A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Summary

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.  

Abstract

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Introduction

Recent cancer research has made significant advancements in identifying genetic mutations, molecular changes and possible treatments for a variety of brain tumors. Despite this progress, brain tumors remain one of the top causes of cancer-related mortality for adults and children. Limiting factors in brain tumor research include the restricted availability of primary patient samples and cell lines and the difficulty in replicating the unique and heterogeneous brain microenvironment in accessible experimental systems. For many brain tumors the conditions required to maintain tumor cells over time are not yet known. Even for brain tumors that can be grown in cell suspen....

Protocol

Ethics Statement: The following procedure involving animal subjects were done in accordance with the National Institutes of Health guidelines and were approved by the Dana-Farber Cancer Institutional Animal Care and Use Committee. All human subjects work was reviewed by the Institutional Review Board Committees of the Brigham and Women's Hospital and Dana-Farber Cancer Institute, and by Stanford University for appropriate use, that informed consent was obtained from all subjects when required, and appropriate waiver of consent requirements was obtained for minimal risk studies.

Timeline of Slice Culture Protocol:

Results

This section exemplifies the type of results to be expected from utilizing the brain tumor/organotypic slice co-culture to investigate regional microenvironment preference as well as to test new therapies. We show that the assay is designed to replicate the microenvironment for brain tumors, as the tissue organization and proliferative state of the slice is maintained (Figure 3). We also demonstrate that an increase in the number of tumor cells on the slice over time may partially be due to the migration.......

Discussion

This protocol describes how brain tumor cells can be fluorescently labeled and plated on a sagittal brain section of a P6 mouse and then monitored for one week in culture. This brain tumor/organotypic slice co-culture assay can be used to determine the effect of regional microenvironment on tumor cell number and may also be used as a system for measuring the efficacy of new drug treatments on human tumor growth. Previous studies have used a similar strategy to assess the role of brain micro-environment on neural precurso.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Invitrogen	17504044
Glucose	Invitrogen	17502048
Pennicillin Streptomycin	Life Technologies	15140-122
HBSS	Life Technologies	14185-052
B-27	Life Technologies	17504-044
N2	Life Technologies	17502-048
Glutamax	Life Technologies	35050061
Neurobasal-A- Medium minus phenol red	Invitrogen	12349015
Low Melting Point Agarose	Promega	V2111
Slice Culture Inserts	Milipore	PICM0RG50
laminin	Invitrogen	23017015
Cm-DiI	Invitrogen	V22888
EDU (Labeling and Detection)	Life Technologies	c10337
Microspheres	Life Technologies	F-21010
Vibratome	Leica
Confocal Microscope	Nikon Eclipse Ni C2si
ImageJ software
5 mm Cover Glasses	Fisher Scientific	64-0700 (CS-5R)