Simon Kjær Hermansen

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

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.

MicroRNA biomarkers in glioblastoma

Recent research suggests that deregulation of microRNAs (miRNAs) is involved in initiation and progression of many cancers, including gliomas and that miRNAs hold great potential as future diagnostic and therapeutic tools in cancer. MiRNAs are a class of short non-coding RNA sequences (18–24 nucleotides), which base-pair to target messenger RNA (mRNA) and thereby cause translational repression or mRNA degradation based on the level of complementarity between strands. Profiling miRNAs in clinical glioblastoma samples has shown aberrant expression of numerous miRNAs when compared to normal brain tissues. Understanding these alterations is key to developing new biomarkers and intelligent treatment strategies. This review presents an overview of current knowledge about miRNA alterations in glioblastoma while focusing on the clinical future of miRNAs as biomarkers and discussing the strengths and weaknesses of various methods used in evaluating their expression.

miR-21 Is Linked to Glioma Angiogenesis: A Co-Localization Study.

MicroRNA-21 (miR-21) is the most consistently over-expressed microRNA (miRNA) in malignant gliomas. We have previously reported that miR-21 is upregulated in glioma vessels and subsets of glioma cells. To better understand the role of miR-21 in glioma angiogenesis and to characterize miR-21-positive tumor cells, we systematically stained consecutive serial sections from ten astrocytomas for miR-21, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), phosphatase and tensin homolog (PTEN), octamer-binding transcription factor 4 (Oct4), sex-determining region Y box 2 (Sox2) and CD133. We developed an image analysis-based co-localization approach allowing global alignment and quantitation of the individual markers, and measured the miR-21 in situ hybridization signal against the immunohistochemical staining of the six different markers. miR-21 significantly co-localized with the hypoxia- and angiogenesis-associated markers HIF-1α (p=0.0020) and VEGF (p=0.0096), whereas the putative miR-21 target, PTEN, was expressed independently of miR-21. Expression of stem cell markers Oct4, Sox2 and CD133 was not associated with miR-21. In six glioblastoma cultures, miR-21 did not correlate with the six markers. These findings suggest that miR-21 is linked to glioma angiogenesis, that miR-21 is unlikely to regulate PTEN, and that miR-21-positive tumor cells do not possess stem cell characteristics.

Expression and prognostic impact of matrix metalloproteinase-2 (MMP-2) in astrocytomas

Astrocytomas are the most frequent primary brain tumors in adults, and despite aggressive treatment patients often experience recurrence. Survival decreases with increasing tumor grade, and especially patients with grade IV glioblastoma have poor prognosis due to the aggressive character of this tumor. Matrix metalloproteinase-2 (MMP-2) is an extracellular matrix degrading enzyme which has been shown to play important roles in different cancers. The aim of this study was to investigate the expression and prognostic potential of MMP-2 in astrocytomas. Tissue samples from 89 patients diagnosed with diffuse astrocytoma, anaplastic astrocytoma and glioblastoma were stained immunohistochemically using a monoclonal MMP-2 antibody. The MMP-2 intensity in cytoplasm/membrane was quantified by a trained software-based classifier using systematic random sampling in 10% of the tumor area. We found MMP-2 expression in tumor cells and blood vessels. Measurements of MMP-2 intensity increased with tumor grade, and MMP-2 expression was found to be significantly higher in glioblastomas compared to normal brain tissue (p<0.001), diffuse astrocytomas (p<0.001) and anaplastic astrocytomas (p<0.05). MMP-2 expression was associated with shorter overall survival in patients with grade II-IV astrocytic tumors (HR 1.60; 95% CI 1.03–2.48; p = 0.036). In glioblastoma, high MMP-2 was associated with poorer prognosis in patients who survived longer than 8.5 months independent of age and gender (HR 2.27; 95% CI 1.07–4.81; p = 0.033). We found a positive correlation between MMP-2 and tissue inhibitor of metalloproteinases-1 (TIMP-1), and combined MMP-2 and TIMP-1 had stronger prognostic value than MMP-2 alone also when adjusting for age and gender (HR 2.78; 95% CI 1.30–5.92; p = 0.008). These findings were validated in bioinformatics databases. In conclusion, this study indicates that MMP-2 is associated with aggressiveness in astrocytomas and may hold an unfavorable prognostic value in patients with glioblastoma.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

AIM Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. MATERIALS & METHODS Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. RESULTS MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. CONCLUSION MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

A 4-miRNA signature to predict survival in glioblastomas

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06–23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93–33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45–0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Abstract 195: The tumor cell compartment holds the unfavorable prognostic value of miR-21 in gliomas

High-grade gliomas are some of the most lethal forms of human cancer and new clinical biomarkers and therapeutic targets are highly needed. MicroRNAs (miRNAs), a group of short noncoding RNAs, hold great potential as new biomarkers and targets as they are commonly deregulated in a variety of diseases including gliomas. MicroRNA-21 (miR-21) is the most consistently over-expressed miRNA in several cancers including gliomas and is therefore very promising as a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in gliomas, paraffin embedded glioma tissue samples from 193 patients with grade I, II, III and IV tumors were analyzed by in situ hybridization (ISH) using LNA-DNA chimeric probes. We found miR-21 expression in tumor cells and tumor-associated blood vessels, whereas no expression was seen in adjacent normal brain parenchyma. Using advanced image analysis we obtained quantitative estimates reflecting the miR-21 expression levels. The miR-21 levels correlated significantly with grade (p = 0.027, rs = 0.161, 95% CI, 0.015 to 0.301) with the highest levels measured in grade IV gliomas. Furthermore, quantitative estimates obtained for the tumor cells alone were significantly associated with poor prognosis in all tumors (p = 0.012) and grade III and IV tumors alone (p = 0.005). This was independent of known clinical parameters (age, grade and sex). In conclusion, we have shown that miR-21 is constricted to tumor cells and tumor blood vessels and that tumor cell miR-21 represent a valuable prognostic biomarker for the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 195. doi:1538-7445.AM2012-195

Three-Dimensional In Vitro Models in Glioma Research - Focus on Spheroids

In the field of glioma research, in vitro models are widely used to investigate tumor biology as well as tumor response to chemotherapy and radiation. There is an increasing need to improve these in vitro models in order to meet the new challenges arising in drug discovery. It is thus important that development of new drugs is based on the latest knowledge about glioma biology such as for example the recent discovery of tumor stem cells (Reya et al., 2001). When investigating glioblastomas in vitro – and especially the supposed tumor stem cells – three dimensional multicellular spheroid models have recently come into focus. The aim of this chapter is to review the development as well as the most recent aspects of the three-dimensional glioma in vitro models focusing on glioma spheroids. The implementation of these models in current and in future in vitro glioma research will be discussed putting emphasis on the themes described below. Cell lines cultured as monolayers have been the in vitro model of choice for many years (Ponten & Macintyre, 1968). However, the three-dimensional aspect came into focus in the 1970’s, where scientists started to grow tumor cells from cell lines as multicellular spheroids (Yuhas et al., 1977). Over the years the spheroid model has been improved by deriving spheroids from cells obtained from dissociated primary glioblastoma tissue (Mackillop et al., 1985) as well as by using organotypic primary spheroids derived from small tumor fragments (Bjerkvig et al., 1990). In general, most in vitro studies are performed with cells cultured in conventional serumcontaining medium. Recently – as the tumor stem cell theory has evolved – the culturing medium has come into focus. It has thus been demonstrated that the use of serum-free medium for culturing of cell line-derived spheroids preserved the in vivo-like features as well as the tumor stem cell-like phenotype suggesting crucial importance of the use of serum-free medium in tumor stem cell research (Lee et al., 2006). Identification of the glioma stem cells is still a matter of discussion. The most used marker in the field has been the cell surface marker CD133. Expression of this putative tumor stem cell marker in gliomas has been studied in several papers demonstrating clusters or niches of CD133 positive tumor cells as well as CD133 positive single cells dispersed in the tumor

Abstract 1156: MicroRNA-21 in gliomas: An in situ hybridization study

High-grade gliomas are some of the most lethal forms of human cancers. In glioblastomas, which are the most frequent and malignant type of glioma, microRNA-21 (miR21) has recently been suggested to be strongly upregulated. Until now, miR21 expression analysis has mainly been carried out using PCR methods, hence little is known about the cellular localization of miR21 in gliomas. Studies of other cancers have demonstrated miR219s prognostic value as well as roles in invasion, growth and anti-apoptosis. This suggests that miR21 is a potential therapeutic target as well as a promising novel prognostic marker in gliomas. The aim of the present study was therefore to investigate the expression of miR21 in gliomas and correlate it with tumor grade and overall survival using the new possibilities of locked nucleic acid (LNA) based in situ hybridization. We used 193 formalin-fixed paraffin embedded astrocytic and oligodendroglial brain tumors of increasing grade (WHO I-IV). Sections from these tumors were stained by a robust in situ hybridization assay using high-affinity LNA probes that specifically detect miR21. The expression of miR21 was revealed as a blue chromogenic staining reaction. Using advanced image analysis, the area of the staining reaction was measured by a trained pixel classifier on images recorded by stereological principles. The optimal sampling fraction was estimated to be 10%. The results showed that the miR21 signal was localized to blood vessels and populations of dispersed single cells. Moreover, the characteristic gemistocytic tumor cells seemed to display a distinct staining reaction. The miR21 signal increased with tumor grade and a very high individual signal variation was observed, especially among glioblastomas. When comparing the miR21 signal with overall survival, a high signal was associated with shorter survival, but in multivariate analysis this was not independent of grade. In conclusion miR21 is localized in both tumor cells and blood vessels in gliomas. A high miR21 expression appears to be associated with tumor grade and shorter survival underlining the importance of miR21 in glioma biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2011-1156

Abstract 3369: Discordant immunohistochemical expression patterns of four different CD133 antibody clones in glioblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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 antibody clones recognizing different epitopes with different glycosylation status, confuses the field. In this study, we sat out to highlight if current discordant CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Paraffin embedded sections of glioblastoma, kidney, pancreas and placenta tissue as well as glioblastoma and retinoblastoma cell lines were stained with four different CD133 antibody clones and analyzed using light microscopy. Ten consecutive sections of glioblastomas were analyzed with each of the four CD133 clones using quantitative stereology. Results revealed presence of CD133+ niches in glioblastomas, often in close relation to blood vessels, using all four antibody clones. The distribution of identified niches did, however, rarely correspond among each antibody clone. Staining of glioblastoma single and niche cells was predominantly cytoplasmatic, which is opposed to the membranous staining observed in epithelial cells in kidney, pancreas and placenta tissues. Quantitative stereology revealed vast dissimilarities regarding fractions of CD133+ niches and single cells among the CD133 antibody clones. Generally, the fraction of CD133 positivity identified by clone W6B3C1 was significantly higher than CD133 fractions identified by clones AC133, ab19898 and C24B9. Furthermore, clone W6B3C1 was the only clone to stain tumor blood vessels. In conclusion, we report that using different CD133 antibodies for immunohistochemical identification of CD133+ cells on paraffin sections will most likely cause dissimilar results. Thus, direct comparison of CD133 studies using different primary CD133 antibodies should be performed with caution. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3369.