Circulating Glioma Model for Chimeric Antigen Receptor T-Cell Therapy Investigations

Summary

The present protocol establishes a circulating glioma model by the lateral ventricle injection to investigate the therapeutic effect of chimeric antigen receptor T-cell therapy in vivo.

Abstract

Circulating tumor cells in cerebrospinal fluid (CSF) are a significant factor in tumor recurrence and intracranial metastasis in glioma. Many studies are attempting to identify effective strategies to target and eliminate the circulating tumor in CSF. Chimeric antigen receptor (CAR) T-cell therapy has been frequently used for various circulating tumors. However, research is limited due to the lack of a suitable circulating glioma model. Here, a circulating glioma model was developed and applied in CAR T-cell therapy investigations. This model was constructed using the lateral ventricle injection, which is widely used in the local treatment of intracranial disease. The glioma cells were injected into the lateral ventricle to form the intracranial primary tumor. The tumors that were shed from the lateral ventricle were used to indicate the circulating tumor in CSF. The model is easy to build and can be applied in various studies on the CAR T-cell therapy of circulating glioma. As a result, the circulating glioma model provides a unique glioma metastasis model for effective CAR T-cell therapy studies of the circulating tumor.

Introduction

Glioma is the most common primary malignant brain tumor in adults^1,2. Current treatments, including surgery, radiotherapy, and chemotherapy, show limited efficacy, with a median survival of only 14-15 months^3,4,5. Circulating glioma cells in CSF play a crucial role in tumor recurrence and intracranial metastasis^6,7. Hence, there is an urgent need to develop more effective treatment strategies to eliminate the circulating glioma cells.

Protocol

The animal experiments in this study were all approved and supervised by the Institutional Review Board and the Animal Ethics Committee of Nanjing Medical University (IACUC-1905048). C57BL/6J female mice, aged 6-8 weeks old, were used for the present study. The reagents and the equipment used are listed in the Table of Materials.

1. Animal preparation

1. House the mice in a barrier, 12 h/12 h light/dark cycle, ambient temperature of 24 °C, and relative humidity of 50% cage.
2. Weigh the mice, and anesthetize them (following institutionally approved protocols) with an intraperitoneal injection of 50 mg/k....

Results

The schematic diagram of the circulating glioma model for CAR-T therapy is shown in Figure 1. CAR-T cells were injected into the lateral ventricle using a microsyringe to demonstrate their therapeutic effect. This method allows direct delivery of the CAR-T cells to the brain, targeting the circulating glioma cells. The injection procedure was performed on day 11, following the implantation of GL261 cells into the mouse lateral ventricle. At various time point.......

Discussion

The circulating glioma model established in this study using lateral ventricle injection provides a significant advancement, offering several advantages over existing models. The lateral ventricle injection technique is highly reproducible, enabling consistent tumor modeling in a relatively short time frame. This makes it a valuable tool for researchers seeking to investigate tumor cell dissemination into the CSF and assess the effectiveness of therapies, such as CAR-T cells. Unlike traditional intravenous CAR-T infusion.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absorbable surgical suture	Shanghai Pudong Jinhuan Medical Products Co.,Ltd	R611
CAR-T	Creative Biolabs
D-Luciferin potassium salt	MCE	HY-12591B
GL261-Luc-Gfp	Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd.	ZQ0932
In vivo bioluminescent imaging system	Tanon	Tanon ABL X6
Laboratory animal shaver	Beyotime Biotechnology	FS600
Mice	Animal Core Facility of Nanjing Medical University
Microinjection pump	RWD	R462
Microsyringe	Hamilton	87943
Mini cranial drill	RWD	78001
Pentobarbital sodium	ChemSrc	57-33-0
Stereotaxic apparatus	RWD	68043
Xylazine	ChemSrc	7361-61-7