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 astrocytic tumor (WHO Grade 4) usually occurs in the cerebral hemispheres of adults, and can also occur in young children and infants. Its growth is rapid and infiltrative, and diagnostic pathological features include nuclear pleomorphism, microvascular proliferation, and necrosis^2,3. For adults with newly diagnosed GBM, median survival rarely extends beyond 12 months¹, with generally poor responses to all therapeutic modalities. We noted that there are many functional and genetic similarities shared by somatic stem cells and cancer cells, and that the molecular pathways that regulate normal brain development are often dysregulated in cancer. In applying stem cell biology paradigms to the study of brain tumors, we were the first researchers to prospectively identify and purify a subpopulation of cells from human GBMs which exhibited the stem cell properties of proliferation, self-renewal, and differentiation in vitro⁴ and in vivo⁵. We applied culture conditions and assays originally used to characterize normal neural stem cells (NSCs) in vitro ^6,7 to multiple pediatric and adult brain tumors, and enriched for these stem-like cells by cell sorting for the neural progenitor cell surface marker CD133^8,9. The CD133+ brain tumor fraction contained cells that had a much higher frequency of tumor initiation than the CD133- fraction in NOD-SCID mouse brains^5,10. This formally established that only a rare subset of brain tumor cells with stem cell properties are tumor-initiating, earning them the name “brain tumor initiating cells” or “BTICs”. The novel identification of BTICs provides new insights into human brain tumorigenesis, giving strong support for the cancer stem cell hypothesis^10-13 as the basis for many solid tumors, and establishes a novel cellular target for more effective cancer therapies^14-20. Therapies that focus on killing the bulk of the tumor may miss the rare stem-like fraction, allowing the tumor to continue to grow. Therapies that focus on killing the cancer stem cell may provide better treatment and prognosis for patients with brain tumors.

In order to study BTIC populations, we have refined our culture protocols to specifically select for cell populations within human brain tumors that possess stem cell properties. Serum-free, neural stem cell (NSC) medium allows for the maintenance of an undifferentiated stem cell state, and the addition of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) allows for the proliferation of multi-potent, self-renewing, and expandable human tumorspheres. Here, we describe the methods involved in processing of primary brain tumors and culturing them in NSC medium to enrich for BTIC populations. We have called our experimental model system “BTIC patient isolates” to emphasize the fact that these cells are only minimally cultured under stem cell conditions to select for stem cell populations. Subsequent immunolabelling of BTIC populations for key stem cell markers such as CD133 and CD15 and flow cytometry analysis is also described. We then discuss the limiting dilution analysis, which aids in studying the self-renewal potential of BTICs. Finally, we explore the gene expression analysis of these rare cells by sorting single cells onto AmpliGrid slides and performing single cell RT-PCR. These techniques are also applicable to other brain tumors such as medulloblastoma, ependymoma and pediatric gliomas.

Protocol

1. Culture of Brain Tumor Tissue 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. <…

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 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