Charlotte Aaberg-Jessen

Immunohistochemical expression of stem cell, endothelial cell, and chemosensitivity markers in primary glioma spheroids cultured in serum-containing and serum-free medium

OBJECTIVETo investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas. METHODSParaffin sections of the original surgical specimens, primary glioma spheroids, and U87 derived spheroids were stained immunohistochemically with the stem cell markers CD133, podoplanin, Sox2, Bmi-1, and nestin; the endothelial cell markers CD31, CD34, and Von Willebrand Factor (VWF); the chemosensitivity markers P-glycoprotein and tissue inhibitor of metalloproteinases-1 (TIMP-1); and glial fibrillary acidic protein, neural cell adhesion molecule CD56, and the proliferation marker Ki67. RESULTSScoring of the immunohistochemical stainings showed that the expression of CD133 and all other markers included was preserved in primary spheroids, confirming the in vivo-like nature of these spheroids. Spheroids in SFM better mimicked the in vivo phenotype with significantly more CD133, CD34, VWF, P-glycoprotein, TIMP-1, and Ki67 compared with SCM. CONCLUSIONIn this first study of the influence of SFM on primary glioma spheroids, the conditions favored an in vivo-like phenotype with increased expression of CD133. More vascular structures were found in SFM, suggesting that the close relationship between blood vessels and tumor stem-like cells was better preserved in this medium.

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.

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.

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.

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.

Estimation of Tumor Volumes by 11C-MeAIB and 18F-FDG PET in an Orthotopic Glioblastoma Rat Model

Brain tumor volume assessment is a major challenge. Molecular imaging using PET may be a promising option because it reflects the biologically active cells. We compared the agreement between PET- and histology-derived tumor volumes in an orthotopic glioblastoma rat model with a noninfiltrating (U87MG) and an infiltrating (T87) tumor phenotype using 2 different radiotracers, 2 different image reconstruction algorithms, parametric imaging, and 2 different image segmentation techniques. Methods: Rats with U87MG- and T87-derived glioblastomas were continuously scanned with PET for 1 h starting immediately after the injection of 11C-methylaminoisobutyric acid (11C-MeAIB). One hour later, 18F-FDG was injected, followed by a 3-h dynamic PET scan. Images were reconstructed using 2-dimensional ordered-subsets expectation maximization and 3-dimensional maximum a posteriori probability (MAP3D) algorithms. In addition, a parametric image, encompassing the entire tumor kinetics in a single image, was calculated on the basis of the 11C-MeAIB images. All reconstructed images were segmented by fixed thresholding of maximum voxel intensity (VImax) and mean background intensity. The agreement between PET- and histology-derived tumor volumes and intra- and interobserver agreement of the PET-derived volumes were evaluated using Bland–Altman plots. Results: By PET, the mean U87MG tumor volume was 35.0 mm3 using 18F-FDG and 34.1 mm3 with 11C-MeAIB, compared with 33.7 mm3 by histology. Corresponding T87 tumor volumes were 122.1 mm3 using 18F-FDG, 118.3 mm3 with 11C-MeAIB, and 125.4 mm3 by histology. None of these volumes were significantly different. The best agreement between PET- and histology-derived U87MG tumor volumes was achieved with 11C-MeAIB, MAP3D reconstruction, and fixed thresholding of VImax. The intra- and interobserver agreement was high using this method. For T87 tumors, the best agreement between PET- and histology-derived volumes was obtained using 18F-FDG, MAP3D reconstruction, and fixed thresholding of mean background intensity. The agreement using 11C-MeAIB, parametric imaging, and fixed thresholding of VImax was slightly inferior, but the intra- and interobserver agreement was clearly superior. Conclusion: Estimation of tumor volume by PET of noninfiltrating brain tumors was accurate and reproducible. In contrast, tumor volume estimation by PET of infiltrating brain tumors was difficult and hard to reproduce. On the basis of our results, PET evaluation of highly infiltrating brain tumors should be further developed.

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 1214: TIMP-1 and the TIMP-1 interacting cell surface protein CD63 in cultured astrocytoma derived spheroids: expression and co-expression with stem cell markers

In previous studies we have shown that the immunohistochemical expression of TIMP-1 (tissue inhibitor of metalloproteinases-1) and CD63 increases with tumor grade in astrocytomas grade II-IV. Moreover, we demonstrated that a high TIMP-1 protein expression in glioblastomas was associated with a shorter overall patient survival. Recent studies have suggested that TIMP-1 can prevent chemo-induced apoptosis and in a study, using human breast epithelial cells (MCF10A), it was demonstrated that the interaction of TIMP-1 and CD63 is necessary for the TIMP-1 anti-apoptotic pathway. The aim of the present study was to investigate the expression of TIMP-1 and CD63 in cultured organotypic multicellular spheroids (OMS) and cell line spheroids (CLS) derived from astrocytomas in order to assess spheroid models for future studies involving TIMP-1, CD63 and chemo-resistance. By investigating the spheroids immunohistochemically, we wanted to elucidate if TIMP-1 and CD63 are co-expressed within the spheroids and whether they are expressed by tumor stem like-cells, since these cells are known to be more resistant to chemotherapeutic treatment. In the present study OMS from nine astrocytomas and CLS from three commercial astrocytoma cell lines were included as well as the glioblastoma tumor stem cell line SJ-1 made in our laboratory. In general high levels of CD63 protein was detected in all the original tumors, whereas TIMP-1 was moderately expressed. TIMP-1 and CD63 expression in OMS was similar to the expression in the original tumors. TIMP-1 was expressed at low to moderate levels in CLS, whereas CD63 was expressed by all tumor cells in all spheroids. TIMP-1/CD63 double immunofluorescence staining was performed on selected OMS and CLS showing tumor cells expressing both proteins. Furthermore, double staining was performed with TIMP-1 and the stem cell markers CD133, nestin and Sox2 demonstrating that a population of the tumor stem like-cells expressed TIMP-1. In conclusion, this study shows that spheroid models can be used in future studies investigating the role of TIMP-1 and CD63 in chemo-induced apoptosis. Furthermore, these results indicate that a fraction of the tumor stem like-cells could be protected by TIMP-1/CD63 interaction. 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 1214.