Abstract 3934: Bcl2-expressing quiescent type B neural stem cells in the SVZ are resistant to concurrent temozolomide/X-irradiation

Abstract

Abstract The subventricular zone (SVZ) is the largest source of neural stem cells (NSCs) in the adult brain. Emerging research indicates that NSCs within the SVZ may be cells of origin for WHO grade IV astrocytoma (glioblastoma, GBM)1. GBM consists of multiple fractions of proliferative and/or quiescent stem-like cells that are thought to be lineally related. GBM located adjacent to the SVZ are very resistant to standard of care concurrent temozolomide (TMZ)/X-irradiation (XRT) therapy, a consequence, it is hypothesized, of their NSC origin2. An important, unanswered question is the origin of this resistance. While a significant effort has been undertaken to study proliferating cells, the origins of quiescent cell resistance are not well understood. Normal NSCs adjacent to the SVZ are mainly quiescent. We rationalized that a fundamental understanding of the response of quiescent NSCs to TMZ/XRT would be informative and aid in our understanding of GBM resistance. For 5 consecutive days cohorts of C57BL/6 mice were administered TMZ (0 or 50 mg.kg i.p.). One hr later 0 or 2 Gy was administered to the brain. Transcardial perfusion was performed on day 6 for half the mice. The remaining mice received adjuvant TMZ (100 mg/kg) or vehicle on days 19-22 and transcardial perfusion was performed on day 82. 10 µm coronal brain sections were obtained and immunostained for well characterized markers of type B NSCs (GFAP and Sox2) and type A neuroblasts (Dcx). Immunofluorescence was imaged using a Leica Aperio Versa 200 slide scanning microscope. Cell Profiler software was used to quantify type B and A cells in the SVZ in all cohorts. Proliferating type A cells were exquisitely sensitive to 5 days of concurrent TMZ/XRT treatment whereas quiescent NSCs located within 30 µm of a dorsal or dorsolateral ventricle were very resistant. NSCs in mice exposed to concurrent and adjuvant therapy were also resistant and importantly, able to repopulate type A cells to sham/control levels. 53BP1 foci formation, a surrogate for DNA DSBs, was quantified in Sox2- and Dcx-expressing cells using confocal microscopy following a single TMZ/XRT exposure. Foci formation, measured 6 min to 24 hrs after TMZ/XRT, was not statistically different between cell types (P>0.05). Because TMZ/XRT induced an apoptotic response in A but not in B cells, as marked by cleaved Caspase-3 staining, we investigated expression of Bax and Bcl2 on a per cell basis. Bax expression was not significantly different for type A or B cells (P>0.05). In contrast, type B NSCs expressed 5-fold more Bcl2 than type A neuroblasts (P< 0.001). In conclusion, we demonstrate that type A neuroblasts are sensitive to TMZ/XRT but can be repopulated by inherently resistant type B NSCs given sufficient time. The resistance of quiescent NSCs to TMZ/XRT is associated with high basal expression of anti-apoptotic proteins. 1Lee et al Nature 2018, 560:243-47; 2Smith et al J Neurooncol 2016, 128:207-16 Citation Format: Brent D. Cameron, Geri Traver, Joseph T. Roland, Daniel Dean, Levi Johnson, Kelli L. Boyd, Rebecca A. Ihrie, Jialiang Wang, Michael L. Freeman. Bcl2-expressing quiescent type B neural stem cells in the SVZ are resistant to concurrent temozolomide/X-irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3934.