Analysis of tumor cell migration on electrospun nanofibers ... of tumor cell... · and cell...
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THE OHIO STATE UNIVERSITY COMPREHENSIVE CANCER CENTER - ARTHUR G. JAMES CANCER HOSPITAL AND RICHARD J. SOLOVE RESEARCH INSTITUTE Analysis of tumor cell migration on electrospun nanofibers identifies STAT3 as a pro-migratory target in gliomas †Jessica De Jesus 1,2 , †Paula Agudelo 2 , S. Williams 3 , M.O. Nowicki 3 , P. K. Li 4 , E.A. Chiocca 3 , J. Johnson 1 , J. J. Lannutti 1,5 , S. E. Lawler 3 and M.S. Viapiano 2,3 A hallmark of malignant gliomas is their ability to disperse through neural tissue, leading to long-term failure of all current therapies. Most assays to study glioma cell motility follow models of invasion developed for epithelial cancers. However, glioma cells disperse preferentially along unique anatomical pathways in the CNS. Here, we have set up and characterized the migration of glioma cells on nanofiber- based scaffolds, which reproduce, in part, the neural topography and lead glioma cells to exhibit morphology and motile behavior observed in neural tissue. Glioma cells migrating on aligned fibers show a specific upregulation of genes associated to the JAK-STAT pathway. Accordingly, STAT3 activation was detected in cells and specimens cultured in aligned fibers, but not random fibers, and cell migration along aligned fibers was significantly reduced with STAT3 inhibitors. This model can be used to test anti-invasive drugs and to investigate pro-invasive mechanisms that may not be readily identified with other in vitro models. Summary U251 glioma cells cultured on random fibers exhibit morphology similar to cells cultured on conventional plastic surfaces, but cells grown on aligned fibers retained an elongated morphology and moved along the fiber axis. - Nanofiber Solutions made the nanofiber-based scaffolds which were produced from poly-ε-caprolactone electrospun onto multiwell culture dishes, forming random or aligned fibers. - Human glioma cells were cultured in suspension to form aggregates, stained with membrane-permeable dyes, and imaged on the nanofibers over a 24-h period. Tumor specimens were obtained from glioma-initiating cells implanted intracranially in nude mice. - ImageJ software was used to calculate the maximum distance of migration of the cells cultured on nanofibers. - Pharmacological inhibitors were used to test whether migration of glioma cells on fibers was dependent on acto- myosin contraction. - Expression of STAT3 and STAT3 gene signature were assayed by quantitative RT-PCR and Western blotting. Targeting of STAT3 was achieved with pharmacological inhibitors and siRNA. U251 aggregates treated overnight with the myosin-II inhibitor blebbistattin showed signficantly reduced dispersion on aligned fibers (A and B). Cells were much less sensitive to myosin-II inhibition on a classical migration model (Transwell assay) (C), and were not affected by blebbistatin on a conventional scratch assay (D). A) A Nanofiber Solutions 24-well plate with 0.19 mm-thick clear polystyrene backing plate, coated with electrospun nanofibers. The double-headed arrow indicates the direction of fiber alignment. B) Scanning electron microscopy of random (top) and aligned (bottom) electrospun fibers. Methodology Nanofiber-based scaffolds Migration on nanofibers depends on myosin-II but not on actin stress fibers Migration on nanofibers correlates with a JAK/STAT signature A and B) Representative images of U251 cell aggregates on aligned fibers stained with membrane cell tracker (green) and with the actin-binding protein phalloidin (red). C and D) Cell stretching and migration on nanofibers is similar in control conditions (DMSO, C) or in presence of the actin-stress fiber inhibitor cytochalasin-D (2 μM, D). E) Cells migrating on plastic were stopped at low concentrations of cytochalasin-D (0.2 μM), while they were only affected by 10-fold higher concentrations of the drug on nanofibers and effectively stopped only at 100-fold higher concentrations. Inhibition of STAT3 reduces cell migration on nanofibers STAT3 inhibition reduces dispersion of glioma explants on nanofibers Above: GFP-expressing intracranial tumors derived from GBM9 glioma- initiating cells were dissected, cleaned, and cultured on aligned nanofibers. A) Representative image of a tumor specimen showing adjacent tissue and dispersed cells. B) Fluorescence picture of the border from a tumor specimen, showing the detail of dispersed cells. Right: Tumor specimens were cultured as above in presence of Stat-3 inhibitors. Cell dispersion out of the tumor core was significantly inhibited (LLL12) or abolished (stattic). ** p<0.01, *** p<0.001 by two-way ANOVA. Supported by: • NIH ARRA/SBIR (GRT00021234) • NSF STTR Phase I (IIP-1010406) • OSU Center for Clinical and Translational Science • OSU Comprehensive Cancer Center 1 Nanofiber Solutions, LLC www.nanofibersolutions.com 2 Center for Molecular Neurobiology, OSU College of Medicine 3 Department of Neurological Surgery, OSU College of Medicine 4 Division of Medicinal Chemistry, OSU College of Pharmacy 5 Department of Materials Science and Engineering, OSU College of Engineering † These authors contributed equally to this work. A) Inhibition of STAT3 using two different inhibitory compounds reduced the dispersion of glioma cells (U87, U251) and glioblastoma- derived stem cells (GBM8, GBM9) on aligned nanofibers. Inhibitors tested included Stattic (0- 2 μM) and the OSU-synthesized compound LLL12 (0-2 μM). B) Western blot showing the effect of LLL12 on cells cultured on aligned fibers. Overnight incubation with LLL12 reduced STAT3 phospho- rylation in a dose-dependent manner. C) Inhibition of STAT3 phosphorylation with LLL12 or Stattic did not affect cell motility in the conventional scratch assay. Quantitative RT-PCR analysis showed selective upregulation of JAK- STAT associated genes on cells migrating on aligned fibers.
Transcript of Analysis of tumor cell migration on electrospun nanofibers ... of tumor cell... · and cell...
THE OHIO STATE UNIVERSITY COMPREHENSIVE CANCER CENTER - ARTHUR G. JAMES CANCER HOSPITAL AND RICHARD J. SOLOVE RESEARCH INSTITUTE
Analysis of tumor cell migration on electrospun nanofibers identifies STAT3 as a pro-migratory target in gliomas
†Jessica De Jesus1,2, †Paula Agudelo2, S. Williams3, M.O. Nowicki3, P. K. Li4, E.A. Chiocca3, J. Johnson1, J. J. Lannutti1,5, S. E. Lawler3 and M.S. Viapiano2,3
A hallmark of malignant gliomas is their ability to dispersethrough neural tissue, leading to long-term failure of allcurrent therapies. Most assays to study glioma cell motilityfollow models of invasion developed for epithelial cancers.However, glioma cells disperse preferentially along uniqueanatomical pathways in the CNS. Here, we have set up andcharacterized the migration of glioma cells on nanofiber-based scaffolds, which reproduce, in part, the neuraltopography and lead glioma cells to exhibit morphology andmotile behavior observed in neural tissue.
Glioma cells migrating on aligned fibers show a specificupregulation of genes associated to the JAK-STAT pathway.Accordingly, STAT3 activation was detected in cells andspecimens cultured in aligned fibers, but not random fibers,and cell migration along aligned fibers was significantlyreduced with STAT3 inhibitors.
This model can be used to test anti-invasive drugs and toinvestigate pro-invasive mechanisms that may not bereadily identified with other in vitro models.
Summary
U251 glioma cells cultured on random fibersexhibit morphology similar to cells cultured onconventional plastic surfaces, but cells grownon aligned fibers retained an elongatedmorphology and moved along the fiber axis.
- Nanofiber Solutions made the nanofiber-based scaffoldswhich were produced from poly-ε-caprolactone electrospunonto multiwell culture dishes, forming random or alignedfibers.- Human glioma cells were cultured in suspension to formaggregates, stained with membrane-permeable dyes, andimaged on the nanofibers over a 24-h period. Tumorspecimens were obtained from glioma-initiating cellsimplanted intracranially in nude mice.- ImageJ software was used to calculate the maximumdistance of migration of the cells cultured on nanofibers.- Pharmacological inhibitors were used to test whethermigration of glioma cells on fibers was dependent on acto-myosin contraction.- Expression of STAT3 and STAT3 gene signature wereassayed by quantitative RT-PCR and Western blotting.Targeting of STAT3 was achieved with pharmacologicalinhibitors and siRNA.
U251 aggregates treated overnight with the myosin-II inhibitor blebbistattinshowed signficantly reduced dispersion on aligned fibers (A and B).
Cells were much less sensitive to myosin-II inhibition on a classical migration model (Transwellassay) (C), and were not affected by blebbistatin on a conventional scratch assay (D).
A) A Nanofiber Solutions 24-well plate with0.19 mm-thick clear polystyrene backingplate, coated with electrospun nanofibers.The double-headed arrow indicates thedirection of fiber alignment. B) Scanningelectron microscopy of random (top) andaligned (bottom) electrospun fibers.
Methodology
Nanofiber-based scaffolds
Migration on nanofibers depends on myosin-II but not on actin stress fibers
Migration on nanofibers correlates with a JAK/STAT signature
A and B) Representative images of U251cell aggregates on aligned fibersstained with membrane cell tracker(green) and with the actin-bindingprotein phalloidin (red).
C and D) Cell stretching and migrationon nanofibers is similar in controlconditions (DMSO, C) or in presenceof the actin-stress fiber inhibitorcytochalasin-D (2 μM, D).
E) Cells migrating on plastic werestopped at low concentrations ofcytochalasin-D (0.2 μM), while theywere only affected by 10-fold higherconcentrations of the drug onnanofibers and effectively stoppedonly at 100-fold higherconcentrations.
Inhibition of STAT3 reduces cell migration on nanofibers
STAT3 inhibition reduces dispersion of glioma explants on nanofibers
Above: GFP-expressing intracranial tumors derived from GBM9 glioma-initiating cells were dissected, cleaned, and cultured on alignednanofibers. A) Representative image of a tumor specimen showingadjacent tissue and dispersed cells. B) Fluorescence picture of theborder from a tumor specimen, showing the detail of dispersed cells.
Right:Tumor specimens werecultured as above in presenceof Stat-3 inhibitors. Celldispersion out of the tumorcore was significantly inhibited(LLL12) or abolished (stattic).
** p<0.01, *** p<0.001 bytwo-way ANOVA.
Supported by:• NIH ARRA/SBIR (GRT00021234)• NSF STTR Phase I (IIP-1010406) • OSU Center for Clinical and Translational Science• OSU Comprehensive Cancer Center
1 Nanofiber Solutions, LLC www.nanofibersolutions.com
2 Center for Molecular Neurobiology, OSU College of Medicine
3 Department of Neurological Surgery, OSU College of Medicine
4 Division of Medicinal Chemistry, OSU College of Pharmacy
5 Department of Materials Science and Engineering, OSU College
of Engineering
† These authors contributed equally to this work.
A) Inhibition of STAT3 using twodifferent inhibitory compoundsreduced the dispersion of gliomacells (U87, U251) and glioblastoma-derived stem cells (GBM8, GBM9)on aligned nanofibers.Inhibitors tested included Stattic (0-2 μM) and the OSU-synthesizedcompound LLL12 (0-2 μM).
B) Western blot showing the effectof LLL12 on cells cultured onaligned fibers. Overnight incubationwith LLL12 reduced STAT3 phospho-rylation in a dose-dependentmanner.
C) Inhibition of STAT3phosphorylation with LLL12 orStattic did not affect cell motility inthe conventional scratch assay.
Quantitative RT-PCR analysis showed selective upregulation of JAK-STAT associated genes on cells migrating on aligned fibers.
