An efficient method for derivation and propagation of glioblastoma cell lines that conserves the molecular profile of their original tumours

Abstract

A growing body of evidence suggests that glioma stem-like cells are more representative of their parent tumours when cultured under defined serum-free conditions with the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF). However, culturing these cells as free-floating spheroids can result in difficulty in efficiently deriving and propagating cell lines. We have combined neurosphere and monolayer culture techniques to improve the efficiency with which cells can be derived from clinical tumour samples under defined serum-free conditions.

We have applied our protocol to consecutive samples of glioblastoma to show that they can form experimental tumours that recapitulate many of the histological features of the parent tumour. We go on to show that the tumour initiating cells also retain the cytogenetic abnormalities of the parent tumour.

Finally we examined the cell lines for expression of markers associated with neural stem cells. Our results confirm the expression of transcription factors associated with neural patterning and specification including Sox2, Olig2, Pax6 and Nkx2.2. We went on to establish that these factors were also expressed in the parent tumour indicating that their expression was not a function of our culture conditions.

The Cambridge Protocol is an efficient method of deriving stem-like tumour initiating cells from glioblastoma. Improving the efficiency of derivation will facilitate the improvement of in vitro and in vivo model systems to study disease mechanisms, screen drugs and develop novel therapeutic approaches in the future.

Introduction

Adult murine neural stem cells (NSC) can be derived and clonally propagated as free-floating spheroid bodies under defined serum-free (SF) conditions with mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Reynolds and Weiss, 1992, Richards et al., 1992). This spheroid approach has also been applied to human embryonic and human adult NSC cultures with limited long-term propagation (Moe et al., 2005, Murray and Dubois-Dalcq, 1997, Nunes et al., 2003, Sanai et al., 2004, Svendsen et al., 1998). Several reports have demonstrated that glioma cultures under SF conditions as spheres select for tumour-initiating cells (TICs) with a phenotype and genotype closer to that typical of primary tumours compared to traditional serum-derived cell lines and culture techniques (De Witt Hamer et al., 2008, Lee et al., 2006). However, the reported efficiency of derivation is less than 50% of samples (Galli et al., 2004, Gunther et al., 2008). These observations emphasise the need to develop techniques to improve our ability to derive and propagate cell lines from clinical samples on a patient-by-patient basis, to avoid waste of precious clinical material and facilitate clinical translational research.

Tumour spheroids comprise a complex microenvironment that is difficult to interrogate or propagate. Monolayer cultures have been successfully applied to murine and human embryonic stem cell, fetal cortical and adult NSC cultures (Conti et al., 2005, Gage et al., 1995, Jessberger et al., 2007, McKay, 1997). Therefore, we hypothesised that by combining primary spheroid and monolayer culture techniques we could reliably and reproducibly derive human glioma cell lines. We tested our hypothesis using clinical material from the commonest class of intrinsic malignant human brain tumours the astrocytomas.