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Cancer Research

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

Published: February 24th, 2021

DOI:

10.3791/62213

1University Bordeaux, INSERM, LAMC, U1029, 33600, Pessac, France, 2Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France, 3Alvéole, Bordeaux, France, 4University Bordeaux, CNRS, IBGC, UMR5095, 33000, Bordeaux, France
* These authors contributed equally

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 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.

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 ....

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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 .......

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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.......

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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.......

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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).

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Name Company Catalog Number Comments
(3-aminopropyl) triethoxysilane Sigma 440140-100ML The amino group is useful for the bioconjugation of mPEG-SVA
96-well round-bottom plate Sarstedt 2582624 Used to prepare spheroids
Accutase Gibco A11105-01 Stored at -20 °C (long-term) or 4 °C (short-term), sphere dissociation enzyme
B27 Gibco 12587 Stored at -20 °C, defrost before use
Basic Fibroblast Growth Factor Peprotech 100-18B Stored at -20 °C, defrost before use
Countess Cell Counting ChamberSlides Invitrogen C10283 Used to cell counting
Coverslips Marienfeld 111580 Cell culture substrate
Dessicator cartridges Sigma Z363456-6EA Used to reduce mosture during (3-aminopropyl) triethoxysilane treatment
DPBS 10x Pan Biotech P04-53-500 Stored at 4 °C
Fiji software, MTrack2 macro ImageJ Used to analyze pictures
Flask 75 cm² Falcon 10497302
HBSS Sigma H8264-500ML
Heparin sodium Sigma H3149-100KU Stored at 4 °C
Laminin 114956-81-9 Promotes neuronal adhesion
Leonardo software loading of envisioned micropatterns
MetaMorph Software  Molecular Devices LLC NA Microscopy automation software
Methylcellulose Sigma M0512 Diluted in NBM for a 2% final concentration
Neurobasal medium Gibco 21103-049 Stored at 4 °C
Nikon TiE (S Fluor, 20x/0.75 NA) inverted microscope equipped with a motorized stage 
Penicillin - Streptomycin Gibco 15140-122 Stored at 4 °C
PLPP Alveole PLPPclassic_1ml Photoinitiator used to degrade the PEG brush
Poly(ethylene glycol)-Succinimidyl Valerate (mPEG-SVA) Laysan Bio VA-PEG-VA-5000-5g Used as an anti-fouling coating
PRIMO Alveole PRIMO1 Digital micromirror device (DMD)-based UV projection apparatus
Trypan blue 0.4% ThermoFisher T10282 Used for cell counting
Trypsin-EDTA Sigma T4049-100ML Used to detach adherent cells

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