Abstract LB-140: Investigating cancer stem cells in glioblastoma initiation and recurrence

Abstract

Abstract In this study, I examine the unique features of quiescent cancer stem cells (CSCs) in glioblastoma initiation and recurrence in genetically engineered mouse models. A nestin-TK-GFP transgene was used to label CSCs with GFP and to render them susceptible to ganciclovir (GCV) treatment in a fully penetrant mouse model of GBM (M7: hGFAP-Cre; Nf1fl/+; p53fl/fl; Ptenfl). Using food-mediated GCV delivery (GCV chow) to optimize the drug delivery, Doublecortin-positive and NG2-positive cells derived from the nestin-TK-GFP transgene labeled neural stem cells were significantly reduced after six weeks. In addition, on GCV chow the tumor-bearing mice containing the nestin-TK-GFP transgene live significantly longer than the ones without the transgene. Isolation and transplantation of the GFP+/GFP- tumor cells demonstrated that the GFP+ cells have enhanced tumorigenic potential over the GFP- cells. These results support the hypothesis that the nestin-TK-GFP transgene targets the CSCs that are responsible for the sustained GBM growth. In the M7 model, the hGFAP transgene has a broader expression, and thus broader Cre targeting, than the nestin transgene. To eradicate the problem of differing scopes of Cre expression versus targeting of CSCs, I generated a new transgene (CGD: nestin-CreERT2-H2BeGFP-hDTR) that enables Cre-mediated recombination, specific cell labeling and targeted cell ablation. The CGD is designed to express a fusion protein that consists of CreERT2, H2B-eGFP, and hDTR (human diphtheria toxin receptor). Containing P2A linkers between the three gene cassettes, the fusion protein can be efficiently cleaved into three active. I have screened for a transgenic line that can induce GBM formation upon tamoxifen induction. Six to eight week-old mice with the following genetic configuration: CGD; Nf1fl/+; p53fl/+; Ptenfl/+, were given one dose of tamoxifen and all mice died of high grade gliomas three to six months later. The analysis of the CGD-induced tumors demonstrated that the CGD transgene labels tumor cells that are relatively quiescent, compared to the highly proliferative Ki67+ cells. Thus our CGD transgene labels putative CSCs within the tumor. In this model, the same cells initiating tumor formation (Cre-expressing) are also expressing H2B-eGFP and hDTR. Thus, all the tumor cells are derived from the CGD transgene and all the CSCs should be labeled with the H2B-eGFP protein. This dramatically facilitates the sorting of the tumor-initiating cells. Transplantation of the GFP+/GFP- cells in these tumors revealed that the quiescent GFP+ cells are more tumorigenic than the proliferating GFP- cells. Furthermore, diphtheria toxin-mediated ablation of the hDTR-expressing cells significantly elongates the lives of tumor-bearing mice. To mimic the endogenous environment for GBM growth, wild-type mice were also used to prove the differential tumorigenic ability of the GFP+/- cells. These results validate the essential roles of CSCs in GBM initiation and recurrence. Citation Format: xuanhua xie, Xiuping Zhou, German B. Sánchez, Luis F. Parada. Investigating cancer stem cells in glioblastoma initiation and recurrence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-140. doi:10.1158/1538-7445.AM2015-LB-140