Robert J. Winn

Leptin Promotes Glioblastoma

The hormone leptin has a variety of functions. Originally known for its role in satiety and weight loss, leptin more recently has been shown to augment tumor growth in a variety of cancers. Within gliomas, there is a correlation between tumor grade and tumor expression of leptin and its receptor. This suggests that autocrine signaling within the tumor microenvironment may promote the growth of high-grade gliomas. Leptin does this through stimulation of cellular pathways that are also advantageous for tumor growth and recurrence: antiapoptosis, proliferation, angiogenesis, and migration. Conversely, a loss of leptin expression attenuates tumor growth. In animal models of colon cancer and melanoma, a decline in the expression and secretion of leptin resulted in a reduction of tumor growth. In these models, positive mental stimulation through environmental enrichment decreased leptin secretion and improved tumor outcome. This review explores the link between leptin and glioblastoma.

Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique.

Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.

Glioblastoma Derived Exosomes Induce Apoptosis in Cytotoxic T Cells Through a Fas Ligand Mediated Mechanism

Introduction Glioblastoma multiforme deploys a number of weapons to thwart the immune system. Within the tumor microenvironment, tumor infiltrating T cells fall victim to cellular and soluble mediators of apoptosis. In prostate and colorectal cancer models, exosomes can induce T cell apoptosis. Exosomes are tiny, membrane bound vesicles that are released from a cell. They contain functional mRNA and protein and have cell surface molecules representative of their parent cell. It is not known if GBM derived exosomes can also mediate apoptosis. In this study, the role of GBM derived exosomes in T cell apoptosis is explored.

Abstract 5406: Glioblastoma derived exosomes induce apoptosis in T cells

Intro: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and is associated with an average lifespan of 15 months after diagnosis. One contributing factor to this poor prognosis is the immune protection afforded by the tumor microenvironment. One method of immune evasion poorly characterized is the effect of tumor released exosomes. Methods: FasL gene expression was determined using reverse transcription polymerase chain reaction (RT-PCR) in three GBM cells lines: LN-229, U-138 MG and T98G. Exosomes were isolated from serum free supernatants by differential ultracentrifugation. Exosomes were quantified using a protein concentration assay. A3 Jurkat T cells were treated with quantified exosomes, recombinant FasL or untreated. After 24 hours a TUNEL assay was performed to investigate the potential apoptotic effects of GBM-derived exosomes. Results: FasL is expressed in LN-229, U-138 MG and T98G cell lines as determined by RT-PCR. After treating A3 Jurkat T cells with exosomes it appeared apoptosis was induced as compared to an untreated control as determined by DNA fragmentation. Discussion: Studies using prostate and colon cancer cell lines showed that FasL was expressed in tumor derived exosomes. When T cells were co-cultured with exosomes apoptosis was induced in a FasL-dependant manner. Our lab previously demonstrated that GBM-derived exosomes are able to inhibit T cell proliferation. These current data suggests that GBM-derived exosomes induce apoptosis in T cells. 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 5406. doi:1538-7445.AM2012-5406

Abstract 3358: 1α,25-dihydroxyvitamin D3 inhibits the proliferation of cultured glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is a common, aggressive type of brain tumor with a median survival of only fifteen months. Brain tumor stem cells (BTSCs) within GBM tumors resist standard treatments, initiate recurrence, and pose a significant challenge for GBM treatment. This study examined Gli1 expression and proliferation of GBM cells in vitro to evaluate the effects of 1α,25-dihydroxyvitamin D3 (vitamin D3). Vitamin D3 is a safe, natural inhibitor of the hedgehog signaling pathway–a mechanism essential to BTSC function. Immunocytochemistry demonstrated that both established GBM cell lines and GBM-derived BTSCs expressed Gli1, indicating hedgehog signaling pathway activity within the cell populations. Vitamin D3 treatment significantly reduced cell proliferation, both in GBM cell lines and GBM-derived BTSCs. Because of active vitamin D39s environmental instability, an in vivo model might provide a better indication of its anti-tumor effects. Ultimately, vitamin D3 may enhance standard GBM treatments by inhibiting hedgehog-expressing cells. 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 3358. doi:1538-7445.AM2012-3358