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RESEARCH PRODUCT

TMOD-36. PRECISE INVESTIGATION OF CANCER STEM CELLS IN A MOUSE GLIOBLASTOMA MODEL

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Cancer stem cells (CSCs) have been shown to play a critical role in glioblastoma (GBM) pathogenesis. However, a precise and thorough understanding of these cells is still lacking. Here we design a novel mouse model to label, purify, and study cancer stem cells in vivo. Firstly we generate and characterize a new transgene to label neural stem/progenitor cells in the subventricular zone (SVZ) with GFP, and drive expression of CreERT2 and human diphtheria toxin receptor in the same cells (CGD: nestin-CreERT2-H2BeGFP-hDTR). Following analysis with both bulk and single cell RNA sequencing of the SVZ tissue demonstrate its faithful expression in the stem/progenitor cell compartment. We then crossed the transgene with floxed alleles of the top three mutated tumor suppressor genes in GBM patients. This genetic configuration (CGD-M4: CGD; Nf1(fl/+);p53(fl/+); Pten(fl/+)) efficiently promotes brain tumors initiated from wild-type neural stem/progenitor cells. Investigation of the tumors showed the CGD-GFP+ cells are quiescent in vivo, yet form more spheres in vitro than the GFP- cells. Further analysis with serial transplantation assays demonstrated the GFP+ cells are more competent to develop tumors and continuously maintain a quiescent population in the transplanted tumors. Diphtheria toxin treatment eliminates the CGD-GFP+ cells through the hDTR receptor in the transgene and greatly reduces the tumor bulk. The conventional chemotherapeutic drug, temozolomide, benefits only GFP- cell transplanted mice but not GFP+ cells. These cells are collected for RNA sequencing to identify genes associated with the CGD-GFP+ cells. Following CRISPR candidate screen reveals genes essential for the CGD-GFP+ cells. This study provides an unprecedented mouse model to study CSCs, which demonstrates their essential role in GBM initiation, recurrence, and drug resistance.