Summary Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). As hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2α and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to nonstem tumor cells and normal neural progenitors. In tumor specimens, HIF2α co-localizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns and HIF2α may represent a promising target for anti-glioblastoma therapies. Significance Recent evidence supports the presence of cancer stem cell populations that contribute to tumor progression through preferential resistance to radiation and chemotherapy, and promotion of tumor angiogenesis, invasion, and metastasis. Therefore, the elucidation of molecular regulators of cancer stem cells may translate into improved anti-neoplastic therapies. Our work demonstrates that cancer stem cells derived from glioblastomas differentially respond to hypoxia with a distinct induction of HIF2α. We find that HIFs are critical to cancer stem cell maintenance and angiogenic drive, and that expression of HIF2α is significantly associated with poor glioma patient survival. These data further suggest that anti-angiogenic therapies can be designed to target cancer stem cell specific molecules involved in neoangiogenesis, including HIF2α and its regulated factors.

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy. (Cancer Res 2006; 66(16): 7843-8)

Bevacizumab and irinotecan is an effective treatment for recurrent glioblastoma multiforme and has moderate toxicity.

Purpose: Recurrent grade III-IV gliomas have a dismal prognosis with minimal improvements in survival seen following currently available salvage therapy. This study was conducted to determine if the combination of a novel antiangiogenic therapy, bevacizumab, and a cytotoxic agent, irinotecan, is safe and effective for patients with recurrent grade III-IV glioma. Experimental Design: We conducted a phase II trial of bevacizumab and irinotecan in adults with recurrent grade III-IV glioma. Patients with evidence of intracranial hemorrhage on initial brain magnetic resonance imaging were excluded. Patients were scheduled to receive bevacizumab and irinotecan i.v. every 2 weeks of a 6-week cycle. Bevacizumab was administered at 10 mg/kg. The dose of irinotecan was determined based on antiepileptic use: patients taking enzyme-inducing antiepileptic drugs received 340 mg/m 2 , whereas patients not taking enzymeinducing antiepileptic drugs received 125 mg/m 2 . Toxicity and response were assessed. Results:Thirty-two patients were assessed (23 with grade IV glioma and 9 with grade III glioma). Radiographic responses were noted in 63% (20 of 32) of patients (14 of 23 grade IV patients and 6 of 9 grade III patients).The median progression-free survival was 23 weeks for all patients (95% confidence interval, 15-30 weeks; 20 weeks for grade IV patients and 30 weeks for grade III patients). The 6-month progression-free survival probability was 38% and the 6-month overall survival probability was 72%. No central nervous system hemorrhages occurred, but three patients developed deep venous thromboses or pulmonary emboli, and one patient had an arterial ischemic stroke. Conclusions: The combination of bevacizumab and irinotecan is an active regimen for recurrent grade III-IV glioma with acceptable toxicity.