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* 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 ....
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 .......
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.......
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.......
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).
....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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