A restricted cell population propagates glioblastoma growth after chemotherapy

Glioblastoma multiforme is the most common primary malignant brain tumour, with a median survival of about one year. This poor prognosis is due to therapeutic resistance and tumour recurrence after surgical removal. Precisely how recurrence occurs is unknown. Using a genetically engineered mouse model of glioma, here we identify a subset of endogenous tumour cells that are the source of new tumour cells after the drug temozolomide (TMZ) is administered to transiently arrest tumour growth. A nestin-DTKIRES-GFP (Nes-DTK-GFP) transgene that labels quiescent subventricular zone adult neural stem cells also labels a subset of endogenous glioma tumour cells. On arrest of tumour cell proliferation with TMZ, pulse-chase experiments demonstrate a tumour re-growth cell hierarchy originating with the Nes-DTK-GFP transgene subpopulation. Ablation of the GFP cells with chronic ganciclovir administration significantly arrested tumour growth, and combined TMZ and ganciclovir treatment impeded tumour development. Thus, a relatively quiescent subset of endogenous glioma cells, with properties similar to those proposed for cancer stem cells, is responsible for sustaining long-term tumour growth through the production of transient populations of highly proliferative cells. We have extensively studied a series of mouse strains harbouring conditional alleles of the tumour suppressors Nf1, p53 (also known as Trp53) and Pten, which spontaneously develop malignant gliomas with 100% penetrance, and have identified the source cells of these tumours as deriving from the subventricular zone (SVZ). Thus, we proposed that adult neural stem cells (NSCs) are the likely source of these tumours. We wished to determine whether a Nes-DTK-GFP transgene, originally devised to mark adult NSCs, would also mark endogenous glioma cells. Elements of the nestin gene can drive transgene expression specifically in adult NSCs (Fig. 1a). The transgene also harbours a cassette containing a modified version of the herpes simplex virus (HSV) thymidine kinase (DTK), allowing for temporally regulated ablation of dividing neural progenitors by systemic ganciclovir (GCV) administration, and an IRES-GFP cassette to mark Nes-DTK-expressing cells in the absence of GCV. The Nes-DTK-GFP transgenic mice showed the expected expression in both glial fibrillary acidic protein (GFAP)-positive adult NSCs and early doublecortin (DCX)-positive neural progenitor cells (NPCs) in the major adult NSC niche: the SVZ of the lateral ventricle (Fig. 1b, c). To validate that the GCV-activated Nes-DTK-GFP transgene could effectively eliminate endogenous neural stem/progenitor cells, 1-month-old transgenic mice were treated with GCV for 2 weeks. In control mice, the rostral migratory stream (RMS), formed by NPCs migrating from the SVZ to the olfactory bulb, is visualized by Nissl staining. In contrast, the RMS was severely diminished in Nes-DTK-GFP mice after GCV treatment (Fig. 1b, bottom). Consistent with this observation, DCX immunostaining indicated the absence of NPCs in the SVZ of GCV-treated Nes-DTK-GFP animals (Fig. 1c, bottom). Because, in the presence of GCV, HSV TK targets only proliferating cells, the GFAP NSCs that remained quiescent were unaffected, although they also express the transgene, as indicated by GFP (Fig. 1c, bottom). Thus, our Nes-DTK-GFP transgene is specifically expressed in SVZ quiescent