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Solid tumors are often characterized by insufficient oxygen supply (hypoxia), as a result of inadequate vascularization, which cannot keep up with the rapid growth rate of the tumor. Tumor hypoxia is a negative prognostic and predictive factor and is associated with a more aggressive phenotype in various tumor entities. Activation of the hypoxic response in tumors, which is centered around the hypoxia-inducible transcription factors (HIFs), has been causally linked to neovascularization, increased radio- and chemoresistance, altered cell metabolism, genomic instability, increased metastatic potential, and tumor stem cell characteristics. Thus, the hypoxic tumor microenvironment represents a main driving force for tumor progression and a potential target for therapeutic interventions. Here, we describe several methods for the analysis of tumor hypoxia and the hypoxic response in vivo in tumor xenograft models. These methods can be applied to various tumor models, including brain tumor xenotransplants, and allow simultaneously determining the extent and distribution of hypoxia within the tumor, analyzing HIF levels by immunohistochemistry and immunoblot, and quantifying the expression of HIF target genes in tumor tissue. The combination of these approaches provides an important tool to assess the role of the hypoxic tumor microenvironment in vivo.

Hypoxia is a common feature of solid tumors, which controls multiple aspects of cancer progression. One important function of hypoxia and the hypoxia-inducible factors (HIF) is the maintenance of cancer stem-like cells (CSC), a population of tumor cells that possess stem cell-like properties and drives tumor growth. Among the changes promoted by hypoxia is a metabolic shift resulting in acidification of the tumor microenvironment. Here, we show that glioma hypoxia and acidosis functionally cooperate in inducing HIF transcription factors and CSC maintenance. We found that these effects did not involve the classical PHD/VHL pathway for HIF upregulation, but instead involved the stress-induced chaperone protein HSP90. Genetic or pharmacologic inactivation of HSP90 inhibited the increase in HIF levels and abolished the self-renewal and tumorigenic properties of CSCs induced by acidosis. In clinical specimens of glioma, HSP90 was upregulated in the hypoxic niche and was correlated with a CSC phenotype. Our findings highlight the role of tumor acidification within the hypoxic niche in the regulation of HIF and CSC function through HSP90, with implications for therapeutic strategies to target CSC in gliomas and other hypoxic tumors. Cancer Res; 76(19); 5845-56. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cultured tumor cells are a central tool in cancer research and have provided fundamental insights in tumor biology. Recent evidence, however, indicates that classically established cell lines from different tumors, including glioblastoma, do not fully reflect the genotypes and phenotypes of the respective primary tumors. By contrast, primary cells, isolated from human tumor samples and maintained in serum-free spheroid cultures at low passage under defined growth factor conditions, reproduce key aspects of tumor cell physiology much more faithfully. Among the tumor cell characteristics that are better represented in primary glioblastoma cell cultures is the self-renewal and differentiation potential of the tumor cells. Indeed, a large body of evidence from the past decade indicates that glioblastomas and other tumors are composed of a hierarchy of heterogeneous types of cells, which are generated and maintained by cells that share characteristics of stem cells. This cancer stem cell/tumor initiating cell population is optimally preserved and maintained in primary glioblastoma cultures. Here, we describe a method for the isolation and culture of primary tumor cells from human glioblastomas in serum-free conditions, which allows the routine generation and proper maintenance of tumor cells as spheroid cultures. Such primary tumor cultures can serve as a model of choice for the study of the mechanisms behind key aspects of glioblastoma biology, including tumorigenicity, stem cell hierarchy, invasion, and therapeutic resistance.