Name: co-culture of glioblastoma stem-like cells on patterned neurons to study migration and cellular interactions
Uploaded: 2021-02-24T14:30:00
Description: Watch this Scientific Journal Video about Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions at JoVE.com

This work was supported by Fondation ARC 2020, Ligue Contre le Cancer (Comite de la Gironde), ARTC, Plan Cancer 2021, INCA PLBIO. Alveole is supported by Agence Nationale de la Recherche (Grant Labex BRAIN ANR-10-LABX-43). Joris Guyon is a recipient of fellowship from the Toulouse University Hospital (CHU Toulouse).

Informed written consent was obtained from all patients (from the Haukeland Hospital, Bergen, Norway, according to local ethics committee regulations). This protocol follows the guidelines of Bordeaux University human and animal research ethics committees. Pregnant rats were housed and treated in the animal facility of Bordeaux University. Euthanasia of an E18-timed pregnant rat was performed by using CO2. All animal procedures have been done according to the institutional guidelines and approved by the local ...

<ol>
	<li>Shergalis, A., Bankhead, A., Luesakul, U., Muangsin, N., Neamati, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+challenges+and+opportunities+in+treating+glioblastoma.">Current challenges and opportunities in treating glioblastoma.</a> Pharmacology Reviews. 70, 412-445 (2018).</li><li>Scherer, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forms+of+growth+in+gliomas+and+their+practical+significance.">The forms of growth in gliomas and their practical significance.</a> Brain. 63, 1-35 (1940).</li><li>Zagzag, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-+and+vascular+endothelial+growth+factor-induced+stromal+cell-derived+factor-1&#945;/CXCR4+expression+in+glioblastomas.">Hypoxia- and vascular endothelial growth factor-induced stromal cell-derived factor-1&#945;/CXCR4 expression in glioblastomas.</a> American Journal of Pathology. 173, 545-560 (2008).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+white+matter+tracts+by+glioma+stem+cells+is+regulated+by+a+NOTCH1-SOX2+positive-feedback+loop.">Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.</a> Nature Neurosciences. 22, 91-105 (2019).</li><li>Venkataramani, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+synaptic+input+to+glioma+cells+drives+brain+tumour+progression.">Glutamatergic synaptic input to glioma cells drives brain tumour progression.</a> Nature. 573, 532-538 (2019).</li><li>Venkatesh, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+synaptic+integration+of+glioma+into+neural+circuits.">Electrical and synaptic integration of glioma into neural circuits.</a> Nature. 573, 539-545 (2019).</li><li>Boy&#233;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CXCR3/LRP1+cross-talk+in+the+invasion+of+primary+brain+tumors.">The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.</a> Nature Communications. 8, 1571 (2017).</li><li>Daubon, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+complex+role+of+thrombospondin-1+in+glioblastoma+development.">Deciphering the complex role of thrombospondin-1 in glioblastoma development.</a> Nature Communications. 10, 1146 (2019).</li><li>Gritsenko, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p120-catenin-dependent+collective+brain+infiltration+by+glioma+cell+networks.">p120-catenin-dependent collective brain infiltration by glioma cell networks.</a> Nature Cell Biology. 22, 97-107 (2020).</li><li>Guyon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+spheroid+model+for+glioblastoma.">A 3D spheroid model for glioblastoma.</a> Journal of Visualized Experiments: JoVE. (158), (2020).</li><li>Strale, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiprotein+Printing+by+Light?Induced+Molecular+Adsorption.">Multiprotein Printing by Light?Induced Molecular Adsorption.</a> Advanced Materials. , (2015).</li><li>Rao, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+white+matter+tract+topography+using+core-shell+electrospun+nanofibers+to+examine+migration+of+malignant+brain+tumors.">Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors.</a> Biomaterials. 34, 5181-5190 (2013).</li><li>Visweshwaran, S. P., Maritzen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+3D+cellular+chemotaxis+assay+and+analysis+workflow+suitable+for+a+wide+range+of+migrating+cells.">A simple 3D cellular chemotaxis assay and analysis workflow suitable for a wide range of migrating cells.</a> MethodsX. 6, 2807-2821 (2019).</li><li>Qian, H., Sheetz, M. P., Elson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+particle+tracking.+Analysis+of+diffusion+and+flow+in+two-dimensional+systems.">Single particle tracking. Analysis of diffusion and flow in two-dimensional systems.</a> Biophysics Journal. 60, 910-921 (1991).</li><li>Pasturel, A., Strale, P. -. O., Studer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+common+hydrogels+into+3D+cell+culture+templates.">Tailoring common hydrogels into 3D cell culture templates.</a> Advance Healthcare Materials. 9, 2000519 (2020).</li><li>Dolmetsch, R., Geschwind, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+brain+in+a+dish:+the+promise+of+iPSC-derived+neurons.">The human brain in a dish: the promise of iPSC-derived neurons.</a> Cell. 145, 831-834 (2011).</li><li>Clark, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-cultures+with+stem+cell-derived+human+sensory+neurons+reveal+regulators+of+peripheral+myelination.">Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.</a> Brain. 140, 898-913 (2017).</li><li>Linkous, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+patient-derived+glioblastoma+with+cerebral+organoids.">Modeling patient-derived glioblastoma with cerebral organoids.</a> Cell Reports. 26, 3203-3211 (2019).</li><li>Han, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfering+with+long+non-coding+RNA+MIR22HG+processing+inhibits+glioblastoma+progression+through+suppression+of+Wnt/&#946;-catenin+signalling.">Interfering with long non-coding RNA MIR22HG processing inhibits glioblastoma progression through suppression of Wnt/&#946;-catenin signalling.</a> Brain. 143, 512-530 (2020).</li></ol>

Glioblastomas extensively invade the parenchyma by using different modes: co-option of surrounding blood vessels, interstitial invasion, or invasion on WMTs18. This latter mode is not well characterized in the literature because of the difficulty in finding suitable in vitro or in vivo models related to WMT invasion. Here, a simplified model has been proposed in which cultured rodent neurons were patterned on laminin-coated surfaces, and fluorescent GBM stem-like cells were seede...

Primary malignant gliomas, including GBMs, are devastating tumors, with a medium survival rate of 12 to 15 months reported for GBM patients. Current therapy relies on large tumor mass resection and chemotherapy coupled with radiotherapy, which only extends the survival rate by few months. Therapeutic failures are intimately related to poor drug delivery across the blood-brain barrier (BBB) and to invasive growth in perivascular spaces, meninges, and along WMTs1. Perivascular invasion, also called vascular co-option, is a well-studied process, and the molecular mechanisms are beginning to be elucidated; however, the process of GBM cell invasion along WMTs is not well understood. Tumor cells migrate into the healthy brain along Scherer&#39;s secondary structures2. Indeed, almost one century ago, Hans-Joachim Scherer described the invasive routes of GBM, which are now referred to as perineuronal satellitosis, perivascular satellitosis, subpial spread, and invasion along the WMT (Figure 1A).
Some chemokines and their receptors, such as stromal cell-derived factor-1&#945; (SDF1&#945;) and C-X-C motif chemokine receptor 4 (CXCR4), but not vascular endothelial growth factor (VEGF), seem to be implicated in WMT invasion3. More recently, a transcellular NOTCH1-SOX2 axis has been shown to be an important pathway in WMT invasion of GBM cells4. The authors described how GBM stem-like cells invade the brain parenchyma on partially unmyelinated neurons, suggesting the destruction of myelin sheaths by GBM cells. A milestone was reached in 2019 when three articles wereconsecutively published in Nature journal, underlining the role of electrical activity in glioma development5,6. Seminal work by Monje and collaborators shed light on the central role of electric activity in the secretion of neuroligin-3, which promotes glioma development.
Winkler and collaborators described connections between GBM cells (microtubes) being crucial in invasive steps, and lately, interactions between GBM cells and neurons via newly described neuroglioma synapses. Those structures favor glutamatergic stimulation of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors located at the GBM cell membrane, which promotes tumor development and invasion. Tumor cell invasion is a central process in the dissemination of metastases or distant secondary foci, as observed in GBM patients. Several factors have been identified to be important in GBM invasion such as thrombospondin-1, a transforming growth factor beta (TGF&#946;-regulated matricellular protein, or the chemokine receptor CXCR3)7,8.
Here, a simplified biomimetic model has been described for studying GBM invasion, in which neurons are patterned on tracks of laminin, and GBM cells are seeded onto it, as single-cells or as spheroids (Figure 1B). The two experimental settings are aimed at recapitulating invasion on neurons, which is observed in GBM9,10,11. Such models have been developed in the past as aligned nanofiber biomaterials (core-shell electrospinning) that allow studying cell migration by modulating mechanical or chemical properties12. The co-culture model described in this article allows a better understanding of how GBM cells escape on neurons by defining new molecular pathways involved in this process.

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			<td>(3-aminopropyl) triethoxysilane</td>
			<td>Sigma</td>
			<td>440140-100ML</td>
			<td>The amino group is useful for the bioconjugation of mPEG-SVA</td>
		</tr>
		<tr>
			<td>96-well round-bottom plate</td>
			<td>Sarstedt</td>
			<td>2582624</td>
			<td>Used to prepare spheroids</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A11105-01</td>
			<td>Stored at -20 &#176;C (long-term) or 4 &#176;C (short-term), sphere dissociation enzyme</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Countess Cell Counting ChamberSlides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Used to cell counting</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Marienfeld</td>
			<td>111580</td>
			<td>Cell culture substrate</td>
		</tr>
		<tr>
			<td>Dessicator cartridges</td>
			<td>Sigma</td>
			<td>Z363456-6EA</td>
			<td>Used to reduce mosture during (3-aminopropyl) triethoxysilane treatment</td>
		</tr>
		<tr>
			<td>DPBS 10x</td>
			<td>Pan Biotech</td>
			<td>P04-53-500</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fiji software, MTrack2 macro</td>
			<td>ImageJ</td>
			<td></td>
			<td>Used to analyze pictures</td>
		</tr>
		<tr>
			<td>Flask 75 cm&#178;</td>
			<td>Falcon</td>
			<td>10497302</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H8264-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Sigma</td>
			<td>H3149-100KU</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td></td>
			<td>114956-81-9</td>
			<td>Promotes neuronal adhesion</td>
		</tr>
		<tr>
			<td>Leonardo software</td>
			<td></td>
			<td></td>
			<td>loading of envisioned micropatterns</td>
		</tr>
		<tr>
			<td>MetaMorph Software&#160;</td>
			<td>Molecular Devices LLC</td>
			<td>NA</td>
			<td>Microscopy automation software</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>M0512</td>
			<td>Diluted in NBM for a 2% final concentration</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nikon TiE (S Fluor, 20x/0.75 NA)</td>
			<td></td>
			<td></td>
			<td>inverted microscope equipped with a motorized stage&#160;</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PLPP</td>
			<td>Alveole</td>
			<td>PLPPclassic_1ml</td>
			<td>Photoinitiator used to degrade the PEG brush</td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)-Succinimidyl Valerate (mPEG-SVA)</td>
			<td>Laysan Bio</td>
			<td>VA-PEG-VA-5000-5g</td>
			<td>Used as an anti-fouling coating</td>
		</tr>
		<tr>
			<td>PRIMO</td>
			<td>Alveole</td>
			<td>PRIMO1</td>
			<td>Digital micromirror device (DMD)-based UV projection apparatus</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>ThermoFisher</td>
			<td>T10282</td>
			<td>Used for cell counting</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
			<td>Used to detach adherent cells</td>
		</tr>
	</tbody>
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co-culture of glioblastoma stem-like cells on patterned neurons to study migration and cellular interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Patterned neurons co-cultured with fluorescent GBM cells were prepared as described in the protocol section, and tracking experiments were performed. GBM cells quickly modified their shape while migrating on the neurons (Figure 1B: panel 6 and Video 1). Cells migrated along the neuronal extensions, in a random motion (Video 1). Fluorescent GBM cells and non-fluorescent neurons can be easily distinguished, and this allowed the tracking of cel...

Watch this Scientific Journal Video about Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions at JoVE.com

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

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