Response of primary glioblastoma cells to therapy is patient specific and independent of cancer stem cell phenotype.

Glioblastoma multiforme (GBM) displays molecular heterogeneity among patients and within individual tumors. Inter- and intratumoral heterogeneity is a major confounding factor for achieving durable therapeutic response. Intratumoral heterogeneity at the cellular level includes cell subpopulations referred to as cancer stem cells (CSCs), which have properties similar to neural progenitors, such as the ability to differentiate into multiple CNS cell lineages.1,2 CSC-enriched cultures derived from primary GBM can be propagated in vitro as neurospheres in suspension2,3 or as adherent monolayers,4 as well as in vivo as xenografts.3,4 Interestingly, the ability of dissociated primary tumors to establish viable CSC suspension cultures has been associated with worse overall survival for patients from whom the cultures were derived,5,6 suggesting that the tumor CSC component is a significant contributor to tumor malignancy. Enhancer of zeste homolog 2 and signal transducer and activator of transcription 3, which both show elevated expression in GBM, preferentially interact in CSCs, and this interaction appears to help maintain a state of stemness.7 CSCs and non-CSCs cultured from the same tumor also exhibit differences in their histone profiles, though how the epigenetic differences relate to differences in culture phenotypes such as drug response is unknown.8 Such paired non-CSC and CSC cultures allow controlled comparisons of genotypically similar but phenotypically distinct cells for molecular, biologic, and therapeutic response characteristics. Here we use these cultures to directly address the hypothesis that CSCs are more resistant than non-CSCs to therapy in a genetically controlled setting. The standard of care for GBM patients is resection, followed by chemotherapy and radiation therapy (XRT). The most commonly used chemotherapeutic agent is temozolomide (TMZ), an orally delivered DNA alkylator that crosses the blood–brain barrier and undergoes spontaneous conversion to the active form 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (ie, MTIC).9 The overall survival of GBM patients who receive TMZ correlates with the methylation status of O6-DNA methylguanine-methyltransferase (MGMT), a DNA repair protein that preferentially removes the TMZ-induced methyl group adduct at O6-guanine.10 In addition to MGMT, GBM may be inherently resistant to TMZ or may develop increased resistance during the course of TMZ therapy. A testable hypothesis to account for GBM TMZ resistance is that it is conferred by tumor CSC subpopulations and that CSCs undergo preferential expansion during or after treatment.11 This resistance could be the result of both intrinsic factors such as increased drug efflux and extrinsic factors such as hypoxic microenvironments.12 A similar mechanism accounted for GBM resistance to radiation therapy.13 However, there is disagreement regarding the relative importance of the GBM CSC component to therapeutic resistance, as indicated by reports suggesting that CD133+ CSC populations may be more sensitive to TMZ14 or XRT15 than tumor-matched CD133–14 or serum-derived but unmatched tumor cultures that are depleted of CSCs.15 Genetic differences between cultures were not controlled for in each case, nor did these studies examine response to combined XRT and TMZ treatment. To directly address the influence of CSCs on GBM response to standard-of-care therapy in a genetically controlled manner, we established CSC-enriched and non-CSC culture pairs from 10 patients with primary GBM. To augment these analyses, we established and tested CSC cultures from an additional 15 primary GBM patients.