Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large-and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.angiogenesis | glioma | metabolism | perfusion G lioblastomas (GBMs) are highly vascularized brain tumors and are therefore attractive targets for anti-angiogenic therapies (1). In particular, vascular endothelial growth factor (VEGF) has been identified as a critical regulator of angiogenesis, and currently a number of clinical trials targeting the VEGFsignaling pathways are under development (2, 3). Bevacizumab (bev), a humanized anti-VEGF antibody, has shown promising results in exploratory phase II trials of recurrent GBM. Alone or in combination with irinotecan, it is well tolerated and shows a high radiological response rate and possibly an increase in median progression-free survival compared with historical controls (4-7), although no impact on overall survival has been reported (8). However, these results are based on small patient cohorts and, because anti-angiogenic agents directly affect vessel permeability, the imaging response assessment based on contrast enhancement (CE) is highly ambiguous (9). Indeed, a direct antitumor effect of bev has remained elusive and the infiltrative part of the tumor may even increase (10,11). In addition to a lack of robust clinical data, the cellular and molecular consequences of anti-VEGF treatment have not been outlined (12). Detailed information on how bev affects ...

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor-initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood-brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real-time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression. ' 2007 Wiley-Liss, Inc.Key words: CD133; brain cancer; angiogenesis; cancer stem cell; xenograft At present, there is a search for tumor cell subpopulations that may be responsible for tumor initiation and progression. Such cells have been termed cancer stem cells and are defined by their capacity to self-renew, express stem cell markers and to initiate tumors in vivo. 1,2 Potential cancer stem cells have been identified in leukaemias, 3-5 breast, 6 prostate, 7 bone, 8 colon and brain cancer. [9][10][11][12][13] In some cases, these tumor-initiating cells have been distinguished from the non-tumor-initiating ones based on expression of cell surface markers. For instance, it has been shown that only CD44 1 / CD24 2 /Lineage 2 breast cancer cells are tumorgenic in animals. 6 In malignant brain tumors, CD133 has been suggested to be a cancer stem cell marker 11,14 since only CD133 positive cells from brain tumor biopsy material were able to initiate brain cancer in a mouse model. 14 Prominin-1 (PROM-1), also called CD133, is a protein with several isoforms of unknown physiological or pathological function, and is localized both in the cytoplasm and at the cell surface. 15,16 It is expressed by human neural stem cells and has been proposed to have a function in central nervous system (CNS) development. 17 It is also express...

Hypoxia is a critical hallmark of solid tumors and involves enhanced cell survival, angiogenesis, glycolytic metabolism, and metastasis. Hyperbaric oxygen (HBO) treatment has for centuries been used to improve or cure disorders involving hypoxia and ischemia, by enhancing the amount of dissolved oxygen in the plasma and thereby increasing O2 delivery to the tissue. Studies on HBO and cancer have up to recently focused on whether enhanced oxygen acts as a cancer promoter or not. As oxygen is believed to be required for all the major processes of wound healing, one feared that the effects of HBO would be applicable to cancer tissue as well and promote cancer growth. Furthermore, one also feared that exposing patients who had been treated for cancer, to HBO, would lead to recurrence. Nevertheless, two systematic reviews on HBO and cancer have concluded that the use of HBO in patients with malignancies is considered safe. To supplement the previous reviews, we have summarized the work performed on HBO and cancer in the period 2004–2012. Based on the present as well as previous reviews, there is no evidence indicating that HBO neither acts as a stimulator of tumor growth nor as an enhancer of recurrence. On the other hand, there is evidence that implies that HBO might have tumor-inhibitory effects in certain cancer subtypes, and we thus strongly believe that we need to expand our knowledge on the effect and the mechanisms behind tumor oxygenation.

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing 13C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of 13C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, l-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1352-5) contains supplementary material, which is available to authorized users.

Conflict of interest: DG is named as an inventor on a patent application relating to the Mab203E1H5 (EP18155716) antibody filed by the University of Bergen.