Dissecting the InterSubstrate Navigation of Migra

Authors: Sonja Mertsch Patrick Oellers Michael Wendling Werner Stracke Solon Thanos

Publish Date: 2013/02/24

Volume: 48, Issue: 1, Pages: 169-179

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Abstract

A hallmark of gliomas is the growth and migration of cells over long distances within the brain and proliferation within selected niches indicating that the migrating cells navigate between complex substrates We demonstrate in the present study a differential preference for migration that depends on Rhoassociated coil kinase ROCK signaling using the alternating Bonhoeffer stripe assay Membrane fractions from nonmyelinated and myelinated brain areas from female rats purified myelin also from female rats and commercial extracellular matrix were used as substrates with each substrate being tested against the others The human tumor cell lines exhibited a clear preference for extracellular matrix over all other substrates and for myelinated over nonmyelinated tissue ROCK signaling was different when cells were cultured on either substrate The ROCK inhibitor Y27632 significantly attenuated and neutralized the preference for extracellular matrix and myelin indicating that ROCK controls the substrate selectivity The findings of this study pave the way for navigationtargeted therapeuticsGlioblastoma multiforme is the most common primary brain tumor with an incidence of 3–5 per 100000 population 1 The prognosis has only slightly improved recently with a current median survival of 146 months today 2 3 One of the first hallmarks of glioblastoma is nonresectable diffuse invasion into healthy brain parenchyma which leads to recurrences in virtually every patient even when surgery and chemo/radiotherapy are applied 2 3 4 A second hallmark is the longdistance migration from the primary tumor often along white matter tracts indicating a high affinity for substrates constituting white matter 4 5 6 Given the densely packed nature of cerebral tissue it is difficult to distinguish between the different affinities of tumor cells toward particular components such as myelin neuronal tissue extracellular matrix ECM or capillaries A migrating glioma cell may simultaneously encounter gray matter white matter and ECM and exhibit different affinities to these componentsCommonly used in vitro migration models such as the radial migration assay wound healing assay and Boyden chamber assay use components of the ECM as migration substrates 7 8 9 Despite many achievements in the field of cell migration using these models the resulting improved understanding of glioma migration 4 has had only a minor impact on therapy These models have revealed that Rhoassociated coil kinases ROCKs 1 and 2 ROCK1 and ROCK2 respectively are important molecules controlling glioma migration that act downstream of the small GTPase RhoA and functions by phosphorylating proteins such as myosin II focal adhesion kinase LIM kinase myosin light chain phosphatase and myosin light chain kinase These various ROCKmediated phosphorylation events organize the actin–myosin cytoskeleton 9 10 ROCKS are important regulators of microfilament structure and they have been reported to both inhibit and promote cell migration in different cancer cells and in various migration assays 10 11 12 In a brain slice assay the inhibition of ROCK by fasudil immobilize glioma cells in intracranial xenograft models preventing glioma progression 13 Thus the migration modes of gliomas vary within the complex brain environment with some being ROCK dependent and others being ROCK independent Live cell imaging of different substrates remains a challenge regarding the tradeoff between in vivo complexity and spatiotemporal resolution of cell migrationTo generate such a true decisiontaking situation for cells facing and crossing the interface between two substrates we investigated migration preferences using a modification of the alternating stripe assay developed in the Bonhoeffer laboratory for studying axonal guidance henceforth referred to as the Bonhoeffer assay to study glioma cell migration 14 15 This device presents two alternating substrates to the cultured cells We used five different substrates that were tested in pairs in every possible combinationHuman glioblastoma cell lines U87MG A172 D54MG and 86HG39 all cell lines are kind gifts of V Senner Institute of Neuropathology Muenster Germany were cultured in Dulbecco’s modified Eagle’s minimal essential medium DMEM containing 10  fetal calf serum 100 U/mL penicillin and 100 μg/mL streptomycin sulfate at 37 °C in an incubator with a 5  CO2 atmosphere all cell culture reagents were purchased from PAA Linz AustriaSchematic diagrams showing the experimental setup for creating alternating membrane stripes from different tissues and coculturing with U87MG cells a Rat b chick c extracellular matrix Biomatrix d purified myelin from rat cortex e immunoblot to test the content of myelin using CNPase in different substrates rat retina rat brain chicken retina and purified myelin used for the stipe assay f corresponding housekeeping control calnexin g stripe assay setup with cells h coculture of stripe assay with cells i depiction of a stochastic preference in migration with a 5050 distribution j depiction of a 9010 preferential distribution of cellsAs stripe components we chose homogenized rat retina to represent CNS tissue which is easy to isolate and free of myelin and oligodendrocytes to represent gray matter For white matter we chose embryonic chick retina which in contrast to rat retina contains myelin Fig 1e f To reveal whether purified myelin has effects which differ from those of homogenized white or gray matter we also isolated myelin from perinatal rat brains Fig 1e f For extracellular matrix we chose the commercially available Biomatrix Serva The retinas of perinatal rats postnatal day 10 and White Leghorn chicken embryos embryonic day 10 were explanted and collected into icecold homogenization buffer HB containing a protease inhibitor cocktail Complete EDTA free Roche Basel Switzerland HB+ The tissue was homogenized first by titration through a 1000μL pipette tip and then through a G27× 075in needle The cell membranes were separated using sucrose gradient centrifugation 150 μL of a 5  v/v sucrose solution was pipetted on top of 350 μL of a 50  w/w sucrose solution and finalized with 800 μL of tissue homogenate These setups were centrifuged at 5525 rcf for 7 min at 4 °C and the white rings between the sucrose layers were collected in 1000 μL of precooled phosphatebuffered saline PBS pH 74 containing the protease inhibitor cocktail henceforth referred to as PBS+ In a second centrifugation 5525 rcf for 7 min at 4 °C the remaining sucrose was washed out excess liquid was removed and the pellets were resuspended in 1000 μL of PBS+ and stored in liquid N2 Myelin fragments were prepared from white matter tracts obtained from rat brains at postnatal day 10 as described above Biomatrix EHS solution a compound containing basement membrane components such as laminin type IV collagen entactin and heparan sulfate was also used

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