Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Summary

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Abstract

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.

To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.

The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

Introduction

Brain tumors are among the most aggressive and heterogeneous cancers known in humans. Although their earlier detection and diagnosis have been facilitated by modern neuro-imaging technology, we still lack curative therapies for many brain tumors, particularly for diffuse, invasive ones or those located deep in the brain.

Brain tumors represent the leading cause of cancer mortality in children due to their highly aggressive and often incurable nature. Glioblastoma (GBM), the most common primary brain tumor in adults, is one of the most aggressive human cancers, feared for its uniformly fatal prognosis¹. This highly malignant astro....

Protocol

1. Culture of Brain Tumor Tissue

1. Add 200 μl thawed Liberase (Roche Applied Science) to 15 ml of artificial CSF (aCSF- see Table 1) and place into 37 °C water bath. Liberase TM is a mix of proteolytic enzymes used to dissociate primary tissue samples, as well as cultured tumorspheres. Unlike Trypsin-EDTA, the Liberase method preserves the surface antigen CD133. For a tissue sample of about 0.5 cm³, we use 200 μl of Liberase. If the tissue is smaller, we use 100 ul.
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Discussion

The cancer stem cell hypothesis¹⁰, based on work in leukemia²¹, breast cancer¹¹ and brain cancer ^4,5, suggests that only a relatively small fraction of cells in the tumor, termed cancer stem cells, possess an ability to extensively proliferate and self-renew. Most of the tumor cells lose the ability to proliferate and self-renew as they differentiate into cells that become the phenotypic signature of the tumor. Finding the key cells in the brain tumor population that are able t.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number
1:1 DMEM:F12	Invitrogen	11320-082
N2 supplement	Invitrogen	17502-048
1M HEPES	Wisent	330-050-EL
Glucose	Invitrogen	15023-021
N-acetylcysteine	Sigma Aldrich	A9165-25g
Neural survival factor -1 (NSF-1)	Lonza Clonetics	CC-4323
Epidermal growth factor (EGF)	Sigma Aldrich	E9644
Basic fibroblast growth factor (bFGF)	Invitrogen	PHG0261
Leukemia inhibitory factor (LIF)	Millipore	LIF1010
Antibiotic/mycotic	Wisent	450-115-EL
Liberase TM	Roche	05 401 119 001
Ammonium chloride solution	Stem Cell Technologies	07850