Abstract 2390: The normal brain microenvironment reduces the radiosensitivity of glioblastoma stem-like cells and identifies additional targets for radiosensitization
Authors: Rath Barbara Helen , Wahba Amy , Camphausen Kevin , Tofilon Philip 
Published: August 2015

Towards developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in co-culture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions, yet had no effect on the established cell line U251. The critical lesion mediating radiation-induced cell death is the DNA double-strand break (DSB). Because γH2AX nuclear foci correspond to radiation-induced DSBs and their dispersal correlates with DSB repair, we determined the initial level of radiation-induced γH2AX foci in GSCs cultured with or without astrocytes. In irradiated co-cultures, the initial level of radiation-induced γH2AX foci was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA DSBs. These findings indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of co-culture media identified a number of bioactive molecules not present under standard culture conditions, including IL-6. The gene expression profiles of GSCs grown in co-culture were also significantly different as compared to GSCs grown alone. For the GSC transcripts increased after co-culture with astrocytes 6 biological functions (containing at least 45 molecules) were identified including Cell Death and Survival, which contained signaling molecules previously associated with radioresponse. Further, pathway analysis identified multiple upstream regulators of affected transcripts that were significantly increased in co-culture. The top regulator molecule was IL-6, which was consistent with elevated levels of this cytokine in co-culture media. Additional upstream regulators included cytokines and growth factors, consistent with a paracrine based effect of astrocytes on GSC gene expression, as well as the transcription factors NFκB and STAT3, which are subject to regulation by cytokines/growth factors. These analyses were consistent with an astrocyte mediated modification in GSC phenotype and moreover, suggested a number of potential targets for GSC radiosensitization that were unique to co-culture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under co-culture conditions and when grown as orthotopic xenografts. These data further suggest that this co-culture system may also provide an approach for identifying additional targets for GBM radiosensitization.

Citation Format: Barbara Helen Rath, Amy Wahba, Kevin Camphausen, Philip Tofilon. The normal brain microenvironment reduces the radiosensitivity of glioblastoma stem-like cells and identifies additional targets for radiosensitization. [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 2390. doi:10.1158/1538-7445.AM2015-2390