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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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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Materials

NameCompanyCatalog NumberComments
(3-aminopropyl) triethoxysilaneSigma440140-100MLThe amino group is useful for the bioconjugation of mPEG-SVA
96-well round-bottom plateSarstedt2582624Used to prepare spheroids
AccutaseGibcoA11105-01Stored at -20 °C (long-term) or 4 °C (short-term), sphere dissociation enzyme
B27Gibco12587Stored at -20 °C, defrost before use
Basic Fibroblast Growth FactorPeprotech100-18BStored at -20 °C, defrost before use
Countess Cell Counting ChamberSlidesInvitrogenC10283Used to cell counting
CoverslipsMarienfeld111580Cell culture substrate
Dessicator cartridgesSigmaZ363456-6EAUsed to reduce mosture during (3-aminopropyl) triethoxysilane treatment
DPBS 10xPan BiotechP04-53-500Stored at 4 °C
Fiji software, MTrack2 macroImageJUsed to analyze pictures
Flask 75 cm²Falcon10497302
HBSSSigmaH8264-500ML
Heparin sodiumSigmaH3149-100KUStored at 4 °C
Laminin114956-81-9Promotes neuronal adhesion
Leonardo softwareloading of envisioned micropatterns
MetaMorph Software Molecular Devices LLCNAMicroscopy automation software
MethylcelluloseSigmaM0512Diluted in NBM for a 2% final concentration
Neurobasal mediumGibco21103-049Stored at 4 °C
Nikon TiE (S Fluor, 20x/0.75 NA)inverted microscope equipped with a motorized stage 
Penicillin - StreptomycinGibco15140-122Stored at 4 °C
PLPPAlveolePLPPclassic_1mlPhotoinitiator used to degrade the PEG brush
Poly(ethylene glycol)-Succinimidyl Valerate (mPEG-SVA)Laysan BioVA-PEG-VA-5000-5gUsed as an anti-fouling coating
PRIMOAlveolePRIMO1Digital micromirror device (DMD)-based UV projection apparatus
Trypan blue 0.4%ThermoFisherT10282Used for cell counting
Trypsin-EDTASigmaT4049-100MLUsed to detach adherent cells

References

  1. Shergalis, A., Bankhead, A., Luesakul, U., Muangsin, N., Neamati, N. Current challenges and opportunities in treating glioblastoma. Pharmacology Reviews. 70, 412-445 (2018).
  2. Scherer, H. J. The forms of growth in gliomas and the....

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