Tumor hypoxia has been attributed to play a crucial role in tumorigenesis and therapeutic resistance. Recently, it has been suggested that hypoxia leads to and maintains the undifferentiated state of tumor stem cells, thereby contributing to chemoresistance. The aim of the present study is to investigate the influence of hypoxia on the protein expression of a panel of stem cell and chemoresistance markers using in vivo-like multicellular tumor spheroids derived from a glioblastoma short-term culture with tumor stem cell properties (SJ-1) as well as a conventional glioblastoma cell line (U87). Spheroids were formed in 21% and 1% O(2) in serum-free medium. The immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67), and stem cell markers (CD133, podoplanin, Bmi-1, nestin, Sox-2) as well as markers related to chemoresistance (MGMT, TIMP-1, Lamp-1, MRP1, MDR-1). As spheroids derived in hypoxia were smaller than in normoxia, a set of experiments was included in which the culturing time of hypoxic spheroids was extended to obtain equally sized spheroids. The results showed that expression of HIF-1α and HIF-2α was increased in hypoxia, whereas Ki-67 was reduced. Expression of stem cell markers CD133, podoplanin, Bmi-1, and nestin was increased in hypoxia, whereas Sox-2 was increased in SJ-1 only. TIMP-1 and Lamp-1 were increased in both SJ-1 and U87. In conclusion, the tumor cell phenotype related to stemness, and thereby potentially to chemoresistance, seems to depend on the oxygen tension, suggesting that development of therapeutic strategies targeting tumor stem cells should take oxygen tension into account.

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different CD133 antibody clones possibly recognizing different CD133 splice variants with epitopes of different glycosylation status confuses the field. The aim was to investigate if current inconsistent CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Ten glioblastomas were immunohistochemically stained with four different CD133 antibody clones (AC133, W6B3C1, C24B9, and ab19898) and analyzed by quantitative stereology. Moreover, the CD133 staining pattern of each antibody clone was investigated in kidney, pancreas, and placenta tissue as well as in glioblastoma and retinoblastoma cultures and cell lines. All antibody clones revealed CD133+ niches and single cells in glioblastomas, but when using different clones, their distribution rarely corresponded. Morphology of identified single cells varied, and staining of various tissues, cultures, and cells lines was also inconsistent among the clones. In conclusion, the authors report inconsistent CD133 detection when using different primary CD133 antibody clones. Thus, direct comparison of studies using different antibody clones and conclusions based on CD133 immunohistochemistry should be performed with caution.

Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2'-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G-negative tumors.

Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed that the primary glioma spheroid area was constant or decreasing after implantation, with a clear increase in the number of invading cells over time. In contrast, the U87MG spheroid area increased after implantation, with no convincing tumor cell invasion. High levels of Bmi-1 and nestin were found in all spheroids, whereas high levels of Sox2 and low to moderate levels of CD133 were only found in the primary spheroids. In conclusion, the invasion of gliomas is preserved using primary glioma spheroids. Some stem cell features are preserved as well, making this model useful in drug development elucidating both invasion and cancer stemness at the early in vitro level.