In Vivo Glioblastoma Tumor Modeling via Stereotaxic Injection in Mice for Tumor Progression Studies

Summary

Glioblastoma research involves challenges in the understanding of its progression and assessment of the tumor microenvironment. This protocol uses stereotaxic injection of brain cancer cells in mice to establish precise in vivo modeling of brain cancer, enabling detailed studies of tumor biology and therapeutic responses.

Abstract

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently use murine in vivo models to study cancer progression and tumor growth, as these models recapitulate critical aspects of the tumor microenvironment, immune responses, and the appearance of classical GB pathology. Here, we describe a detailed protocol for performing stereotaxic injections to establish GB tumor models in immunocompetent C57BL/6 mice. Stereotaxic surgery enables rapid and precise delivery of GB cells grown as 2D cultures or spheroids into the brain using anatomical landmarks without the need for image guidance. Specifically, GL261 murine cell lines are injected into the striatum, allowing subsequent assessment of behavior and physiology. On day 21 post surgery, animals are euthanized following perfusion and their brains extracted and fixed. Hematoxylin-eosin staining on brain slices enables measurement of tumor area and volume, while immunohistochemistry enables imaging of tumor proliferation markers. This paper provides detailed protocols for a reliable approach to generate in vivo GB models for studying tumor growth and cancer progression. Additionally, this method is adaptable for other GB cell lines, transgenic mice, or patient-derived xenografts broadening its applicability for GB research.

Introduction

GB is a highly aggressive brain tumor and the most frequent malignant neoplasm of the central nervous system, distinguished by its rapid proliferation and deep infiltration into adjacent brain regions¹. Despite the implementation of standard therapeutic approaches, including surgery, radiotherapy, and chemotherapy, the prognosis for GB remains dismal². Patients have a median survival of less than 2 years³, with limited advancements in treatments or therapeutic options over the past decades⁴. This highlights the need for a more comprehensive understanding of the molecul....

Protocol

All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal ethics protocol for the use of animals in this study was approved by the Committee on the Ethics of Animal Experiments of the University of Chile. This protocol used 8-12-week-old C57BL/6 immunocompetent mice housed at 23 ± 2 °C with a humidity of 35 ± 5% under a 12:12 h light-dark cycle with access to food and water ad libitum. This section outlines the protocol for establishing the in vivo GB model. Figure 1 provides an overview of the experimental wor....

Results

The protocol provided here successfully establishes an in vivo GB model evidenced by the presence of unilateral motor problems and the presence of highly proliferative and infiltrative tumors. The following representative results confirm the effectiveness of the method and highlight the possible sources of variability.

GB tumor growth is associated with an increase in the monitoring score during the last 4-5 days with no evidence of major weight loss (Figure 2A

Discussion

GB remains highly lethal and resistant to treatment due to its ability to evade chemotherapy and radiotherapy¹⁹. The lack of effective therapies for GB has sustained interest in developing new treatments and uncovering new pathogenic pathways. This underscores the need for animal models that accurately reproduce the characteristics of these brain tumors, particularly their treatment resistance, tissue invasion, and patient anticancer immune responses. Therefore, it is crucial to use models that mi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3,3′-Diaminobenzidine (DAB)	Merck	D5905
ABC-HRP	VECTOR	PK-4000
Alcoholic eosin Y	Merck	102439
Automatic cell counter Countess II	Thermofisher Scientific	AMQAF1000
B-27 supplement 50x	Thermofisher Scientific	17504044
Biotinylated Goat Anti-Rabbit IgG (H+L)	Santa Cruz	(BA-1000-1.5)
C57BL/6 mice	Jackson Lab	664
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye	Thermofisher Scientific	C10340
Coverlips	Fisher Scientific	12323128
DMEM/F-12 supplement	Gibco	10565018
Dulbecco's modified Eagle's medium (DMEM)	Gibco	1965092
EdU (5-ethynyl-2′-deoxyuridine)	Thermofisher Scientific	E10187
Entellan	Merck	107961
Gel boost	Clear	72-04-5022
GL261	N/A	N/A	Gift from Dr.Eric Chevet
Glass slides	Fisher Scientific	11562203
Harris hematoxilyn	Merck	HHS32
Heating mantle	N/A	N/A
hiFBS	Gibco	A5209501
Hydrochloric acid	Merck	01-01-7646
Hydrogen peroxide	DifemCare	2402069
ImageJ software
Isofluorane	Lunan Pharma	N/A
Leica CM1510s cryostat	RANKIN	CM1510S
L-Glutamine	Gibco	25030081
Low-Profile disposable blades	N/A	N/A
Mayer’s hematoxylin	Merck	51275
Microsrynge	Hamilton	87930
Mixantin Plus	Drag Pharma	N/A
Mouse Recombinant  Epidermal Growth Factor (EGF)	PeproTech	315-09-500UG
Mouse recombinant Fibroblast Growth Factor (FGF)	PeproTech	450-33-50UG
N2 supplement 100x	Thermofisher Scientific	A1370701
Naxpet Ketoprophen	Drag Pharma	N/A
Nicotears gel	Nicolich	N/A
Non essential aminoacids (MEM-NEA)	Gibco	11140050
Paraformaldehyde	Merck	30525-89-4
PBS	N/A	N/A
Penicilin/streptomycil	Gibco	15140122
Positive charged slides	Bioland	BNAO1
ProLong Gold Antifade Mountant	Thermofisher Scientific	P36930
QuPath software
Rabbit Anti-Ki67	Abcam	ab15580
Scissors	N/A	N/A
Sodium Azide	Merck	S2002
Sodium tetraborate	Merck	221732
Stereotaxic frame	Stoelting	51730
Surgical blades N°15
Surgical scalpel	ChannelMED
Sutures	TAGUM	N/A
Syringes 1 mL and 27G Needle	Cranberry	AAJECR01
Tissue-TEK O.C.T	Sakura Finetek	25608-930
Triton X-100	Merck	9036-19-5
Trypsin/EDTA	Gibco	25200056
Tweezers	N/A	N/A
V Fraction Bovine Serum Albumin	Merck	0604-29-8
Veterinary dremel
Xylene	Merck	95-47-6