Abstract B44: Therapeutic response of primary glioblastoma cancer stem cells relative to patient-matched non-stem tumor cells
Authors: Fouse Shaun D. , Nagarajan Raman P. , Nakamura Jean , James C. David , Chang Susan , Costello Joseph F. 
Published: June 2012

Background: Populations of cells within glioblastoma multiforme (GBM) tumors that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. We asked whether early passage primary CSC cultures respond differently than patient-matched non-CSC cultures to both standard-of-care (Temozolomide (TMZ) and Radiation (XRT)) and experimental epigenetic therapy targeting histone deactylases (HDACs).

Experimental Design: We established primary cultures of CSCs as adherent glioma neural stem cell and patient-matched non-CSC monolayer cultures in serum from 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with single or multiple physiological doses of TMZ (5–50uM), with or without XRT (2Gy). To examine the relative efficacy of the HDAC inhibitor vorinostat, the paired cultures were treated with physiological doses (1–2uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur and for cell viability using CellTiter-Glo. MGMT methylation was assessed by methylation-specific PCR and genome-wide methylation analysis was performed on IIIumina Infinium arrays.

Results: The majority of patient-derived primary cultures, including CSC and non-CSC cell lines, are resistant to clinically relevant doses of TMZ. This result is in line with the general lack of response in most but not all GBM patients. Furthermore, the in vitro response to TMZ correlated with the methylation status of MGMT, a robust prognostic marker of survival in GBM patients treated with TMZ. Surprisingly, we observed for two pairs of patient-matched cultures that the CSC cultures were more responsive to single and multi-fraction XRT treatment than the non-CSCs. To further understand this difference in response in patient-matched cultures, we performed genome-wide DNA methylation analysis and found over 1500 genes with differential methylation between CSC and non-CSC cultures, including PTEN and CDKN2A. We examined the status of CD133, a marker of stemness, for our CSC and non-CSC cultures and found that cultures that were enriched for CD133+ cells were not more resistant to either TMZ or XRT. Finally, we examined whether vorinostat, an HDAC inhibitor currently in clinical trials with TMZ/XRT for GBM patients, could sensitize cultures to standard of care therapy. Clinically relevant doses of vorinostat led to decreased cell viability but did not appear to have a synergistic effect when combined with either TMZ or XRT.

Conclusions: Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in less time than the interval between surgery and onset of adjuvant therapy. Furthermore, we find that for our patient-matched cultures, the CSC population is not uniformly more resistant to therapy in vitro than the non-CSC population. If the in vitro response mirrors patient response in vivo, the in vitro cultures might be beneficial for making clinical decisions on a patient-specific basis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B44.