Abstract 5851: Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation
Authors: Timme Cindy R. , Shah Marzia , Camphausen Kevin , Tofilon Philip J. 
Published: July 2017

Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness, phenotypic heterogeneity and radioresistance. As an approach to investigating the relationship between these aspects of GBM biology and tumor cell proliferation, we used the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase, to identify proliferating cells in orthotopic brain tumor xenografts. Specifically, CD133+ NSC11 GSCs were implanted into the right striatum of nude mice; at various times after tumor cell implantation CldU was delivered in a series of daily intraperitoneal (IP) injections. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the mouse brain. NSC11 tumors are first detectable using bioluminescent imaging (BLI) at day 21 post-implantation. After 4 daily injections of CldU beginning on Day 21, approximately 60% and 30% of tumor cells were CldU positive in the white matter (corpus callosum) and grey matter (striatum), respectively. However, when CldU injections were initiated on Day 28 post-implantation, approximately 80% of cells were positive in both tissue locations after 4 doses, which corresponds to a rapid tumor growth phase according to bioluminescent imaging. Tumor cells were also detected in the left hemisphere and had lower percentages of CldU positive cells as compared to the right hemisphere (site of implantation), which illustrates the extensive invasive capacity of this GSC line and is consistent with the “grow or go” concept whereby GBM cells switch between proliferative and migratory states. NSC11 cells also displayed the hallmark GBM cell migration along white matter tracts. To determine the pattern of proliferating tumor cell after irradiation, NSC11 brain tumor xenografts received three daily doses of 12Gy, which results in an approximate 10-day survival advantage, followed by a series of 4 daily IP injections of CldU. After the 4 doses of CldU, approximately 2% of tumors cells were CldU positive in the white matter of the 3x12Gy-treated tumors and no proliferating cells were detected in the grey matter. These values can be compared to 50% and 40% CldU positive cells in the white and grey matter, respectively, of the sham irradiated tumors. When the CldU injections were initiated 4 days post-3x12Gy, discrete pockets of CldU positive tumor cells began appearing in both white and grey matter from 7-8 days after the completion of radiation treatment. These pockets of proliferating cells may reflect radioresistant subpopulations and provide a framework to further characterize radioresistance based on phenotype and/or microenvironmental niche.

Citation Format: Cindy R. Timme, Marzia Shah, Kevin Camphausen, Philip J. Tofilon. Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5851. doi:10.1158/1538-7445.AM2017-5851