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BACKGROUND: Both enhanced SPARC expression and the loss of p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival and inhibit apoptosis. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. METHODS: p53-null and Sparc-null mice were bred to obtain heterozygotes. These were crossed to obtain p53-null/Sparc-null neonates, which served as the source of p53-null/Sparc-null astrocytes for comparison to genetically-matched, control p53-null/Sparc-WT astrocytes. In vitro, Sparc loss was assessed using colony formation in soft agar, proliferation assay, flow cytometry, and Annexin V analysis. Orthotopic xenograft implantation was used to assess Sparc loss on tumor growth, proliferation, collagen deposition, and macrophage/microglia recruitment. RESULTS: In vitro, Sparc loss in p53-null astrocytes resulted in increased cell proliferation (2.7-fold; p < 0.01), but decreased cell survival (WT = 7.2% vs. null = 18.56% apoptotic cells) and a longer cell cycle recovery (2 days) after serum-withdrawal. Furthermore, Sparc loss suppressed growth in soft agar. Histomorphological analysis of CD68 and PAS ± diastase staining of tumor sections demonstrated that Sparc loss promoted macrophage/microglial activation (day 7 = 1.35-fold and day 50 = 1.93-fold; p < 0.001) and phagocytic morphology, respectively. At 7 days, Sparc-null tumors had decreased tumor cell survival, proliferation (MIB-1 for WT = 7.1% vs. null = 0.1%; p = 0.0345), and reduced tumor size (WT = 0.839 mm2 vs. null = 0.199 mm2; p = 0.0091). In addition, Sparc loss altered collagen deposition (WT = collagen bundles vs. null = long fiber structure). CONCLUSIONS: Our results indicate that Sparc loss significantly reduced the ability of p53-null astrocytes to recover from stress in vitro, and to survive and proliferate in vivo. This inability to grow in vivo was associated with an increase in macrophage/microglial activation and phagocytosis. Therefore, we conclude that the loss of p53 in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance. We propose that in the background of p53 loss/mutation, therapeutically suppressing SPARC alone, or in combination with other therapies, should promote the ability of patient's immune system to eradicate the tumor. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

We investigated whether cilengitide could amplify the antitumor effects of radiotherapy in an orthotopic rat glioma xenograft model. Cilengitide is a specific inhibitor of alphav series integrins, and acts as an antiangiogenic. U251 human glioma cells express alphavbeta3 and alphavbeta5 integrins. We used in vitro assays of adhesion and growth of tumor and endothelial cells to evaluate cytotoxicity and the potential for cilengitide to enhance radiation toxicity. Treatment was then evaluated in an orthotopic model to evaluate synergy with therapeutic radiation in vivo. In vitro, cilengitide blocked cell adhesion, but did not influence the effects of radiation on U251 cells; cilengitide strongly amplified radiation effects on endothelial cell survival. In vivo, radiotherapy prolonged the survival of U251 tumor-bearing rats from 50 to over 110 days. Cotreatment with cilengitide and radiation dramatically amplified the effects of radiation, producing survival over 200 days and triggering an enhanced apoptotic response and suppression of tumor growth by histology at necropsy. Signaling pathways activated in the tumor included NFkappab, a documented mediator of cellular response to radiation. Because cilengitide has a short plasma half-life (t((1/2)) approximately 20 min), antiangiogenic scheduling typically uses daily injections. We found that a single dose of cilengitide (4 mg/kg) given between 4 and 12 hr prior to radiation was sufficient to produce the same effect. Our results demonstrate that blockade of alphav integrins mediates an unanticipated rapid potentiation of radiation, and suggests possible clinical translation for glioma therapy.

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell-derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell-derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.

In this study we investigated epigenetic modifications such as DNA methylation, histone acetylation and histone methylation in the regulation of heparanase expression in glioblastoma. We found that heparanase promoters are differentially methylated among three glioblastoma cell lines; however, all these cells expressed baseline levels of heparanase. 5-Aza-2'-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, revoked heparanase expression in all the examined cells. Trichostatin A (TSA), a histone deacetylase inhibitor, activated heparanase expression in promoter unmethylated LN229 and T98G cells but not in promoter methylated U251n cells. To identify the mechanisms of heparanase induction by 5-Aza-dC, heparanase expression-related transcription factors were examined. No detected transcription factors (EGR1, Ets1, GABPα and Sp1) were found to be induced either by 5-Aza-dC or TSA. Furthermore, we found that 5-Aza-dC increased acetylation of histone H3 and di-methylation of histone H3 lysine K4 (H3K4me2) in LN229 and T98G cells. The increased histone acetylation and H3K4me2 were also observed in heparanase-expressing tumor tissues by immunohistochemistry staining. Additionally, we found that nuclear factor κB (NFκB) p65 but not NFκB p50 was correlated with heparanase expression, which could be expressed both by neoplastic cells and angiogenesis-related neovessel cells. However, we did not observe any regulatory mechanism between heparanase and NFκB p65 via transient transfection of their cDNA in T98G and U251n cells. We concluded that heparanase expression is associated with histone modifications and promoter DNA methylation plays a role in the control of gene silencing. Overexpression of both heparanase and NFκB p65 may be the result of excessive histone modifications.

SPARC (secreted protein acidic and rich in cysteine) is expressed in all grades of astrocytoma, including glioblastoma (GBM). SPARC suppresses glioma growth but promotes migration and invasion by mediating integrin and growth factor receptor-regulated kinases and their downstream effectors. PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is commonly lost in primary GBMs, negatively regulates proliferation and migration by inhibiting some of the same SPARC-mediated signaling pathways. This study determined whether PTEN reconstitution in PTEN-mutant, SPARC-expressing U87MG cells could further suppress proliferation and tumor growth but inhibit migration and invasion in SPARC-expressing cells in vitro and in vivo, and thereby prolong survival in animals with xenograft tumors. In vitro, PTEN reduced proliferation and migration in both SPARC-expressing and control cells, with a greater suppression in SPARC-expressing cells. PTEN reconstitution suppressed AKT activation in SPARC-expressing and control cells but suppressed the SHC-RAF-ERK signaling pathway only in SPARC-expressing cells. Importantly, coexpression of SPARC and PTEN resulted in the smallest, least proliferative tumors with reduced invasive capacity and longer animal survival. Furthermore, direct inhibition of the AKT and SHC-RAF-ERK signaling pathways suppressed the proliferation and migration of SPARC-expressing cells in vitro. These findings demonstrate that PTEN reconstitution or inhibition of signaling pathways that are activated by the loss of PTEN provide potential therapeutic strategies to inhibit SPARC-induced invasion while enhancing the negative effect of SPARC on tumor growth.

Cancer stem cells (CSCs) are characterized by their self-renewing potential and by their ability to differentiate and phenocopy the original tumor in orthotopic xenografts. Long-term propagation of glioblastoma (GBM) cells in serum-containing medium results in loss of the CSCs and outgrowth of cells genetically and biologically divergent from the parental tumors. In contrast, the use of a neurosphere assay, a serum-free culture for selection, and propagation of central nervous system-derived stem cells allows the selection of a subpopulation containing CSCs. Gliosarcoma (GS), a morphological variant comprising approximately 2% of GBMs, present a biphasic growth pattern, composed of glial and metaplastic mesenchymal components. To assess whether the neurosphere assay would allow the amplification of a subpopulation of cells with “gliosarcoma stem cell” properties, capable of propagating both components of this malignancy, we have generated neurospheres and serum cultures from primary GS and GBM surgical specimens. Neurosphere cultures from GBM and GS samples expressed neural stem cell markers Sox2, Musashi1, and Nestin. In contrast to the GBM neurosphere lines, the GS neurospheres were negative for the stem cell marker CD133. All neurosphere lines generated high-grade invasive orthotopic tumor xenografts, with histological features strikingly similar to the parental tumors, demonstrating that these cultures indeed are enriched in CSCs. Remarkably, low-passage GS serum cultures retained the expression of stem cell markers, the ability to form neurospheres, and tumorigenicity. The GS experimental tumors phenocopied the parental tumor, exhibiting biphasic glial and mesenchymal components, constituting a clinically relevant model to investigate mesenchymal differentiation in GBMs.

Glioblastomas are heterogeneous tumors displaying regions of necrosis, proliferation, angiogenesis, apoptosis and invasion. SPARC, a matricellular protein that negatively regulates angiogenesis and cell proliferation, but enhances cell deadhesion from matrix, is upregulated in gliomas (Grades II–IV). We previously demonstrated that SPARC promotes invasion while concomitantly decreasing tumor growth, in part by decreasing proliferation of the tumor cells. In other cancer types, SPARC has been shown to influence tumor growth by altering matrix production, and by decreasing angiogenesis via interfering with the VEGF-VEGFR1 signaling pathway. We therefore examined whether the SPARC-induced decrease in glioma tumor growth was also, in part, due to alterations in matrix and/or decreased vascularity, and assessed SPARC-VEGF interactions. The data demonstrate that SPARC upregulates glioma matrix, collagen I is a constituent of the matrix and SPARC promotes collagen fibrillogenesis. Furthermore, SPARC suppressed glioma vascularity, and this was accompanied by decreased VEGF expression and secretion, which was, in part, due to reduced VEGF165 transcript abundance. These data indicate that SPARC modulates glioma growth by altering the tumor microenvironment and by suppressing tumor vascularity through suppression of VEGF expression and secretion. These experiments implicate a novel mechanism, whereby SPARC regulates VEGF function by limiting the available growth factor. Because SPARC is considered to be a therapeutic target for gliomas, a further understanding of its complex signaling mechanisms is important, as targeting SPARC to decrease invasion could undesirably lead to the growth of more vascular and proliferative tumors. © 2008 Wiley-Liss, Inc.

Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Therefore, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival and inhibit apoptosis during glioma formation. This study determined whether the loss of Sparc in astrocytes that are null for p53 (p53-null/Sparc-null) would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation (15-33%, p Citation Format: Stacey L. Thomas, Chad R. Schultz, Ezekiell Mouzon, William A. Golembieski, Nancy Lemke, Laila M. Poisson, Jorge A. Gutierrez, Sandra Cottingham, Sandra A. Rempel. Loss of Sparc in p53-null astrocytes alters collagen deposition and promotes macrophage activation and tumor phagocytosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2015-2363

The high-mobility group–box transcription factor sex-determining region Y–box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications.

Background and Objective: Glioblastoma (GBM) is the most aggressive primary brain tumor. GBM cells are phenotypically plastic and can present cancer stem cell (CSC) properties, such as the ability to self-renew and differentiate into the cells comprising the bulk of the tumor, phenocopying the parental tumor in mouse xenografts. GBM CSCs express neural stem cell (NSC) markers, including Sox2, a HMG-box transcription factor that regulates the expression of genes associated with self-renewal and differentiation. Culturing dissociated GBM tumors in serum-containing medium typically results in loss of the CSC phenotype and downregulation of Sox2, while serum-free neurosphere medium (NM) is used for selection and propagation of CSCs. We have previously identified a GBM specimen (GS1) which, contrary to the norm, retained Sox2 expression in low passage serum culture, along with the ability to dedifferentiate into neurospheres in vitro, and to form tumors in rodents (deCarvalho et al., Stem Cells 2010, 28:181-190). Here we investigate the endogenous expression of Sox2 in GBM biopsies, xenograft tumors, and cultured cells, and test the hypothesis that Sox2 may be a driver of the cancer stem cell phenotype in GBMs. Experimental Approaches: Endogenous expression of Sox2, downstream targets, and cell lineage markers, was determined by immunohistochemistry. miRNAs targeting Sox2 were stably expressed in GBM cells. The effect of Sox2 knockdown on clonogenicity, global gene expression, proliferation in vitro, tumor formation and morphology was determined. Results: Sox2 expression was heterogeneous within and among the 26 GBM surgical specimens tested, ranging from 1% to over 80% positive nuclei. Robust Sox2 expression was observed in over 80% of 56 GBM orthotopic xenografts analyzed. Sox2 positive cells co-expressed mesenchymal (vimentin), neural (nestin), and astrocytic (GFAP) markers. Knocking down Sox2 expression significantly impaired the ability of GS1 cells to form neurospheres, without affecting cell proliferation, significantly altered the expression of known target genes, such as Nestin, and genes in various signaling pathways, and affected xenograft tumor formation. Sox2 protein levels in GBMs were further regulated by post-transcriptional mechanisms. Conclusions: Sox2 is part of the signature gene set for the proneural GBM subclass. Our results show that Sox2 protein expression is ubiquitous in GBM tumors and xenografts, independent of patient survival or treatment status. Sox2 is a master regulator of embryonic and neural stem cell phenotype, and our data contributes to the view that it may have an important role in the plasticity of GBM cells in acquiring CSC phenotype, with possible implications for the pathology of GBMs. 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 427. doi:1538-7445.AM2012-427

Global molecular profiling efforts are leading to the identification of prognostic gene expression signatures for gliobastoma. Recently, several extracellular matrix (ECM) components typically expressed in mesenchymal tissues, have been shown to be overexpressed in a subgroup of high grade gliomas from poor prognosis patients, including the highly related small leucine-rich repeat proteoglycans (SLRPs) biglycan and decorin. These proteins were originally isolated from cartilage and are widely expressed in differentiated mesenchymal tissues, but their involvement in glioblastoma pathology is not understood. Biglycan expression in glioblastoma surgical specimens, cancer stem cells, and xenografts was assessed by RNA microarray, real-time PCR, Western blot and immunohistochemistry, and compared to decorin. Over 80% of the glioblastoma surgical specimens expressed both proteoglycans in the pericellular location of endothelial cells. In neoplastic cells, while decorin was observed in the pericellular location of discrete regions of about 40% of the samples, biglycan localized to the nucleus in most tumors analyzed. It has been previously reported that biglycan possess a nuclear localization signal, but in vivo nuclear localization in a glial tumor has not been shown. The unglycosylated core biglycan protein was ubiquitous, while the glycosylated form was less uniformly distributed among the tumor samples. Glioblastoma cancer stem cell enriched neurosphere cultures derived from the tumor specimens did not express biglycan or decorin in vitro. However, biglycan was re-expressed upon tumor formation, as nuclear staining was observed in the orthotopic tumor xenograft initiated by the neurospheres. Using the Rembrandt data base (http://caintegrator-info.nci.nih.gov/rembrandt) we observed a robust inverse correlation of biglycan expression in GBMs with survival. The nuclear localization in GBMs suggests an unconventional role for this proteoglycan in the pathology of this malignancy. 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 4155.