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...

There is increasing evidence that the growth and spread of cancers is driven by a small subpopulation of cancer stem cells (CSCs) - the only cells that are capable of long-term self-renewal and generation of the phenotypically diverse tumour cell population. Current failure of cancer therapies may be due to their lesser effect on potentially quiescent CSCs which remain vital and retain their full capacity to repopulate the tumour. Treatment strategies for the elimination of cancer therefore need to consider the consequences of the presence of CSCs. However, the development of new CSC-targeted strategies is currently hindered by the lack of reliable markers for the identification of CSCs and the poor understanding of their behaviour and fate determinants. Recent studies of cell lines derived from oral squamous cell carcinoma (OSCC) indicate the presence of subpopulations of cells with phenotypic and behavioural characteristics corresponding to both normal epithelial stem cells and to cells capable of initiating tumours in vivo. The present review discusses the relevance to OSCC of current CSC concepts, the state of various methods for CSC identification, characterization and isolation (clonal functional assay, cell sorting based on surface markers or uptake of Hoechst dye), and possible new approaches to therapy.

BackgroundGlioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model.Methodology/Principal FindingsWe used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect.Conclusions/SignificanceIn conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.

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