Abstract C230: Osteopontin as a driving force of glioma-related inflammation and a candidate for therapeutic targeting in malignant gliomas?

Abstract

Novel concepts of anticancer therapies focus on targeting tumor-supportive microenvironment and/or cancer stem-like cells (CSC). We combined two approaches in a quest for novel targets for malignant glioma therapy. Brain macrophages accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and cause immunosuppression. We found that one of glioma- derived factors crucial for activation of the pro-invasive phenotype is osteopontin (OPN, SPP1). OPN expression is up-regulated in many cancers, correlating with malignancy grade and poor patient prognosis. We demonstrated hundreds-fold increased expression of OPN in rat C6 glioma and elevated expression in human glioma cells in comparison to non-transformed astrocytes. Thus, we developed C6 glioma cell clones stably expressing control (shNeg) or OPN specific shRNA. Invasion of control and shOPN expressing glioma cells was evaluated in Matrigel assay after culturing cells for 36 h in the presence or absence of brain macrophages (microglia). Quantification of invading glioma cells, stained with a fluorescent dye was performed by laser scanner cytometry. Knockdown of OPN greatly diminished both basal and microglia-induced invasion of glioma cells. To evaluate contribution of OPN to glioma growth in vivo, we injected shOPN or shNeg glioma clones into the striatum of Wistar rats. Silencing of OPN in glioma cells greatly reduced the tumor volume on 15th day after tumor implantation. Staining for Iba-1 positive microglia/macrophages demonstrated no difference in the number of brain macrophages infiltrating gliomas. However, Iba1-positive cells infiltrating tumors with silenced OPN expression displayed ramified morphology, contrary to amoeboid, fully activated cells infiltrating control tumors. As glioma initiating stem-like cells were suggested to induce immunosuppressive macrophages, we hypothesized that OPN expression may differ in CSC. We employed commonly used procedures to enriched in CSC: rhodamine 123 exclusion and sphere-forming assays. Cells negative for rhodamine 123 were considered as a fraction enriched in CSC and sorted. Spheres grown in neural stem cell medium supplemented with epidermal growth factor and basic fibroblast growth factor were collected at 14th day after seeding. Enrichment in CSC was confirmed by increased expression of the markers of cancer stem cells: Nanog and Oct3/4 in selected cell populations. We demonstrate that human glioma cell populations enriched in CSC exhibit higher expression of OPN than bulk cells. To identify signaling pathways regulating OPN expression, glioma cells were exposed to a panel of signal transduction inhibitors, and OPN mRNA and protein levels were determined by qPCR and ELISA. Those studies showed involvement of NF B and ERK signaling pathways in regulation of OPN expression. We conclude that OPN overexpressed in glioma cells, crucial for both tumor invasion and macrophage activation is a potential promising target for glioma therapy. High expression of OPN in CSC points to its crucial role in glioma pathology. The study was supported by grants N N301 290837 and N N301 786240 from the Polish Ministry of Science and Higher Education Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C230.