Analysis of tumor cell migration on electrospun nanofibers ... of tumor cell... · PDF...

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Transcript of Analysis of tumor cell migration on electrospun nanofibers ... of tumor cell... · PDF...

  • 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--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 M, D).

    E) Cells migrating on plastic werestopped at low concentrations ofcytochalasin-D (0.2 M), 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).

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