The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP ؉ cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133 ؉ . Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133 ؉ ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133 ؉ ependymal cells represent an additional-perhaps more quiescent-stem cell population in the mammalian forebrain.adult neural stem cells ͉ subventricular zone

Purpose To identify mediators of glioblastoma anti-angiogenic therapy resistance and target these mediators in xenografts. Experimental Design We performed microarray analysis comparing bevacizumab-resistant glioblastomas (BRGs) to pre-treatment tumors from the same patients. We established novel xenograft models of anti-angiogenic therapy resistance to target candidate resistance mediator(s). Results BRG microarray analysis revealed upregulation versus pre-treatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pre-treatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase (FAK) and STAT3. We developed two novel xenograft models of anti-angiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pre-treatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met shRNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. Conclusion These findings support the role of c-Met in survival in hypoxia and invasion, features associated with anti-angiogenic therapy resistance; and growth and therapeutic resistance of xenografts resistant to anti-angiogenic therapy. Therapeutically targeting c-Met could prevent or overcome anti-angiogenic therapy resistance.

Purpose To identify mechanisms and mediators of resistance to anti-angiogenic therapy in human glioblastoma. Experimental Design We performed microarray gene expression analysis and immunohistochemistry comparing 21 recurrent glioblastomas progressing during anti-angiogenic treatment with VEGF neutralizing antibody bevacizumab to paired pre-treatment tumors from the same patients. Results Microarray analysis revealed that bevacizumab-resistant glioblastomas (BRGs) had 2 clustering patterns defining subtypes that reflect radiographic growth patterns. Enhancing BRGs (EBRGs) exhibited MRI enhancement, a long-established criterion for glioblastoma progression, and expressed mitogen-activated protein kinases, neural cell adhesion molecule-1 (NCAM-1), and aquaporin 4. Compared to their paired pre-treatment tumors, EBRGs had unchanged vascularity and hypoxia, with increased proliferation. Non-enhancing BRGs (NBRGs) exhibited minimal MRI enhancement but had FLAIR-bright expansion, a newer criterion for glioblastoma recurrence since the advent of anti-angiogenic therapy, and expressed integrin α5, laminin, fibronectin1, and PDGFRβ. NBRGs had less vascularity, more hypoxia, and unchanged proliferation than their paired pre-treatment tumors. Primary NBRG cells exhibited more stellate morphology with a 3-fold increased shape factor and were nearly 4-fold more invasive in matrigel chambers than primary cells form EBRGs or bevacizumab-naïve glioblastomas (P<0.05). Conclusion Using microarray analysis, we found two resistance patterns during anti-angiogenic therapy with distinct molecular profiles and radiographic growth patterns. These studies provide valuable biologic insight into the resistance that has limited anti-angiogenic therapy to date.