An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Summary

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Abstract

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.

Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.

Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.

Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.

The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

Introduction

Glioblastoma multiforme appears as the most aggressive form of brain tumor in adults with a median survival of 12 months and a 5-years survival rate of 5%. Clinical management relies on surgery, radiotherapy and chemotherapy often used in combination. However, the effects of these treatments remain palliative^1-3.

Up to now, most of neuro-oncology studies rely on techniques that are only able to provide a static view and performed on large cohorts of tumor bearing animals sacrificed at different time-points (see for example^4,5). The recent development of follow-up methods based on intravital imaging allows studying glio....

Protocol

All experimental procedures were performed in accordance with the French legislation and in compliance with the European Community Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. The research on animals was authorized by the Direction Départementale des Services Vétérinaires des Bouches-du-Rhône (license D-13-055-21) and approved by the ethical committee of Provence Cote d'Azur n°14 (Project 87-04122012).

1. Spheroids Preparation

1. Preparation of the agarose-coated Petri dishes
   1. Mix 1 g of agarose with 100 ml of cell culture ....

Results

Once the surgical protocol is performed (Figure 1), animals can be observed by means of fluorescent microscopy over weeks until sacrifice. An inflammatory reaction may be observed after the surgery that disappears within one or two weeks. Tumor growth can be observed by various microscopy techniques including fluorescent macroscopy and two-photon microscopy (Figure 2). Example images depicted here were realized on a fluorescence macroscope and a two-photon microscope coupled to a femtose.......

Discussion

This approach allows the use of optical imaging methods to monitor over days and weeks the growth of an orthotopically implanted glioma. The same animal can subsequently be subjected to virtually any brain imaging modality during the course of the pathology; yet the two-photon microscopy specific preparation offers the unique opportunity to achieve subcellular resolution inside the brain of the living animal. Our protocol presents the advantage to enforce the tumor growth into the cerebral parenchyma rather than extra-du.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drill	Dremel (Germany)	398	any high quality surgical bone drill would suffice
Drill burr (#1/4 Carbide Round Burr)	World Precision Instruments (USA)	501860 (#1/4)	also sold by Harvard Apparatus
Tissue scissors	World Precision Instruments (USA)	14395
Dumont tweezers M5S	World Precision Instruments (USA)	501764
Dental cement	GACD (USA)	12-565 & 12-568
Cyanoacrylate	Eleco-EFD (France)	Cyanolit 201
Glass capillaries without filament	Clark Electromedical Instruments (UK)	GC100-15
Microliter syringe (25 µl)	Hamilton (USA)	702
Micromanipulator	World Precision Instruments (USA)	Kite-R
T derivation (3-way stopcock - Luer lock)	World Precision Instruments (USA)	14035-10
Stereotactic frame (mouse adaptor)	World Precision Instruments (USA)	502063
Glass coverslips	Warner Instruments (USA)	CS-5R (64-0700)
Cross-linked dextran gel (Sephadex) G50 Coarse 100-300 µm beads	Available from various suppliers including Sigma (Germany)
Eye ointment	TVM (France)	Ocry-gel
Fluorescence macroscope	Leica MZFLIII (Germany)		also sold by other companies
Two-photon microscope	Zeiss LSM 7MP (Germany)		also sold by other companies (Nikon, …)
Infrared tunable femtosecond laser (Maï-Taï)	Spectra Physics (USA)		also sold by other companies