 1 1 0 0 2 4 36 2 2 48

VAMP 0.5 6 0 0 9 0 2 2 0.5 20.5

Total 50 77.7 54 60 132 80 68 70.6 16 608

FMT-XCT Project Effort Form 2 - indicative efforts per activity type per beneficiary6

Activity Type HMGU

CEA FORTH UCL FIHGM UZH VAMP TOTAL ACTIVITIES

RTD/Innovation activities

WP 2 – XCT development 1 51.7 - - 18 1 6 77.7

WP 3 – FMT theory 12 - 36 6 - - - 54

WP 4 – Inversion with priors 12 6 6 36 - - - 60

WP 5 – FMT XCT integration 96 9.7 6 3 6 2 9 132

WP 6 – Cancer imaging 12 51 6 6 1 4 - 80

WP 7 – Therapy imaging 6 9 8 8 1 36 - 68

WP 8 – FMT vs. PET 6 12.6 6 6 36 2 2 70.6

Total 'research' 149 140 68 59 62 45 19 542

Demonstration activities

WP name

WP name

Etc

Total 'demonstration'

Consortium management activities

WP 1 - Management 42 2 2 1 1 1 1 50

Total ' management' 50

Other activities

WP 9 – Training & dissemination 6 3 2 0.5 2 2 0.5 16

Total 'other' 16

TOTAL BENEFICIARIES 197 145 72 60.5 65 48 20.5 608

6 Please indicate in the table the number of person months over the whole duration for the planned work , for

each work package, for each activity type by each beneficiary

FMT-XCT 201792

38

B.1.3.7 List of milestones and planning of reviews Milestone number

Milestone name WP’s no. Lead beneficiary

Delivery date from Annex I

Comments

1 Consortium Agreement Wp1 P1 0 Signed Document

2 XCT design Wp2 P2 1 Executive committee agreement

3 Optimal Free-space FMT system Wp5 P1 12 Functional Prototype

4 Direct vs. Conventional FMT performance

Wp3 P3 15 Quantitative image analysis & validation on simulated and experimental data

5 XCT dual energy vs. contrast enhancement

Wp2 P5 18 Quantitative image analysis on experimental data

6 Optimal FMT inversion with priors

Wp4 P4 21 Quantitative image analysis & validation on simulated and experimental data

7 Automatic Feature Extraction Wp4 P1 24 Quantitative image analysis on experimental data

8 FMT-XCT Shielding Wp5 P7 24 Experimental measurements

9 FMT-XCT system Wp5 P1 27 Functional prototype

10 FMT-XCT methodology for in-vivo quantification

Wp6 P2 32 Histological verification

11 FMT-XCT methodology for quantified treatment imaging

Wp7 P6 36 Histological verification

12 FMT-XCT methodology for optimal in-vivo imaging

Wp8 P2 42 PET-XCT verification

Tentative schedule of project reviews

Review

no.

Tentative timing, i.e. after

month X = end of a reporting period 7

planned venue

of review

Comments , if any

1 After project month: 1 Kick-off meeting

2 After project month: 12 EC meeting,

mo.12

3 After project month: 24 EC meeting,

mo.24

4 After project month: 36 EC meeting,

mo.36

7 Month after which the review will take place. Month 1 marking the start date of the project, and all dates

being relative to this start date.

FMT-XCT 201792

39

B2. Implementation B 2.1 Management structure and procedures

The key components contributing to the management of the project are: Executive committee

The executive committee consists of the Work Package Leaders and the VAMP General Manager (Holger Bruenner), each also selected by being a thought world leader in his particular field. The Executive committee is responsible for managing all the major items associated with this proposal, including to define, divide and develop the tasks, monitor the progress, manage research teams, take decisions on scientific and technological issues, prepare reports, ensure scientific, work and animal ethics, handle intellectual property and commercialization issues, ensure gender balance etc. It is presided by the coordinator and is assisted by the management group of WP1.

Advisory committee The advisory committee consists of senior external members that are well respected in scientific fields

associated with this proposal. The advisory committee will be composed immediately after the kick-off meeting of this proposal following recommendations by the executive committee. The role of the advisory committee is to advise the executive committee on strategy issues and to enable further visibility of the development to the scientific community and industrial parties. Scientists approached and in the process of being invited include Prof. Markus Schwaiger of TUM University in Germany, to impart clinical outlook, Prof. Clemens Lowik of Leiden University in the Netherlands, to advice on Molecular and small animal imaging and Prof. Critt Moonen to impart application outlook. Honorariums are provisioned in the management package for advisory members, if they are institutionally allowed in their respected institutes.

Advisory committee members will be invited to all consortium and executive committee meetings and dissemination activities and leave to their discretion to attend. To facilitate a true interaction however, the Executive committee will interact with Advisory Committee members on the basis of personal correspondence and private meetings at the site of the Advisory member or in international meetings. Coordinator

The work of the Executive Committee is frequently translated into daily management and representation duties by the co-ordinator.

Executive Committee member Each member will be responsible for the scientific co-ordination of their projects or WP and

administering the budget necessary for performing the tasks relevant to the respective work-package (or project). The Consortium Agreement (CA) will be further defining the specific role of an executive committee member as a work package leader by defining:

- the WP leaders’ exact delegated responsibilities; - their participation and duties in the management (meetings, decisions, reports); - the scope of their power.

This is a focused project and the structure above guarantees a direct and efficient executive strategy. The particulars of the structure are further explained bellow.

FMT-XCT 201792

40

Management roadmap: The management roadmap follows according to the timing (Gantt) chart on page 14. Meetings: The executive committee will privately communicate by group email, telephone and ad-hoc web-based conferences. In addition a quarterly web-based conference for all WP leaders will be setup to review progress and discuss issues and objectives. Also, yearly consortium meetings will be organized as described in Wp 1. The committee will further evaluate progress and propose an action plan for communication of progress with industry in order to facilitate technology transfer, when certain milestones have successfully reached to guarantee successful demonstration as well.

Reports: Yearly WP internal reports and summary of key issues on web-based discussions. Summary consortium reports at months 12,36. Full reports to the EC at month 24 (half-time), 48 (full-time). Overall coordination and decision making 1) Science & Development: The co-coordinator will be responsible for the harmonious execution of the

various work packages and aided by an on-line progress report system and ad-hoc and planned communications and will intervene for any outstanding issues, by contacting the Executive Committee and if necessary seek advice by the Advisory Members. The coordinator will further ensure that key developments are also appropriately communicated to the scientific community and the public.

2) Decision making: Decision making on major issues, such as new possible partners or commercial dissemination etc will be led by the coordinator in conjunction with the executive committee and after seeking when necessary input from Advisory members. Typically, major decisions will seek a unanimous or majority agreement by the Executive Committee members.

3) Electronic communication: An online update tool of the Gannt chart, a discussion list, a progress report submission system, web based conferencing tools and emails are all key means of electronically ensuring appropriate communication between Executive Committee members and WP participants.

4) Financial management will be administered with the involvement of internal auditing systems of public institution partners.

5) Pre-existing IP management: Access of consortium partners to pre-existing IP and know-how will be regulated according to EU guidelines and previous paradigm. The Consortium Agreement will naturally seek an acceptable and beneficial arrangement for all institutions involved.

6) New Intellectual Property will be managed by the inventor consortium partner, in agreement with the executive committee. Prior to the start of the project, a detailed IP agreement will be signed among the partners, regulating responsibility for protecting new IP, ownership and access of other consortium partners to new IP.

7) Cooperation supervision: Each party undertakes to follow the production schedule and budget specified in the technical provisions of the contract. In view of the uncertain character of basic research projects, the production timetables are generally given for information only and do not incur the liability of the parties. However, to limit the risk of uncontrolled time and cost escalation in the project a strict and effective inspection and supervision system managed by the Executive Committee will be created based on the meetings described above via electronic media and once a year in person, frequent technical and financial progress reports (actions completed and results obtained) and ad-hoc meetings and web-based conferences, including the right for the parties to review their position within the cooperative venture as based on clearly stated reasons.

The members of the executive committee are the work-package leaders in this proposal (besides partner VAMP). In this way this proposal maintains an Executive committee that is closely linked and actively participates to the goals of the proposal. This structure facilitates allows a direct communication between partners and a common understanding of goals. Each member of the executive committee further directly supervises or steers his team, and has selected members that further allow the direct communication within work-packages.

FMT-XCT 201792

B 2.2 Beneficiaries

Partner 1 - HMGU

Description of the organization and facilities HMGU, the National Research Center for Environmental Health of the German Government and the State of Bavaria, is a member of the Helmholtz Association of National Research Centers contributing to the foundation of future Medicine and Health Care. It consists of 22 institutes and 3 independent departments. It employs more than 1600 scientists, students and technical stuff and runs a budget of 156mEuro. The HMGU Institute involved in this proposal is the newly founded Institute for Biological and Medical Imaging (IBMI). IBMI is a highly interdisciplinary Institute with focus on the development of imaging technologies in basic research, pre-clinical research and drug discovery and in clinical applications. IBMI employs 18 scientists and engineers with focus on imaging technology development, image processing and reconstruction and the development and use of animal models for developing in-vivo imaging applications with a view towards improving understanding on disease development and treatment and clinical translation. The Institute has direct access to animal models of cancer and close ties to the German Mouse Clinic housed at HMGU. The Institute occupies ~1000m2 of modern laboratory and office space at the HMGU facilities at Neuheberg and ~120m2 of modern office and laboratory space at the city campus of the Technical University of Munich. Main tasks attributed and previous experience IBMI will lead WP5, i.e. the development of the FMT-XCT prototype and corresponding integration of algorithmic developments achieved by the consortium. IBMI will further manage the consortium and lead training and dissemination activities of information and commercialization effort. IBMI director Prof. Ntziachristos has more than 16 years experience in the development of imaging and hybrid systems and has successfully developed several prototypes and imaging methods achieving imaging performance published in PNAS and Nature journals. In addition he is the inventor of 9 patents. From 6 published ones, five have already been licensed and one system has been commercialized. Prof. Ntziachristos brings therefore also the experience necessary to steer the successful commercial dissemination of the technology as well. Dr. Ntziachristos will be aided in managerial tasks by highly experience HMGU administrative personnel and by a dedicated managing expert hired exclusively for WP1, i.e. the management.

Partner 2 CEA

CEA participates here with two different groups. Explicit description of the different groups is given herein

Description of the organization and facilities CEA (Commissariat à l’Energie Atomique) was created in 1945 and is a public research organisation with more than 16,000 researchers, engineers and other employees. Its mission is to develop know-how and assure the technological transfer in a large variety of applications such as in the areas of nuclear energy, biotechnology, environmental protection, microelectronics, optoelectronics and many others. CEA further advises government authorities and industrial partners. Part of the CEA’s technological transfer occurs through its subsidiary company AREVA. CEA is also committed to fundamental research in particle and nuclear physics, astrophysics, molecular and cellular biology, climatology, radiation-matter interaction, and condensed matter physics. A dedicated division, DTBS, with a staff of 130 people has been set up for developing X- and Gamma-ray imaging detectors, the associated electronics and the modelling, simulation and data processing techniques necessary to their development, X- ray tomographic systems and dual energy systems and optical imaging technology applications in the field of medical diagnostics and health care. Description of the Laboratory for Experimental Molecular Imaging (LIME) – [Bertrand Tavitian]. Bertrand Tavitian is the head of the Laboratory for Experimental Molecular Imaging (LIME) of I2BM, the Biomedical Imaging Institute of the CEA, and the head of Inserm-CEA research Unit 803 for In vivo imaging of gene expression. Located near Paris in the Service hospitalier Frédéric Joliot in Orsay, the LIME has a staff of 50 and occupies 500 square meters housing chemistry, molecular and cellular biology and animal facilities together with the major facilities for Molecular Imaging: a 7-line cyclotron, 15 radiochemical hoods, 4 PET cameras including a small animal FOCUS PET, 4 SPECT cameras including a small animal SPECT-CT, a micro-CT and 4 small animal optical imaging systems including the TomoFluo3D from the LETI. The LIME is an active partner of 11 national and 6 European research networks and coordinates the FP6 Network of excellence EMIL (European Molecular Imaging Laboratories), the Paris canceropole multimodality cancer imaging programme, and the French national platform for Imaging in Experimental Oncology (ICE). Main tasks attributed and previous experience The LIME will provide expertise, both in original and standard animal models of cancer, and in original and commercial fluorescent probes,. The ICE platform will be made available for imaging animal cancer models using FMT, XCT and PET. LIME will evaluate fluorescent probes and their capacity to provide quantitative biochemical imaging of tumors. In addition, LIME will further develop its proprietary methodology for fluorine-18 labeling of macromolecules into a combined fluorescent+PET reporter reagent, in order to offer a double modality imaging approach with the same probe. Finally, the presence of a Clinical Pharmacology Group inside the same facility will simplify the eventual translation into clinical applications. Professor Bertrand Tavitian has more than 19 years experience in molecular imaging and has developed several probes and imaging methods for macromolecular imaging. Description of the LETI– [Philippe Rizo]. Main tasks attributed and previous experience LETI will be responsible for the design and development of a micro-CT system and the investigation of improving soft tissue contrast using dual energy (vs contrast agents with the help of P.7) methods. LETI began to develop X-ray systems in1980 initiating development in digital radiography using Gd2O2S converter screens and video cameras. It

FMT-XCT 201792

developed X-ray Scanners for medical and industrial applications and 3D cone beam tomographic systems. In 1990 it began to study dual energy measurements, tomography with incomplete data and tomography of moving objects. These developments have all been transferred or licenced to industrial partners. In 2003 LETI extended its interests to fluorescence imaging with the development of a fluorescence reflectance imaging system (FRI), now transferred to the Cyberstar SA and distributed by Hamamatsu. Since 2005, LETI/DTBS is investigating deep fluorescence imaging and bimodality imaging such as X-ray and fluorescence. The team involved in this project will rely on the skills of two specialists in X-ray image processing, two specialists in diffuse optical imaging and two specialists of optical and X-ray instrumentation.

Partner 3 - FORTH

Description of the organization and facilities FORTH is the leading research centre in Greece housing the following institutes: Institute of Electronic Structure and Laser, Institute of Molecular Biology and Biotechnology, Institute of Applied & Computational Mathematics, Institute of Computer Science, Institute of Mediterranean Studies, Institute of Chemical Engineering & High temperature chemical process, Biomedical Research Institute, and Crete University Press. FORTH has an experience in managing externally funded projects. It runs currently 51 national projects and 180 European projects with a budget of 10.7 and 51.5m€ respectively. FORTH is currently the co-ordinator in 34 projects out of these 180 European projects. In addition to the current projects, FORTH has run 836 national and European projects of a total budget of around 110 m€,.since 1986. Finance and accounting systems of FORTH work with SAP 4.0B implemented since 1/1/2002. Two of FORTH’s Institites are involved in the proposed project:

1. Institute of Electronic Structure and Laser (IESL) 2. Institute of Molecular Biology and Biotechnology (IMBB)

1. Since its formation in 1983, the Institute of Electronic Structure and Laser of the Foundation for Research and Technology- Hellas (IESL-FORTH) has established its presence in the areas of Laser Science, Microelectronics, Polymer Physics, Materials Science and Environmental studies. In IESL-FORTH research activities range from fundamental studies through to applied and technological research while emphasis in scientific excellence is of prime importance. 2. The research conducted at the IMBB places emphasis in the elucidation of basic life processes, in the decoding of the structure and the integrated function of genes in a given organism (genomics-postgenomics) and finally in the discovery of the mechanisms that control the development and function of an organism. Main tasks attributed and previous experience FORTH will be leading WP3 which is related to the development of the appropriate theory for the 360 degrees FMT and a major participant in most of the workpackages of the project, contributing in both the theoretical and experimental developments. FORTH has been one of the leading centres in optical tomography methodologies with pioneering contributions in both the forward and inverse problems as well as novel experimental approaches and prototype systems. In particular FORTH will be involved in developing direct inversion methods in measurements obtained from complex geometries. FORTH will also contribute by performing tests on phantoms as well as animal models and improving the multi-wavelength methodologies employed for the simultaneous detection of multiple fluorophores. Furthermore, FORTH will be participating in the incorporation of the different theoretical models and the validation of the experimental prototypes. Important also in the technique dissemination and training activities, is the development of user-friendly software that can be accessible by users and not only developers.

Partner P4 - UCL

Description of the organization and facilities UCL has an annual turnover of £500M, academic and research staff totaling 4,000, and over 3,000 PhD research students. The department of Computer Science has over 50 academic staff with specialist groups involved in Imaging Science, Computer Graphics, BioInformatics. Intelligent Systems Networking, and Software Systems Engineering. In 2005, the Centre for Medical Imaging (CMIC) was formed jointly between Computer Science and the department of Medical Physics & BioEngineering to create a world class grouping combining excellence in medical imaging sciences with innovative computational methodology, finding application in biomedical research and in healthcare. The research of the group focuses on detailed structural and functional analysis in neurosciences, imaging to guide interventions, image analysis in drug discovery, imaging in cardiology and imaging in oncology with a strong emphasis on e-science technologies. The Centre has very close links with the Faculty of Clinical Sciences, the Faculty of Life Sciences and associated Clinical Institutes, in particular the Institute of Neurology, the Institute of Child Health and the Centre for Neuroimaging Techniques (CNT). Main tasks attributed and previous experience The main tasks for partner 5are WP3 and WP4 with some input into WP1, WP5, WP6 and WP7. We will contribute mathematical and computational techniques for the development of forward and inverse modeling in optical tomography, particularly the development of multimodality reconstruction techniques using anatomical and functional priors and a Bayesian formulation. UCL has been the pioneering centre for the development of diffuse optical tomography. The group has expertise in inverse problems in medical imaging in general, and optical tomography in particular. A software package (TOAST) developed by this group is a sophisticated finite element method (FEM) based software for modelling of several medical imaging modalities and includes non-linear reconstruction methods and Bayesian regularization techniques. It is public-domain and has several hundred users world-wide. Other funding related to this project is provided by EPSRC, MRC, Wellcome Trust and the British Council.

FMT-XCT 201792

Partner 5 - Medical Imaging Laboratory - Hospital Gregorio Marañón (FIHGM)

Description of the organization and facilities The General University Hospital Gregorio Marañón is a public hospital of the Community of Madrid with more than 22 buildings, 1,700 beds and 8,500 workers and takes care of approximately 750,000 inhabitants. It emphasizes on technological excellence as well as the high quality of its professionals. The Laboratorio de Imagen Médica (LIM, Medical Imaging Laboratory) is part of the Experimental Medicine Unit, and has a long standing experience in molecular imaging and imaging system development. The web page (http://www.hggm.es/image) maintains an updated list of projects, publications and instrumentation developments. LIM has assumed a leadership role in medical imaging is Spain, working in close collaboration with the Spanish Research Council and the main Technology Institutes and Medicine Schools. Since 1997 the LIM has been involved in 35 national projects (8.1 M€ total budget) and 6 international projects, further serving several technology transfer contracts with several multinational companies regarding software and hardware development. Starting in 2006, the LIM is the scientific leader of a CENIT project funded by the Spanish Ministry of Industry, and CIBER action for the Ministry of Health and Human Services. Both projects are focused on molecular imaging research with special emphasis in technology development and clinical applications. Before that, the LIM was the main coordinator of a nation-wide medical imaging excellence network comprised by 50 research groups and 29 centers from all around Spain. LIM has developed an imaging suite is equipped with small-animal high-resolution systems for PET, CT, SPECT and optical (reflectance) imaging. A state-of-the-art small-animal 7 Tesla MRI system is being installed and the first images are planned for October 2007. Surgical, microsurgical and animal housing facilities are available on site, as is a molecular biology laboratory equipped with standard instrumentation. We also have a hardware workshop and an electronics laboratory. Main tasks attributed and previous experience LIM participation in this proposal involves the design and development of single and dual-energy XCT and the cross-validation of the FMT-XCT performance with PET-XCT. LIM is a European leader in the development of hybrid, multi-modal systems and is ideally suited for these tasks. LIM has been pioneered and commercially translated hybrid PET-XCT imaging systems and is overall focused on research into medical imaging technology. In addition, it has strong expertise in the application of such devices and methods in biological experiments. LIM consists of a highly multidisciplinary team and further closely collaborates with clinicians and healthcare professionals to address various research and healthcare needs. The methods for multimodal image integration and analysis developed by the LIM are currently applied in functional image interpretation, neurosurgery, radiotherapy, nuclear medicine, and microscopy. These applications require advanced software for image fusion and quantification, and some have been integrated into commercial products.

Partner 6. University of Zürich

Description of the organization and facilities The MRI and optical imaging labs of the Animal Imaging Center located at the ETH Campus Hönggerberg are part of the Institute for Biomedical Engineering (IBT), which is a joint institute of the University of Zürich (UZH) and the Swiss Federal Institute of Technology (ETH). The UZH is the State University of the Kanton of Zürich, while the ETH is a National University of the Swiss Federation. The IBT's focus is the development of imaging technology. Belonging to the Medical Faculty of UZH it fosters immediate links to the biomedical research application.The Institute has a wide spread range of collaborations, with the Neuroscience Center Zürich (UZH/ETH, focus application: neural plasticity and repair), with the Competence Center for Systems Physiology and Metabolic Disease (ETH, focus application: metabolic diseases and cancer), but also with medical faculties (Neurology, Cardiology, Gastro-Intestinal Diseases to name a few). Moreover, the animal imaging group is associated with the ETH Department of Chemistry and Applied Biosciences, which is of great relevance considering the development of targeted probes. Main tasks attributed and previous experience The task of the participant is to research methods for monitoring treatment response at the molecular level and longitudinally using the FMT-XCT platform as it relates to drug discovery and clinical translation. The hybrid imaging method will be used to evaluate both the normal progression of subcutaneous xenografts and orthotopic tumors as well as to assess the efficacy of anti-tumor therapies for breast cancers (general cytostatics, hormonal and target-specific therapy). It will be also analyzed to what extent the combined information from XCT and FMT provide superior information to mere XCT readouts. The group has long-standing experience in evaluating treatment response in animal models of human diseases using imaging methods for a variety of human pathologies including cancer. Tumor related work of the group that was previously within a pharmaceutical industry environment comprised the assessment of anti-angiogenic therapy in orthotopic models using MRI or the effect of MMP inhibition using protease activatable probes in combination with fluorescence imaging. Currently there are two ongoing projects within our group that are closely related to the proposed contribution: The development of an assay demonstrating activation of the HIF pathway, a critical element in tumor development, and secondly, techniques to quantitatively assess neo-vascularization in tumor models, combining multiple modalities with mathematical modeling.

Partner P7 - VAMP

Description of the organization and facilities VAMP GmbH means in full “Verfahren und Apparate der Medizinischen Technik”, which translates to “Methods and devices for the medical physics”. It was founded in 1997 by Prof. Dr. Willi Kalender, PhD as a spin-off of the Institute of Medical Physics of the University Erlangen-Nuremberg. VAMP benefits from the huge amount of experience which was

FMT-XCT 201792

gathered throughout the years at the University, but has ever since built up a reputation of its own as a manufacturer of highly sophisticated micro computed tomography (micro CT) systems as well as a supplier of software for clinical CT applications. Being a SME, VAMP aims not to compete with the major suppliers of clinical CT systems, but specializes in the highly innovative field of applicationspecific solutions and scientific challenges. With currently 13 employees of mainly academic education, VAMP is at the moment establishing a product for pharmaceutical research in the market. The TomoScope® 30s micro CT product line offers an unparalleled scan speed and is targeted mainly at pharmaceutical research. By means of the excellent relationship with various universities and non-academic research facilities, VAMP is able to offer state-of-the-art CT imaging technologies whilst maintaining a reasonable price-to-performance ratio. Main tasks attributed and previous experience The goal for partner 7 is to design, develop and adapt the tomography system for all the other partners to build their parts upon. Our contribution will span the integration of the techniques developed by other groups into an easily usable, robust and rugged, industrial-grade rotating-gantry XCT-FMT combination system. Due to the modular design of our products, there are many components which can be used in the experimental setup, but major parts have to undergo a radical redesign because of the hugely different approach the FMT is using for acquiring pictures as compared to the “conventional” X-ray CT. Being in close contact with research facilities ever since its founding, VAMP offers a highly trained crew of engineers and scientists of PhD level to take care of all the requirements, which will evolve as the project proceeds. Other projects related to this one presented here include the delivery of a dual-source micro-CT system to the DFG (German Research Association) research group 661 “Multimodal Imaging”, a multidisciplinary focus group for exploring the use of combination imaging techniques to the study of various clinical relevant questions as ischemia and tumor marking. The VAMP TomoScope® 30s Duo will there be used as a reference standard to match the newly developed imaging techniques against and is about to be delivered in the upcoming weeks at the time of writing.

Partner P8- UC3M

Description of the organisation and facilities The Carlos III University of Madrid (UC3M) is a public university founded in 1989. UC3M has strived, since its foundation, to make research one of the fundamental pillars of its activity, both for the enhancement of its teaching and for the new knowledge and new areas of research. UC3M, as university, has a national reputation for its research efficiency. It holds second place in participation in the EU Framework Programme (standardized data by number of permanent researchers) and second in average number of publications per permanent researcher in the period 2002-2006 (Web of Science). Recently (Report 2009 of CNEAI-National Commission for Evaluation of Research Activity), UC3M has reached the top of the Spanish universities in research activity.The Biomedical Imaging and Instrumentation Group (BiiG) is part of the Departmento de Bioingeniería e Ingeniería Aeroespacial, Universidad Carlos III de Madrid (UC3M). BiiG is a leading research group in the area of bioengineering, in particular with regard to medical imaging techniques and applications. The group conducts research activities is areas like new imaging technologies, image processing and analysis methodologies, image tomographic reconstruction, their practical applications in biomedicine, and biomedical instrumentation. The research lines also include: Technology development for small-animal imaging, Cardiac imaging and quantification, Magnetic Resonance Imaging, Neuroimage, Multimodality imaging, Equipment design. BiiG has presently more than 1000 m2 including offices, electronics and mechanic workshop, laboratory-operating room for small-animal molecular imaging, and a fully-equipped magnetic resonance imaging suite. The BiiG group collaborates with other UC3M departments such as Electronics, Mathematics, Physics, etc. Moreover, the University established consortia with several Hospitals in the Comunidad de Madrid that allow sharing human resources and infrastructures, such as laboratories, animal housing facilities, operating rooms, laboratory for molecular biology, analysis equipment, etc.All these resources constitute one of the more complete settings at a national scale, which is also available to external researchers by providing support and consulting for their research. BiiG has approximately 40 researchers, to which we have to add the participation of professionals from other departments of the Hospital and the University. Main tasks attributed and previous experience BiiG participation in this proposal involves the design and development of single and dual-energy XCT and the cross-validation of the FMT-XCT performance with PET-XCT. BIIG is a European leader in the development of hybrid, multi-modal systems and is ideally suited for these tasks. BIIG has been pioneered and commercially translated hybrid PET-XCT imaging systems and is overall focused on research into medical imaging technology. In addition, it has strong expertise in the application of such devices and methods in biological experiments. BIIG consists of a highly multidisciplinary team and further closely collaborates with clinicians and healthcare professionals to address various research and healthcare needs. The methods for multimodal image integration and analysis developed by the BIIG are currently applied in functional image interpretation, neurosurgery, radiotherapy, nuclear medicine, and microscopy. These applications require advanced software for image fusion and quantification, and some have been integrated into commercial products.

FMT-XCT 201792

45

B.2.3 Consortium as a whole This proposal brings together world renowned scientists and thought leaders that bring highly complementary expertise. Professor Ntziachristos is a world leader in the development of fluorescence tomography systems and has applied fluorescence tomography techniques in small animal imaging (Nat. Med.8(7):757 2002, Nat. Biotech 23(3):313 (2005), Nat. Biotech) and has constructed and clinically applied in breast cancer patients the first world-wide optical-MRI hybrid system (PNAS). He has strong experience in optical instrumentation and a highly developed overview of the tasks necessary to bring a prototype to in-vivo application, to clinical application including necessary institutional approvals, and commercial dissemination. Therefore he is well qualified for managing this development and applying to this project the next generation of optical technology to achieved superior performance. Prof. Ntziachristos’s work is highly complemented by the group of Dr. Jorge Ripoll. Dr. Ripoll has pioneered several theoretical aspects in developing time-efficient theoretical models for photon propagation in tissues and has been the acting administrator of the Integrated Project for Molecular Imaging under FP6. He therefore brings with him necessary theoretical skills that are essential for the development of a practical algorithm for the forward problem. The “optical development” team is finally solidified by Professor Simon Arridge of University College London. Professor Arridge is one of the inventors of optical tomography with almost 20 years experience in the field of optical inversion. He has developed several algorithmic schemes based on numerical method and brings with him key expertise in numerical modeling of the forward problem and the inversion of optical tomography problems and the use of priors. For more than 10 years Prof. Arridge’s group has made available the TOAST algorithm, which is a public algorithm for optical tomography utilized by several groups in Europe, USA and the rest of the world. Dr. Arridge has applied TOAST to clinical optical imaging of the breast and brings key experience on clinical translation as well.

In terms of the FMT development the core team of Drs. Ntziachristos, Ripoll and Arridge is probably the strongest European team in optical tomography, collectively with more than 150 peer-reviewed publications on the field. These scientists have served as chairs and program chairs in virtually all international conferences on optical technologies and carry long-standing experience in the field. Each group brings the state of the art on each of 1) optical instrumentation, 2) time-efficient forward models and 3) inversion approaches and the use of image priors while it has an excellent grasp of the overall technology to be highly communicative and synergistic.

XCT developments are led by Dr. Phillipe Rizo at LETI, Dr. Manuel Desco and Dr. Vaquero from FIHGM and the VAMP team. Dr. Rizo is a world expert in the development of X-ray CT components and has invented and published several technological and algorithmic advances in X-ray CT. Dr. Rizo and his group bring a highly unique skill set in this development. They are the only group worldwide that while highly efficient in X-ray CT, they have more recently also invested resources into optical imaging. In this role this is a highly important group in this proposal, that brings state of the art X-ray CT technology while having hands on exposure on the particulars of an optical system. Therefore a high degree of understanding and communication can be achieved between the groups of Dr. Rizo and Drs. Ntziachristos, Ripoll and Arridge. Dr. Rizo’s group however, will not simply integrate XCT into FMT, they will research key improvements in soft-tissue differentiation by developing dual-energy XCT as an add-on and compare its performance with contrast enhanced XCT. As such the group contributes in offering a true state of the art system, not only by means of integration but also due to key progress in each of the individual technologies, necessary for offering improved soft tissue differentiation compared to single-energy or no-contrast enhanced XCT, which is information needed for the FMT system, as described in Wp2 and Wp4. Dr. Rizo’s team will work together with group of Professor Desco. Dr. Desco is a world leader in the development of XCT based hybrid systems, including PET-XCT and SPECT-XCT for small animal imaging. He has ample experience also with the commercialization of such systems. Dr. Desco’s contributions in the design and development of a functional XCT system are fundamental herein to the development of this hybrid approach. His participation further minimizes development risk, by avoiding possible pitfalls that PET-XCT and PET-XCT run into and by focusing the development into functionally

FMT-XCT 201792

46

significant aspects, thus accelerating progress. Moreover, Dr. Desco’s team is pivotal in validating the overall hypothesis and motivating idea in this proposal, i.e. that the hybrid XCT-FMT system will reach imaging performance in small animals with similar utility to other ionizing radiation based imaging systems, in particular the powerful PET-XCT. Although this would be considered virtually impossible just a few years ago, as also shown in the recent literature but also in preliminary data herein, current state of the art optical tomography has demonstrated resolution and imaging accuracy that come close or are better than PET. While the PET sensitivity is probably two-orders of magnitude higher in absolute amounts of injected probe, optical methods do not need to inject “tracer” amounts but it is safe to inject several nano-moles of fluorochrome, thus gaining back on sensitivity. Questions like that can be best addressed experimentally and by closely working with Dr. Desco group, who have already developed an XCT-PET system for small animal imaging and carry with them all the necessary experience to provide accurate and realistic comparisons between FMT-XCT with PET-XCT-PET. Obviously the motivation of this comparison is not to prove one technology better than the other, but identify the limits of operation and the different applications that each method can best operate at, so as to provide appropriate “usage indications” to the biomedical community. Finally the XCT development is greatly complemented by the VAMP group. VAMP is backed by strong XCT expertise as well and has been recently developing dual energy systems as well. VAMP will not only provide an industrial grade but will actively participate into the development and later incorporation of an optical XCT system, as decided in the milestones and work closely with the other partners in functionalizing a highly performing prototype.

While the five teams described above build a strong worldwide consortium on technological development, this proposal has further signed on the groups of Drs. Tavitian and Rudin to enable highly adept pre-clinical imaging studies with an outlook to clinical translation as well. Dr. Tavitian is the president of the European Society for Molecular Imaging and the administrator for the European Molecular Imaging Network (EMIL) under FP6. He therefore brings a tremendous outlook on the possible utility and need for use of the FMT-XCT technology. In practical terms, this experience translates to the availability of key animal models and fluorescent probes and reporter technologies in Dr. Tavitian’s laboratory.

Similalry Dr Rudin has assembled a highly efficient team of engineers and physisicts. Dr Rudin, is a professor at ETH and previously employed as an imaging director in Novartis. He is therefore a though leader with exceptional applied experience in the use of imaging methods in pre-clinical research and drug-discovery. A significant success of this proposal will be in its ability to monitor treatment by utilizing molecular and functional contrast generally not available in conventional modes of imaging. In particular, breast cancer treatment and evaluation of new therapeutic regimes can be significsantly accelarted and main-streamed by the development of technologies that allow personalized treatment monitoring. Dr. Rudin brings in this role the necessary overview on therapeutic protocols of breast cancer, with appropriate animal models.

Industrial/commercial involvement

The novel technologies developed within this project will form the basis of intellectual properties that belong to the developers and which will be exploited appropriately by the respective institutions where the developers work. Commercial exploitation will be sought in coordination with appropriate companies and VAMP serves the requirements for such candidacy. More generally however, after 24 months, the consortium will consider including additional companies in order to explore further applications of the technology generated since the association with the high-end industry is in the intentions of the partners. Finally, in addition to research skills, exploitation experience is needed to transform at least some of the research results into tangible and easily identified socio-economic items (such as efficient diagnostic methods, spin-off companies, licensed patents, etc). A considerable number of the partners involved in the project possess such experience. Furthermore, VAMP has a strong track record in developing novel commercial devices for small animal imaging. These skills are a significant strength of this consortium towards exploitation of results and increasing European Competency.

FMT-XCT 201792

47

Sub-contracting Two subcontracts are foreseen now in this proposal, one with investigators at the University of Muenster and the other with the Technical University of Munich. The total amount of the subcontracting costs is 51,000 euro, as follows:

1). Partner 1 (co-coordinator) is sub-contracting in this proposal Dr. Christoph Bremer of Muenster University to bring a “radiology” perspective in the proposal and further enhance and solidify the view towards clinical translation of this proposal. Dr. Bremer is a practicing radiologist and expert in clinical optical breast cancer imaging. He has contacted optical imaging studies together with Schering and IDSI Inc. in the clinic. In parallel he has ample experience in the use of fluorescent agents for breast cancer detection in pre-clinical models and clinically. Dr. Bremer is well published in the use of fluorescent agents in breast cancer models. He is subcontracted (and not included as partner) because although he is expected to offer highly valuable advice on the relevance of our results to clinical breast cancer imaging and steer pre-clinical experiments with an outlook to clinical translation, he is not expected to provide key technical developments herein. Therefore he will work under the Munich group, in close collaboration with Prof. Ntziachristos and his group and he will often travel to Munich to consult on progress and advice on results, in the last two years of the proposal. This subcontract will cover travel expenses and a consulting fee or honorarium at a rate of 600 euro / consultation day or per full day of visit.

2) The Technical University of Munich (TUM) will be subcontracted to perform high-energy imaging experiments as described in Wp8. It is important to perform these experiments in the proximity of the developed FMT-XCT system as further explicated in Wp8. Technical personnel under Professor Ziegler and under Dr. Themelis will be employed under this subcontract, with the overall supervision of Dr. Ntziachristos, and expenses will further cover the experimental costs at the Technical University of Munich. These costs include PET running time at 800 euro/hour utilizing approximately 12 hours, imaging and animal handling personnel fees for local animal handling in the course of the experiment at the Technical University of Munich (~ 3 man-months - 14,000 euro), chemist and probe development fees (~ 0.5 man month and costs of radiotracer development at 500 euro of radiochemistry facility use), animal fees for housing at the Technical University of Munich (800euro). Support of laboratory supplies (3,000 euro) a dedicated on site spectral sensor for correlative optical measurements in the TUM surgical facility (4,800), custom made mounting and cassette development costs for enabling FMT/CT and PET/CT co-registration as described in Wp8 (development performed locally at TUM around the PET system 4,300) and an optical home-build intravital system for operation in the PET room for on-site inspection of fluorescence activity (5,100), while mice are imaged by PET/CT. Due to the relatively small involvement of the Technical University of Munich in this proposal, it was decided to treat this as a subcontract, rather as an equal partner for simplicity purposes. However the contribution of the work performed at TUM is essential in this proposal and reflects to the development of partners 1,2 and 6 and is a key aspect of Wp8.

B 2.4 Resources to be committed

Besides manpower, the proposal asks only for the necessary components to develop a single XCT-FMT prototype (under Wp5) and biological materials and animals and other experimental cost (imaging costs etc) in order to achieve the objectives of the study. The major components requested therefore relate to:

1. low light sensitive CCD camera, 2. An illumination laser and mounting components 3. Components required to develop single or dual energy X-ray CT as per milestone 5, and 4. an industrial grade X-ray CT system.

FMT-XCT 201792

48

A dedicated FMT-XCT system is requested as it is important for the advancement of the technology and for training and demonstration actions. An XCT prototype developed at CEA-LETI under Wp2 will be developed out of LETI’s own available instrumentation. In addition increased plans for training through motility and the locality of the XCT-FMT prototype and the XCT developments request travel funds, in addition to those utilized for travel to scientific meetings and regular consortium meetings. The following tables summarize the budget justification per participant

Partner

Effort and budget justification

P1-HMGU

1. Major management, training and dissemination activity, travel. 2. One scientist and two students to contribute to FMT-XCT development, in-vivo imaging, algorithmic developments in Wp3, Wp4, data analysis in Wp6 – Wp8 and the planned PET-XCT experiments at Munich.

3. A CCD camera, Laser system and mounting components 4. Consumables for animal costs and histological analysis

P2-CEA LETI

1. Small management, training and dissemination portion, travel. 2. One scientist and one technician and a part time student to develop the XCT prototype, perform XCT experiments and enable XCT integration with the FMT system in Wp5 as well as in-vivo animal experiments.

P2-CEA LIME

1. Small management, training and dissemination portion, travel. 2. One scientist and two technicians to develop animal models and probes and participate in in-vivo experiments in Wp6 and Wp7.

3. Consumables associated with animal costs, fluorochrome development and imaging time for validation purposes during probe development.

P3-FORTH 1. Small management, training and dissemination portion, travel. 2. One scientist and one student for algorithmic developments, data analysis of in-vivo and phantom experiments in Wp5-Wp8 and for participation in in-vivo experiments and integration of code under a user friendly scheme.

3. Consumables associated with animal costs performed at FORTH as per Wp7.

P4- UCL 1. Small management, training and dissemination portion, travel. 2. One scientist and one student for algorithmic developments, data analysis of in-vivo and phantom experiments in Wp5-Wp8 and for participation in in-vivo experiments

P5-FIHGM 1. Small management, training and dissemination portion, travel. 2. One scientist and one student to develop phantoms, perform validation experiments and lead the PET-XCT experiments in Wp8.

3. Consumables associated with phantom cost development and imaging costs in Madrid.

P6-UZH 1. Small management, training and dissemination portion, travel. 2. One student and a part time scientist to develop and characterize animal models and perform imaging experiments associated with imaging treatment.

3. Consumables associated with animal costs and imaging time for validation purposes during probe development.

VAMP

1. Small management, training and dissemination portion , travel. 2. One scientist (fuyll time 14 months) and a part time technician to modify a CT gantry and perform measurements and design of the XCT and XVCT-FMT prototypes.

3. Components associated with the development of the gantry and an XCT system.

In addition to this effort, all academic partners commit at least 3 staff members, as per description in Section 2.2 with an average effort of 20-30% to supervise and enable many the tasks described in the work-packages. In addition to these costs significant infrastructure and staff is utilized, with contribution that is not provided by the EU in this proposal. A summary of these resources are as follows:: All additional resources required for this proposal are available through existing infrastructure in the corresponding laboratories as follows:

FMT-XCT 201792

49

P.1 will mobilize necessary optical and biological support infrastructure (optical tables, optical components and tools, animal housing facilities, histology facilities etc) and permanent technical personnel of the Institute to support the development of the FMT-XCT prototype and the corresponding in-vivo studies. In addition he will utilize an existing Linux cluster at IBMI for computational tasks associated with image processing and reconstruction.

P.2. will utilize existing X-ray CT instrumentation at LETI as the basis of a single energy X-ray CT system on which he will add dual energy capacity. CEA-LIME will utilize the small animal facility core to develop the appropriate animal models and fluorochromes, including microscopes, FACS and RT-PCR systems and a large number of chemistry synthesis facilities to provide the appropriate fluorochromes proposed.

P.3 will utilize experimental FMT setups developed for stand-alone small animal imaging in order to produce test measurements for algorithmic optimization and multi-spectral implementation parameters.

P.4 will utilize existing computer networks to perform the necessary computational tasks associated with the algorithmic developments proposed.

P.5. Will similarly utilize extensive small animal imaging and handling facilities associated with the development of animal models and co-relative tests for imaging validation.

P.6 will utilize a previously developed hybrid PET-XCT system for original validation of phantoms in addition to small animal imaging and handling facilities.

P.7 will utilize significant infrastructure in the manufacturing and measurement of XCT industrial grade gantries and systems as well as computational power to test with reconstructions.

These contributions are summarized in the following table

Partner

Major Infrastructure contributed at no cost to EU

P1-HMGU

Optical components and optical measurement systems. Computer network for data analysis and computations. Optical tables etc. Surgical and histological analysis facilities

P2-CEA (LIME and LETI)

X-ray CT components and systems, X-ray measurement devices XCT-PET systems for animal and probe optimization Chemistry synthesis facilities and facilities for histological analysis

P3-FORTH Computer network for computations, Stand-alone FMT system in the vertical geometry Animal facilities

P4- UCL Computer network for computations

P5-FIHGM XCT and PET-XCT systems for correlative and phantom imaging Surgical, microsurgical and Animal facilities, Computer network for computations

P6-UZH 2 optical planar imaging systems and 2 stand-alone tomographic optical for developing and optimizing reporter systems 1 micro CT system (Scanco Medical AG), 2 small animal magnetic resonance imagers (Biospec 94/30 and Pharmascan 47/16 imagers from Bruker BioSpin GmbH), which will be used for comparative/complementary measurements. Animal facilities.

VAMP

Computer network for computations Machine shop and measurement/manufacturing instrumentation for XCT systems

Each partner has mobilized appropriate effort in order to accomplish the objectives put forth in the proposed time frame. Importantly each partner has been selected for bringing a particular expertise into the project. Therefore each of the contributions is highly expert and it is expected that the performance of each partner in the corresponding tasks assigned is highly efficient. By enabling high interaction between partners, it is therefore expected that a highly synergistic development will be accomplished. By ensuring extended training activities, this proposal further enables the dissemination of expertise amongst groups and to other research groups and the industrial sector as well, in order to expand and propagate the information and knowledge base.

FMT-XCT 201792

50

FMT-XCT 201792

51

B3. Potential Impact

B 3.1 Strategic impact

This system advances multi-modality imaging by offering the first FMT-XCT system worldwide. FMT has in the last 3 years advanced at a high performance imaging modality by utilizing non-contact free-space approaches, compared to fiber-based systems that utilized tedious procedures of fiber coupling to tissue and yielded only a small number of measurements that were not sufficient for high quality imaging. Based on this knowledge and performance, this development is an obvious next step that only very recently became technologically feasible; the very first publications for optical tomography that can operate in 360-degree geometries by using direct CCD camera coupling (i.e. high quality measurements) in the absence of fibers or matching fluids was only published in the first quarter of 2007 by P1 and P3. This proposal will utilize this knowledge to build for the first time the best known optical tomography system operating on the free-space principle. In this design it can then seamlessly combined with XCT that also operates with similar geometrical arrangements and analogous instrumentation (i.e. an X-ray source and a flat panel detector). The combination however with XCT technology is crucial in advancing the performance of the imaging method by enabling a highly desired anatomical map to co-register optical and XCT information but also for utilizing the XCT information into the FMT inversion to offer the best yet optical imaging performance. In return, the combination of a highly performing optical imaging method into the XCT system offer to tremendously improve the potential application base for X-ray CT imaging by offering a system that can yield highly versatile functional and molecular contrast to the high quality anatomical contrast of XCT. In addition the research of alternative methods herein to improve soft-tissue contrast (by dual energy approaches or by utilizing contrast agents) offers overall to yield a highly accomplished system with high potential impact. The system can enable new types of therapeutic intervention, by yielding highly accurate information on animal models of disease and quantitatively resolving effects of treatment in-vivo and longitudinally on the same animal. In this role it can facilitate time-efficient and accurate observations of a large number of possible treatment combinations and optimize dose of drugs and radiation as a function of a particular cancer. The technology can also be used in key clinical applications as well, i,e, in breast cancer imaging or arthritic joint imaging. In this role this technology can be used as an efficient imaging tool for personalized medicine, especially since the system can be used for frequent observations of treatment progression and help in patient speficic decision making. There are several possible modes of operation, where accurate X-ray CT scans are only occurring at selected N-points but ultra low dose scans of low resolution are more frequently administered to guide FMT sessions that can more frequently administered due to the use of no-ionizing radiation. Accordance with global EU priorities in FP7

This proposal highly complements EU priorities in advancing the understanding on efficiently promoting good health, to prevent and treat major diseases and to deliver health care. Increasing the competitiveness of European health care biotechnology and medical technology sectors

The proposed technology is ideally suited for large-scale dissemination. This is because it is based on technology that is easily accessible by manufacturers and is biomedical laboratory friendly. In contrast to nuclear imaging methods, this technology does not require the significant investments that accompany the production of radio-nuclei, the associated radio-chemistries and any safety issues. Instead, X-rays can be locally generated by a small form factor X-ray source and be conveniently shielded even when offered in simple bench-top systems. Importantly the key contrast modality in this proposal, i,e fluorescence is a highly available technology in any biological, biotechnology or biomedical lab. This implies that the proposed system, if successful, can change the paradigm of in-vivo imaging by offering true quantitative

FMT-XCT 201792

52

functional and molecular imaging in addition to the high-resolution anatomical imaging to a significantly larger number of the biotechnology and biomedical sectors. Innovation This proposal offers a high degree of innovations by:

1. Offering a never-before developed hybrid system 2. Advancing optical imaging technology beyond the current state of the art 3. Researching optimal, but currently unknown, parameters for incorporating XCT information

into the FMT inversion 4. By researching quantitative methods for offering highly sensitive differentiation of breast

cancer 5. By researching highly elaborate inversion methods and optimal ways in incorporating a-priori

information in the FMT inversion.

B 3.2 Plan for the use and dissemination of foreground

Dissemination actions

This proposal is driven by dissemination. It is one of the fundamental objectives herein to develop a highly performing system for in-vivo imaging that is ideally suited for the biomedical bench and for large-scale propagation. The particular reason is that the components employed in the technology proposed are highly accessible and cost efficient, the system can operate with minimum resource investment (i.e. it does not require highly expensive components and operation/management costs as in the case of hyper-conductive magnets and space, or cyclotron and radiochemistry facilities. It is therefore envisaged that small form factor versions of the prototype build herein will find their place next to the microscope, or photo-spectrometer in any biology lab interested in in-vivo imaging and clinical translation. The partners will further utilize connections to the industry to raise awareness and solicit discussions on commercial propagation. Close contact with policy makers and regulatory bodies on national and EU level will be established to assist future developments and the necessary steps to ensure safety of the technology and the appropriate legislation. Dissemination is essential for success to provide a good communication among consortium members by regular project meetings, joint training courses, workshops, mobility and a dedicated web site. The results of the project are aimed to have a major impetus on medical research in Europe. Therefore a complex strategy of dissemination is planned. The scientific community will be notified about the scientific achievements primarily through numerous scientific publications and conference presentations. The project web site will have a part accessible to the public. Contact with the industry will be achieved by standardising the new model systems for pharmaceutical testing purposes, and by the participating SME. Close contact with policy makers and regulatory bodies on national and EU level will be established to assist future developments and the necessary steps to ensure safety of the technology and the appropriate legislation. Raising public participation and awareness

Dissemination of the results to the general public will be organised by a dedicated public relation personnel working with the co-ordinator. Further international scientific organisations will be involved in the dissemination of the results. Good communication will be helped by a dedicated web site. The final report will include the broader ‘science and society’ implications of the project outputs.

FMT-XCT 201792

53

Technology Transfer and commercialization The developed prototype can be of significant interest to the industry due to the foreseen dissemination (commercialization) potential and the large market that exists. Partners have been already approached or are in discussions with Siemens, GE Healthcare and smaller micro-CT and CT companies for the potential interaction and collaboration in pre-clinical but also clinical imaging as it associated with digital tomo-synthesis for breast cancer healthcare. The executive committee will decide after month 24 on the addition on other partners in accordance also with the CA and EU guidelines. Decisions will take into account the overall performance achieved and act for the best possible commercialization outcome of the technology.

Managing intellectual property

This activity has also been described also in Section 2.1. Intellectual property will actively managed in Wp1 and through the consortium agreement. The participants are all experience in technology commercialization and have already generated IP in their respected fields. However due to the innovative components in this proposal , it is expected that significant new IP will be created in algorithmic and technical issues associated with the development of the proposed hybrid systems. These issues will be explicitly handled in the consortium agreement with focus in maintaining high recognition of the inventor while ensuring appropriate action, also through executive committee meetings, to technology transfer and commercialization propagation of the invented technologies. Overall we expect this proposal to yield a highly commercializable platform, well protected by corresponding IP.

FMT-XCT 201792

54

B4. Ethical issues

No human participation is proposed and no personal data are collected in this proposal. Use of animals: This call requests pre-clinical imaging, as necessary for developing a new imaging modality and creating new knowledge and algorithms. In response, the partners have included preclinical imaging in two key categories:

5. Imaging of pre-clinical animal models of breast cancer (Wp6) 2. Pre-clinical imaging of breast cancer treatment in-vivo with a view towards clinical

translation and personalized medicine. The proposal spends considerable effort to develop phantoms (Wp8) in order to perform much of the imaging experiments required for algorithmic development, system development and imaging optimizing without the use animals. Therefore the use of animals is restricted only to issues that fundamentally relate to developing the system for in-vivo “pre-clinical imaging with a view towards clinical translation”, as requested in the call. We note that non-invasive imaging systems ultimately result in the use of a significant smaller number of animals compared to the current state of the art. This is because processes can be followed in the same animal over time, and therefore conclusions can be attained without the needs to euthanize large cohorts of animals in order to gain insights in the time course of events by statistically piecing together the information.

The use of animals herein is used to 1) provide training sets for algorithmic optimization and 2) examine the performance of the technology towards detecting and characterizing cancer and it treatment, as it further relates to realistic biomedical research needs and the most critical clinical application. The Wp6 and Wp7 tables are summarized bellow. Table 4.1 then summarizes the animal numbers and purpose for all work-packages.

TABLE FROM Wp6: Cell lines , probes and interrogations considered in Wp6

Cell Line Site Probes – em.

wavelength Interrogation & Purpose Partner

MDAMB-231 Mammary/ lung AptamerHM1–690, RGD-770 or CaB-690, MMP-780, Angio-810

Detection limit Quantification of protease levels Accuracy in quantifying growth.

2,3,4

Human erb-B2 + /- Mammary Trastuzumab – AF680 (680) or CaB-690, MMP-770, Angio-810

Accuracy in quantifying receptor levels Accuracy in quantifying growth

1,2,5

PyMT Mammary/ lung VEGF-690,RGD-peptide-770 or CaB-690, MMP-770, Angio-810

Test angiogenic switch. Detection limit of early development and metastasis

2,6,3,4

U87 Brain AptamerU87–690, RGD-770 or CaB-690, MMP-770, Angio-810

Image quality in imaging in the brain. Detection limit, image metastasis

All

[ ‘or’ indicates that half the animal will be imaged with the top line combination and the other half with the bottom line combination]

TABLE FROM Wp7: Cell lines, probes and treatment considered in Wp6

Cell Line Site Probes – emission wavelength

Treatment Partner

MDAMB-231

Mammary/ lung VEGF-690, MMP-780, Annexin-810 or AptamerHM1-690, RGD-770, Annexin-810

Doxorubicin & cyclophosphamide followed by docetaxel

2,6,3,1

HIF-transgenic MDAMB-231

Mammary/ lung Annexin-810, MMP-780 and Oxy/Deoxy Hb intrinsic signatures and mCherry

Doxorubicin & cyclophosphamide followed by docetaxel

6,3,1

Human erb-B2 + /- Mammary Trastuzumab–680,MMP-780,Annexin-810

Doxorubicin & cyclophosphamide with or without Trastuzumab followed by docetaxel

1,2,5

PyMT Mammary/ lung VEGF-690, MMP-780, Annexin-810 Doxorubicin & cyclophosphamide followed by docetaxel

All

[ ‘or’ indicates that half the animal will be imaged with the top line combination and the other half with the bottom line combination ]

FMT-XCT 201792

55

TABLE 4.1: Summary of animals utilized in all work-packages

Wp Animals Purpose Provided by

2 5 Nude mice 5 Balb/c mice

Investigate dual-energy vs. contrast agents performance CEA-LIME

3 5 Nude mice 5 Balb/c mice

Measure auto-fluorescence for multi-spectral algorithm Optimize algorithms for boundary removal Optimize algorithms for direct inversion

FORTH, UZH

5 5 Nude mice 5 Balb/c mice

Test and optimize FMT-XCT performance and acquisition settings in-vivo. HMGU

6 90 Nude mice

Examine the detection limit, quantification of protease levels and accuracy in quantifying growth, using two different fluorescent probe combinations as it relates to optimizing imaging strategies for pre-clinical and clinical imaging. N=12 animals per cell line and per probe plus n=3 control animals per probe plus 12 animals to anticipate unforeseen experimental issues or need to improve statistical certainty in some groups due to high standard deviation observed. Train partners in FMT-XCT in-vivo imaging.

CEA-LIME

6 15 PyMT Trangenic animals

Test angiogenic switch. Detection limit of early development and metastasis with a spontaneous breast cancer model n=15 animals.

CEA-LIME

7 45 nude mice Quantitatively examine FMT-XCT performance in assessing differential treatment. UZH

7 15 PyMT Trangenic animals

Quantitatively examine FMT-XCT performance in assessing treatment in a spontaneous animal model. Train partners in FMT-XCT imaging of treatment response.

CEA-LIME

8 10 nude mice Perform FMT-XCT and PET-XCT of the same mice to quantitatively assess performance metrics of XCT-FMT using PET-XCT as the gold standard.

HMGU

8 10 MDAMB-231 nude mice

Develop and confirm dual-labelled FMT, PET probes. CEA-LIME

A total of 215 animals are requested. There is variability in the animal models requested, because cancer is a heterogeneous disease. Also system performance needs to be tested at different sited of disease because the optical signals (intensity) depend on depth and on surrounding imaging properties and it is a goal of this proposal to develop a method that will perform well both for superficial and deep-seated activity. Finally we expect to obtain different performance when using different fluorescence probes, and this parameter need to be explicitely studied herein in order to yield accurate new knowledge on method capacity. We therefore request to investigate system and algorithmic performance with different cancer types and probes and also as applies to cancer treatment as described in Wp6 and Wp7. All animal handling, imaging, euthanasia and discarding will be performed according to institutional guidelines of the partner’s site where the experiments are performed at. Practical considerations

The average group size for pharmacological studies is N=6 to 12 animals, with the actual group size depending on the magnitude of the drug effect and the inter-animal variability. Here provisions are taken to request a few number of additional animals to anticipate for experimental failure (for example problems with hardware operation during development that may invalidate some imaging sessions). All experiments will be carried out in strict adherence to the legislation of the respective country.

After completion of in-vivo imaging experiments all animals will be euthanized and tissues will be harvested for histological and immuno-histochemical analysis. All not-harvested tissues or post-analysis

FMT-XCT 201792

56

samples will be discarded according to institutional procedures, approved by the corresponding state animal committees where the experiments are performed.

Balb/c mice are generally treated with approved for human application hair removal products to reduce the fur for optical imaging experiments. The process overall takes less than a minute for mouse and is safe and customarily used in optical imaging studies.

Research will be carried out with due concern for the environment, in particular the disposal of all radioactive and chemical waste generated during the course of the programme. European Regulations will be strictly adhered to for the disposal of such waste. No release in the environment of any genetically modified organism in this programme is planned.

The only genetically manipulated organisms involved in these projects are the transgenic mice, cultured cell lines and E.coli used in manufacture of the DNA constructs. All genetic manipulations will be carried out using relevant containment. Thus, all genetic modification procedures will be carried out under suitable conditions, by appropriately trained staff, after risk assessment has been carried out and permission sought in advance (as necessary). Experiments and procedures will be carried out under conditions which comply with the Genetically Modified Organisms (Contained Use) Regulations (1992) the Genetically Modified Organisms Regulations in the Netherlands (COGEM) or other legislation required by EC directive 90/219/EEC on the contained use of genetically modified micro-organisms and local gene laws.

The safety and well-being of all participants in the network is a matter of vital importance and all research workers will undergo the necessary national training courses in laboratory safety before starting their work. The highest standards of safety will be maintained in particular during genetic manipulations which this proposal requires. These techniques will be carried out according to national guidelines in force.

Imaging practices, Pain and Euthanasia All imaging protocols and other imaging procedures

will be performed under anaesthesia for immobilization and will induce no pain. Any surgical implantation of cells will be performed under sterical conditions and similar under anaesthesia. Animals will be daily monitored for levels of health and overall well-being and all animals that are observed to be at any condition that appears to be less than fully healthy will be removed from the studies and immediately euthanized. Similarly, animals with tumors grown beyond 1 cm will be also euthanised. Some animals will undergo longitudinal imaging for a maximum of 4 weeks, with one session per week. Any signs of discomfort will trigger removal of the animal from the session. Mice will be maintained under barrier conditions in which there will be minimal possibility of infection and therefore no discomfort to the animals. Administered XCT dose will be appropriately monitored according to European and Institutional guidelines and no overdose will be allowed in any of the animals. Animals undergoing imaging with radio-isotope tracers will be euthanized the day of administration, within 3 hours after tracer administration. No longitudinal studies will be performed in these animals. Euthanasia will be performed with according to accepted European and Institutional guidelines. In summary All research carried out will respect international codes of practice.The consortium particularly will take into account the opinions of the European Group of Advisers on the Ethical Implications of Biotechnology (1991 –1997) and the opinions of the European Group on Ethics in Science and New technologies (as from 1998). Relevant EC-policy related issues (e.g. Life sciences and biotechnology – A strategy for Europe (COM(2002) 27)), will be taken into account, as well. In addition, all local or national requirements for ethical committee approval will be obtained. The scope of the present project is limited to animal experimentations and creation of a new imaging platform. No immediate effect on the human medical field or society is envisaged within the scope of the project. General Statement: the FMT-XCT will comply with the ethical rules laid down for FP7 projects in all of its activities, and internal and external representation of the Consortium. Partners will conform to all pertinent legislation, conventions and declarations including

FMT-XCT 201792

57

• The Charter of Fundamental Rights of the EU

• Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2001 on the approximation of the laws, regulations and administrative provisions of the Member States relating to the implementation of good clinical practice in the conduct of clinical trials on medicinal products for human use

• Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of individuals with regard to the processing of personal data and on the free movement of such data

• Council Directive 83/570/EEC of 26 October 1983 amending Directives 65/65/EEC,75/318/EEC and 75/319/EEC on the approximation laid down by law, regulation or administrative action relating to proprietary medicinal products

• Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions

• Directive 90/219/EEC of 23 April 1990 on the contained use of genetically modified micro-organisms

• Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC

• Helsinki Declaration in its latest version

• Convention of the Council of Europe on Human Rights and Biomedicine signed in Oviedo on 4 April 1997, and the Additional Protocol on the Prohibition of Cloning Human Beings signed in Paris on 12 January 1998

• UN Convention on the Rights of the Child

• Universal Declaration on the human genome and human rights adopted by UNESCO

Human embryonic stem cells: N/A: no stem cells are employed in this study. Identify the countries where research will be undertaken and which ethical committees and regulatory organisations will need to be approached during the life of the project. France, Germany, Greece, Switzerland, Spain

FMT-XCT 201792

58

B5. Consideration of gender aspects This group is highly sensitive in issues of gender equality and already employs several women scientists and staff. While naturally all hiring and project assignment decisions will categorically contain no discrimination against gender, the group further realizes that there is an imbalance in the representation of women in research and science. To counteract this imbalance, all hiring announcements will contain explicit wording to promote women and minority participation. Correspondingly all hiring committees or decision makers will be similarly advised to make particular efforts to welcome underrepresented groups into applying and into the selection process. We further propose affirmative action to the extent that two applications are otherwise equal, to select the gender that balances better gender issues. However, such measures might not be sufficient to compensate for differences in family situation of researchers. We are planning specific measures, including creating part-time job opportunities within the project, allowing parents (both woman and man) with young children to balance better family and work duties. Training and dissemination activities will follow the same rules; whereas no selection will be based on gender, affirmative action towards selecting gender that balances gender representation in the consortium. The proposal co-ordinator Prof. Ntziachristos has received training in balancing gender and minority participation, as part of training courses in Massachusetts General Hospital and will be available to further advice and ensure that such measures are abundant in the overall group’ decision making. Action plans to ensure gender equality will actively happen in all executive committee meetings by explicitly discussing issues of balancing gender participation and describing the actions taken to ensure such steps by all participants. In addition action will be taken to encourage project leaders to discuss with human resources representatives of their institution on issues of optimally managing work and private life and the group. The group is further prepared to seek the help of social workers on issues of balancing gender-related private and work life in order to ensure a particular stimulating and facilitating work environment independent of gender, by considering special needs that exist among different members of the group, not only gender-wise but more generally as well.