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...

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stemlike cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.glioma ͉ invasiveness ͉ vessel cooption

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-011-0867-2) contains supplementary material, which is available to authorized users.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1101-1) contains supplementary material, which is available to authorized users.