Many cancers feature cellular hierarchies that are driven by tumor-initiating, cancer stem cells (CSCs) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients, and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2+, OLIG2+, and TLX+ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiation therapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displayed histological features, including single cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology.

Normal stem cells reside in functional niches critical for self-renewal and maintenance. Neural and hematopoietic stem cell niches, in particular, are characterized by restricted availability of oxygen and the resulting regulation by hypoxia inducible factors (HIFs). Glioblastoma multiforme (GBM) is the most common malignant brain tumor and also contains high degrees of hypoxia. Heterogeneity within the neoplastic compartment has been well characterized in GBM and may be derived from genetic and epigenetic sources that co-evolve during malignant progression. Recent experimental evidence has supported the importance of hypoxia in cancer stem cell (CSC) niches. We hypothesized that HIFs require epigenetic modifying proteins in order to promote tumor malignancy in GBM. Here we demonstrate that in GBM the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) is induced by hypoxia and enhances hypoxic responses. Loss of MLL1 reduces expression of HIF transcripts and HIF2α protein. Targeting MLL1 by RNA interference inhibited expression of HIF2α and target genes, including VEGF. CSCs expressed higher levels of MLL1 than matched nonstem tumor cells and depletion of MLL1 reduced CSC self-renewal, growth, and tumorigenicity. These studies have uncovered a novel mechanism mediating tumor hypoxic responses linking microenvironmental regulation of epigenetic modifying proteins to cellular heterogeneity and provide rationale for the design of more sophisticated clinical approaches targeting epigenetic regulation.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a median survival of 12–15 months with treatment consisting of surgical resection followed by ionizing radiation (IR) and chemotherapy. Even aggressive treatment is often palliative due to near universal recurrence. Therapeutic resistance has been linked to a subpopulation of GBM cells with stem cell-like properties termed GBM initiating cells (GICs). Recent efforts have focused on elucidating resistance mechanisms activated in GICs in response to IR. Among these, GICs preferentially activate the DNA damage response (DDR) to result in a faster rate of double-strand break (DSB) repair induced by IR as compared to the bulk tumor cells. IR also activates NOTCH and the hepatic growth factor (HGF) receptor, c-MET, signaling cascades that play critical roles in promoting proliferation, invasion, and resistance to apoptosis. These pathways are preferentially activated in GICs and represent targets for pharmacologic intervention. While IR provides the benefit of improved survival, it paradoxically promotes selection of more malignant cellular phenotypes of GBM. As reviewed here, finding effective combinations of radiation and molecular inhibitors to target GICs and non-GICs is essential for the development of more effective therapies.