Overexpression of TIMP-1 and Sensitivity to Topoisomerase Inhibitors in Glioblastoma Cell Lines.

The multifunctional protein - tissue inhibitor of metalloproteinases-1 (TIMP-1) - has been associated with a poor prognosis in several types of cancers including glioblastomas. In addition, TIMP-1 has been associated with decreased response to chemotherapy, and especially the efficacy of the family of topoisomerase (TOP) inhibitors has been related to TIMP-1. As a second line treatment of glioblastomas, the vascular endothelial growth factor (VEGF) antibody bevacizumab is administered in combination with the TOP1 inhibitor irinotecan and glioblastoma cell levels of TIMP-1 could therefore potentially influence the efficacy of such treatment. In the present study, we aimed to investigate whether a high TIMP-1 expression in glioblastoma cell lines would affect the sensitivity to TOP inhibitors, and whether TIMP-1 overexpressing cells would have alterered growth and invasion. We established TIMP-1 overexpressing subclones from two human glioblastoma cell lines. TIMP-1 overexpressing U87MG cells were significantly more resistant than low TIMP-1 expressing clones and parental cells when exposed to SN-38 (TOP1 inhibitor) or epirubicin (TOP2 inhibitor). No significant differences were observed for the TIMP-1 transfected A172 cells. Implantation of both U87MG and A172 spheroids into organotypic brain slice cultures revealed a reduced growth of TIMP-1 overexpressing U87MG spheroids, however, no significant differences in invasion were observed. The present study suggests that TIMP-1 overexpression reduces the effect of TOP inhibitors in glioblastoma. TIMP-1 also appeared to reduce spheroid growth, but did not influence invasion. Whether TIMP-1 plays a role in irinotecan resistance and has a predictive potential in glioblastoma patients remains to be elucidated.

Heterogenic expression of stem cell markers in patient-derived glioblastoma spheroid cultures exposed to long-term hypoxia.

AIM: To investigate the time profile of hypoxia and stem cell markers in glioblastoma spheroids of known molecular subtype. MATERIALS & METHODS: Patient-derived glioblastoma spheroids were cultured up to 7 days in either 2% or 21% oxygen. Levels of proliferation (Ki-67), hypoxia (HIF-1α, CA9 and VEGF) and stem cell markers (CD133, nestin and musashi-1) were investigated by immunohistochemistry. RESULTS: Hypoxia markers as well as CD133 and partially nestin increased in long-term hypoxia. The proliferation rate and spheroid size were highest in normoxia. CONCLUSION: We found differences in hypoxia and stem cell marker profiles between the patient-derived glioblastoma cultures. This heterogeneity should be taken into consideration in development of future therapeutic strategies.

Co-expression of TIMP-1 and its cell surface binding partner CD63 in glioblastomas.

BACKGROUND: We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1. METHODS: CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation. RESULTS: The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2. CONCLUSION: The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness.

Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo.

BACKGROUND: Glioblastoma is the most frequent and most malignant brain tumor with the patients having a median survival of only 14.6 months. Although glioblastoma patients are treated with surgery, radiation and chemotherapy recurrence is inevitable. A stem-like population of radio- and chemoresistant brain tumor-initiating cells combined with the invasive properties of the tumors is believed to be critical for treatment resistance. In the present study, the aim was to investigate the effect of a novel therapeutic strategy using the lysosomotropic detergent siramesine on glioblastomas. METHODS: Standard glioma cell lines and patient-derived spheroids cultures with tumor-initiating stem-like cells were used to investigate effects of siramesine on proliferation and cell death. Responsible mechanisms were investigated by inhibitors of caspases and cathepsins. Effects of siramesine on migrating tumor cells were investigated by a flat surface migration assay and by implanting spheroids into organotypic rat brain slice cultures followed by confocal time-lapse imaging. Finally the effect of siramesine was investigated in an orthotopic mouse glioblastoma model. Results obtained in vitro and in vivo were confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains. RESULTS: The results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal membrane. Co-treatment of the cell lines with inhibitors of caspases and cathepsins suggested differential involvement in cell death. Siramesine caused tumor cell death and reduced secondary spheroid formation of patient-derived spheroid cultures. In the flat surface migration model siramesine caused tumor cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model. CONCLUSIONS: In conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal destabilizing drugs in killing glioblastoma cells, but siramesine was without effect in the organotypic spheroid-brain slice culture model and the in vivo xenograft model.

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.

Highly Effective Auger-Electron Therapy in an Orthotopic Glioblastoma Xenograft Model using Convection-Enhanced Delivery.

Glioblastoma, the most common and malignant primary brain tumor, always recurs after standard treatment. Therefore, promising new therapeutic approaches are needed. Short-range Auger-electron-emitters carry the ability of causing highly damaging radiation effects in cells. The aim of this study was to test the effect of [125I]5-Iodo-2'-deoxyuridine (125I-UdR, a radioactive Auger-electron-emitting thymidine analogue) Auger-therapy on immature glioblastoma spheroid cultures and orthotopic xenografted glioblastoma-bearing rats, the latter by means of convection-enhanced delivery (CED). Moreover, we aimed to determine if the therapeutic effect could be enhanced when combining 125I-UdR therapy with the currently used first-line chemotherapeutic agent temozolomide. 125I-UdR significantly decreased glioblastoma cell viability and migration in vitro and the cell viability was further decreased by co-treatment with methotrexate and/or temozolomide. Intratumoral CED of methotrexate and 125I-UdR with and without concomitant systemic temozolomide chemotherapy significantly reduced the tumor burden in orthotopically xenografted glioblastoma-bearing nude rats. Thus, 100% (8/8) of the animals survived the entire observation period of 180 days when subjected to the combined Auger-chemotherapy while 57% (4/7) survived after the Auger-therapy alone. No animals (0/8) treated with temozolomide alone survived longer than 50 days. Blood samples and post-mortem histology showed no signs of dose-limiting adverse effects. In conclusion, the multidrug approach consisting of CED of methotrexate and 125I-UdR with concomitant systemic temozolomide was safe and very effective leading to 100% survival in an orthotopic xenograft glioblastoma model. Therefore, this therapeutic strategy may be a promising option for future glioblastoma therapy.

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.

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.

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.

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.

Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stability and causes efficient target de-repression: a proof of concept.

Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2'-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1 month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5 days inhibition was superior to 1 day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24 h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed-as an initial proof of concept-that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.

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

UNLABELLED: 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.

Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas.

Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

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.

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.

Effects of chemotherapeutics on organotypic corticostriatal slice cultures identified by a panel of fluorescent and immunohistochemical markers.

Effects of chemotherapeutics on glioma cell lines and spheroids are usually investigated without evaluating the effects of chemotherapeutics on normal brain tissue. To perform such investigations, the aim of this study was to establish a panel of markers for detection of general cell death and more specific neuronal and glial degeneration induced by chemotherapeutics in organotypic rat corticostriatal slice cultures. The slice cultures were exposed to the alkylating agents temozolomide (TMZ) and nimustine (ACNU), the tyrosine kinase inhibitor imatinib mesylate (IM) and the microtubule-destabilizing agent vincristine (VCR). Densitometric measurements of uptake of the fluorescent dye propidium iodide (PI) were used for quantifying cellular degeneration. Moreover, paraffin sections were hematoxylin eosine stained and immunostained for the neuronal marker microtubule-associated protein 2 (MAP2), the astroglial marker glial fibrillary acidic protein (GFAP), and the oligodendroglial marker p25α. The results showed that the supposed clinically relevant drug concentrations were non-toxic. However, a time dependent increase in PI uptake was observed for high drug concentrations, except for TMZ, where no toxicity was observed. Corresponding immunostaining showed loss of MAP2 and increased expression of GFAP and p25α for cultures exposed to 1,000 nM VCR. Cultures exposed to high concentrations of ACNU and IM disintegrated, leaving no tissue for histology. In conclusion, corticostriatal slice cultures and the established panel of markers represent an excellent tool for detecting toxicity induced by chemotherapeutics. Toxicity was not detected at clinical concentrations, but high concentrations with toxic effects were identified suggesting that some of the earlier identified anti-cancer effects are general cytotoxic effects and not specific anti-cancer effects.

Effects of hypoxia on expression of a panel of stem cell and chemoresistance markers in glioblastoma-derived spheroids.

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.

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

OBJECTIVE: To 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. METHODS: Paraffin 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. RESULTS: Scoring 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. CONCLUSION: In 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.

Low expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) in glioblastoma predicts longer patient survival.

In colorectal cancer and breast cancer a high TIMP-1 level has been shown to correlate with a shorter overall patient survival and it has been suggested that TIMP-1 is involved in tumour invasion, proliferation and apoptosis in different types of cancers. TIMP-1 is known to be expressed in gliomas but whether TIMP-1 is a prognostic marker in gliomas has not previously been investigated. In the present study, the TIMP-1 expression was investigated immunohistochemically in 112 formalin-fixed paraffin embedded astrocytomas and related to tumour grade and overall patient survival by scoring the TIMP-1 immunoreactivity of both tumour cells and blood vessels. Moreover, TIMP-1 in situ hybridisation was performed on ten of the glioblastomas. In the vast majority of the tumours TIMP-1 protein was expressed in both tumour cells and blood vessels. In situ hybridisation for TIMP-1 mRNA on glioblastomas confirmed the immunohistochemical expression of TIMP-1. The percentage of TIMP-1 positive tumour cells and blood vessels as well as the staining intensity varied between tumours of the same grade, but the total staining score increased with tumour grade. The multivariate Cox regression test showed that glioblastoma patients with the lowest TIMP-1 expression had a significantly longer overall survival (HR (95% CI) = 3.2 (1.5-6.7), P = 0.004) when compared to the patients with higher TIMP-1 protein expression. In conclusion, this study showed that low TIMP-1 immunohistochemical expression predicts longer overall survival in glioblastoma patients, suggesting a role for TIMP-1 as a biomarker in glioblastoma.