Stine Skov Jensen

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.

Glioma spheroids obtained via ultrasonic aspiration are viable and express stem cell markers: a new tissue resource for glioma research

BACKGROUND Ultrasonic aspirators allow safe, rapid, and accurate removal of brain tumors. However, the tissue fragments removed are used surprisingly little in research. OBJECTIVE To investigate whether such tissue fragments could be cultured as organotypic multicellular spheroids because access to biopsy tissue is often limited. METHODS Tissue fragments obtained by ultrasonic aspiration from 10 glioblastomas and tumor biopsy tissue from 7 of these tumors were cultured in serum-containing and serum-free medium. On culturing, the fragments formed spheroids, which were prepared for histology. Two glioblastoma cell lines from ultrasonic fragments and biopsy tissue were established as well. RESULTS Hematoxylin and eosin staining showed viable glioma spheroids obtained from both ultrasonic and biopsy tissue in both types of medium. Endothelial growth factor receptor and PTEN/chromosome 10 status was found to be preserved in most spheroids (7-8 of 10 tumors), together with the level of glial fibrillary acidic protein, von Willebrand factor, and Ki-67. The levels of stem cell markers CD133, Bmi-1, nestin, and Sox2 also were preserved. The ultrasonic spheroids had higher levels of glial fibrillary acidic protein and von Willebrand factor and lower levels of Bmi-1, nestin, Sox2, and Olig2 compared with conventional biopsy spheroids. For both types of spheroids, the stem cell medium seemed to favor expression of stem cell markers. The established cell lines were capable of both spheroid formation at clonal density and tumor formation in vivo. CONCLUSION Viable organotypic and proliferating spheroids were easily obtained from ultrasonic tissue fragments. The preservation of markers and the establishment of cell lines with tumor-initiating cell properties suggest ultrasonic spheroids as a new tissue resource for glioma research.

Establishment and Characterization of a Tumor Stem Cell-Based Glioblastoma Invasion Model

Aims Glioblastoma is the most frequent and malignant brain tumor. Recurrence is inevitable and most likely connected to tumor invasion and presence of therapy resistant stem-like tumor cells. The aim was therefore to establish and characterize a three-dimensional in vivo-like in vitro model taking invasion and tumor stemness into account. Methods Glioblastoma stem cell-like containing spheroid (GSS) cultures derived from three different patients were established and characterized. The spheroids were implanted in vitro into rat brain slice cultures grown in stem cell medium and in vivo into brains of immuno-compromised mice. Invasion was followed in the slice cultures by confocal time-lapse microscopy. Using immunohistochemistry, we compared tumor cell invasion as well as expression of proliferation and stem cell markers between the models. Results We observed a pronounced invasion into brain slice cultures both by confocal time-lapse microscopy and immunohistochemistry. This invasion closely resembled the invasion in vivo. The Ki-67 proliferation indexes in spheroids implanted into brain slices were lower than in free-floating spheroids. The expression of stem cell markers varied between free-floating spheroids, spheroids implanted into brain slices and tumors in vivo. Conclusion The established invasion model kept in stem cell medium closely mimics tumor cell invasion into the brain in vivo preserving also to some extent the expression of stem cell markers. The model is feasible and robust and we suggest the model as an in vivo-like model with a great potential in glioma studies and drug discovery.

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.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

Comparative studies of TIMP-1 immunohistochemistry, TIMP-1 FISH analysis and plasma TIMP-1 in glioblastoma patients

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with poor prognosis and resistance towards chemotherapy in several cancer forms. In a previous study we found an association between a low TIMP-1 tumor immunoreactivity and increased survival for glioblastoma patients, when compared to moderate and high TIMP-1 tumor immunoreactivity. The aim of the present study was to further evaluate TIMP-1 as a biomarker in gliomas by studying TIMP-1 gene copy numbers by fluorescence in situ hybridization (FISH) on 33 glioblastoma biopsies and by measuring levels of TIMP-1 in plasma obtained pre-operatively from 43 patients (31 gliomas including 21 glioblastomas) by enzyme-linked immunosorbent assay (ELISA). The results showed TIMP-1 gene copy numbers per cell ranging from 1 to 5 and the TIMP-1/CEN-X ratio ranging between 0.7 and 1.09, suggesting neither amplification nor loss of the TIMP-1 gene. The TIMP-1 protein levels measured in plasma were not significantly higher than TIMP-1 levels measured in healthy subjects. No correlation was identified between TIMP-1 tumor cell immunoreactivities and the TIMP-1 gene copy numbers or the plasma TIMP-1 levels. In conclusion, high immunohistochemical TIMP-1 protein levels in glioblastomas were not caused by TIMP-1 gene amplification and TIMP-1 in plasma was low and not directly related to tumor TIMP-1 immunoreactivity. The study suggests that TIMP-1 immunohistochemistry is the method of choice for future clinical studies evaluating TIMP-1 as a biomarker in glioblastomas.

Abstract 4305: Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

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.

Abstract 5563: Comparative studies of TIMP-1 immunohistochemistry, TIMP-1 FISH analysis and plasma TIMP-1 in glioblastoma patients

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with poor prognosis in various cancers as well as with resistance towards chemotherapy. In a previous study we found an association between a high immunohistochemical TIMP-1 protein level and a shorter survival for glioblastoma patients. The aim of the present study was to further evaluate TIMP-1 as a prognostic marker in gliomas by performing TIMP-1 fluorescence in situ hybridization (FISH) and by measuring TIMP-1 in plasma. TIMP-1 FISH analysis was performed on 33 glioblastoma biopsies in order to elucidate if the increased levels of TIMP-1 protein could be explained by gene amplification. Moreover, blood samples were collected from 43 patients (31 gliomas including 21 glioblastomas) prior to brain tumor surgery. The plasma TIMP-1 protein level was measured by ELISA. The results showed TIMP-1 FISH scores between 0.7 and 1.1, suggesting neither amplification nor loss of the TIMP-1 gene. The TIMP-1 level measured in plasma was not significantly higher than the TIMP-1 level measured in healthy matched controls. No correlation was identified between the immunohistochemical TIMP-1 levels and the FISH scores or the plasma TIMP-1 levels. In conclusion, TIMP-1 FISH scores or plasma TIMP-1 levels are without prognostic potential in gliomas. The present study suggests that the high immunohistochemical TIMP-1 protein levels in gliomas are not caused by TIMP-1 gene amplification. Moreover, TIMP-1 in plasma is low and not directly related to the tumor TIMP-1 level found by immunohistochemistry. The study suggests that TIMP-1 immunohistochemistry is the method of choice when using TIMP-1 as a biomarker in gliomas. 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 5563. doi:1538-7445.AM2012-5563

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 568: Effects of irradiation on glioblastoma spheroids cultured in stem cell medium

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Radiotherapy is an important part of the treatment of glioblastomas – the most frequent and malignant primary brain tumor. However, the current treatment remains palliative and new treatments are urgently needed. Since a subpopulation of cells – cancer stem cells – is believed to be particularly resistant to the current treatment and is most likely the cause of tumor recurrence, the use of models preserving cancer stem cell features is of increasing importance. Recent reports have suggested that glioblastoma spheroids derived from short term cultures in stem cell medium preserve the pheno- and genotype better than when cultured in serum containing medium. With this background, the aim of this study was to investigate the response of irradiation of such spheroids as well as spheroids implanted into organotypic corticostriatal slice cultures in order to have a model preserving the invasive feature of glioblastomas. The response to irradiation (2, 5, 10 and 50 Gy) was investigated in spheroids from a short term culture with cancer stem cell-like properties established in our laboratory (C76) and the U87 glioblastoma cell line for comparison. Cell death was measured by uptake of propidium iodide (PI) and release of lactate dehydrogenase (LDH) and moreover, expression of p53 and the proliferation marker (MIB-1) was assessed by immunohistochemical staining. The formation of secondary spheroids after irradiation was evaluated using a limiting dilution assay as well as a spheroid proliferation assay at non-clonal density. Finally, fluorescently labeled C76 spheroids implanted into slice cultures were irradiated and the response on invasion was evaluated by confocal microscopy and anti-human vimentin immunohistochemical staining. For both C76 and U87 spheroids, the results showed a minor increase in PI uptake with increasing irradiation dose but no increase in LDH release. The expression of p53 also increased, whereas MIB-1 was reduced. The spheroid formation capacity in the limiting dilution assay was higher for C76 than U87 but decreased for both C76 and U87 with increasing irradiation dose along with a decrease in the number and size of secondary formed spheroids in the spheroid proliferation assay. The spheroid formation capacity was clearly best preserved for C76, but otherwise the results did not show major differences for C76 and U87. Using the invasion assay, the invasion of C76 spheroids into brain tissue was not affected by irradiation. In conclusion, irradiation of glioblastoma spheroids derived from short term cultures with cancer stem cell-like properties is feasible and easy to use as an in vitro model. The results might suggest that irradiation also affects tumor cells with a more stem-like phenotype although the effects on the unique tumor stem cells need to be further investigated. 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 568. doi:10.1158/1538-7445.AM2011-568