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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.)

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.