Background/Aims: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The defining characteristics of GBM are diffuse infiltration of tumor cells into normal brain parenchyma, rapid growth, a high degree of infiltration of microglia and macrophages, and the presence of necrosis. Microglia/macrophages are frequently found in gliomas and they extensively infiltrate GBM tissue, up to 30% of total tumor mass. However, little is known about the effect of necrotic cells (NCs) on microglia infiltration in GBM and the tumor-infiltrating microglia-induced factors in GBMs. Methods: In this study, to address whether necrosis or necrosis-exposed GBM cells affect the degree of microglia/macrophage infiltration, migration and invasion/infiltration assays were performed. Culture supernatants and nuclear extracts of CRT-MG cells treated or untreated with necrotic cells were analyzed using a chemokine array and electrophoretic mobility shift assay, respectively. Results: The presence of NCs promoted the migration/infiltration of microglia, and GBM cell line CRT-MG cells exposed to NCs further enhanced the migration and infiltration of HMO6 microglial cells. Treatment with NCs induced mRNA and protein expression of chemokines such as Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and Macrophage Inflammatory Protein-3α (CCL20/MIP-3α) in CRT-MG cells. In particular, CCL2/MCP-1 and CCL20/MIP-3α were significantly increased in NC-treated CRT-MG cells. NCs induced DNA binding of the transcription factors Nuclear Factor (NF)-κB and Activator Protein 1 (AP-1) to the CCL2/MCP-1 and CCL20/MIP-3α promoters, leading to increased CCL2/MCP-1 and CCL20/MIP-3α mRNA and protein expression in CRT-MG cells. Conclusion: These results provide evidence that NCs induce the expression of CCL2/MCP-1 and CCL20/MIP-3α in glioblastoma cells through activation of NF-κB and AP-1 and facilitate the infiltration of microglia into tumor tissues.

Background/Aims: Leptin is a hormone expressed by adipose tissue that regulates body energy homeostasis and weight loss by activating leptin receptors in the hypothalamus. Leptin receptors are also expressed in astrocytes. An anti-apoptosis effect of leptin in brain has recently been reported. However, the anti-apoptosis mechanism of leptin in the brain is unknown. Methods: To investigate whether leptin exerts protective effects against glutamateinduced apoptosis in astrocytes, we performed cell viability assays and apoptosis assays using rat primary astrocytes. Intracellular signaling pathways involved in anti-apoptosis effects of leptin were analyzed by immunoblotting together with a leptin mutant (S120A/T121A) with antagonist function and pharmacological inhibitors. Results: We found that glutamateinduced apoptosis in rat primary astrocytes was significantly decreased by treatment with leptin. Leptin inhibited glutamate-induced phosphorylation of ERK1/2 in astrocytes. The leptin S120A/T121A mutant did not inhibit glutamate-induced ERK1/2 phosphorylation and ERK1/2-mediated apoptosis. Conclusions: Collectively, our results provide initial evidence that leptin exerts an anti-apoptotic effect against glutamate toxicity through activation of intracellular signaling pathways which reverse glutamate-induced ERK1/2 phosphorylation in primary astrocytes. Therefore, our findings suggest that leptin might be considered a candidate for potential therapeutic applications in glutamate-induced brain excitotoxicity.

Glioblastoma multiform (GBM), a grade IV astrocytoma, is most lethal and common adult primary intracranial tumor. GBM is characterized by diffuse infiltration into normal brain parenchyma, rapid growth and the presence of necrosis and microglia/macrophage infiltration. Among these properties of GBM, necrosis has been implicated to be a strong predictor of poor prognosis; however, the effect of necrosis on GBM progression is poorly understood at present. In this study, we examined the effect of necrosis on glioblastoma cells by exploring molecular mechanisms underlying gene expression and chemokine expression. Data obtaining from chemokine array and ELISA showed that CRT-MG human gliomablastoma cells secreted several chemokines including MCP-1 and MIP-3α in response to necrotic cells. Expression levels of mRNA and protein of MCP-1 and MIP-3α were also increased by treatment with necrotic cells in CRT-MG in a dose-dependent manner. Necrotic cells induced NF-κB/AP-1 activation and their binding to the MCP-1 and MIP-3α promoter, leading to enhanced MCP-1 and MIP-3α production in GBM cells. Finally, transwell migration assay showed that incubation with necrotic cells significantly enhanced the migration of microglia. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the expression and secretion of MCP-1 and MIP-3α are enhanced in GBM cells and microglia infiltration/migration to the tumor site are facilitated. Citation Format: Yieun Jung, So-Hee Ahn, Hyunju Park, Jiwoo Lim, Jihee Lee Kang, Youn-Hee Choi, Eun Ju Kim. Necrotic cells promote microglia infiltration in glioblastoma through regulating MCP-1 and MIP-3α expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5924. doi:10.1158/1538-7445.AM2017-5924

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. The GBM-specific tumor microenvironment (TME) plays a crucial role in tumor progression, immune escape, local invasion, and metastasis of GBM. Here, we demonstrate that hypoxia, reactive oxygen species (ROS), and differential concentration of glucose influence the expression of cytokines and chemokines, such as IL-6, IL-8, and IP-10, in human glial cell lines. Treatment with cobalt chloride (CoCl2) and hydrogen peroxide (H2O2) significantly increased the expression levels of IL-6, IL-8, and IP-10 in a dose-dependent manner in CRT-MG and U251-MG astroglioma cells, but not in microglia cells. However, we found strikingly different patterns of expression of cytokines and chemokines between H2O2-treated CRT-MG cells cultured in low- and high-glucose medium. These results suggest that astroglioma and microglia cells exhibit distinct patterns of cytokine and chemokine expression in response to CoCl2 and H2O2 treatment, and different concentrations of glucose influence this expression under either hypoxic or oxidant-enriched conditions.

were used. Pharmacological inhibition of HSP90 activity in these cell lines were achieved through geldanamycin and resorcinol derivatives. The response to these inhibitors at different time points was evaluated. Results: Westerns blots indicated that HSP70 and HSP90-a protein expression were increased after 17-AAG, IPI-504, STA-9090 and AUY-922 treatments. EGFR, EML4-ALK and CDK4, the oncogenic client proteins studied, were depleted by the HSP90 inhibitors in the NSCLC cell lines. The strong relationship between client driver protein dependence on Hsp90 and the sensitivity to its inhibition was demonstrated in the HCC827 and H3122 cell lines. Conclusions:The reduction of oncogenic client proteins alongside HSP70 and HSP90-a induction could be used as a validated biomarker signature of HSP90 inhibition in the cell lines studied. Future study will be focused on understanding the biological basis for the differential response to these treatments. Legal entity responsible for the study: Instituo de Biomedicina de Sevilla (IBIS) (CSIC, HUVR, Universidad de Sevilla) Funding: Fundación para la Investigación de Sevilla (FISEVI) Disclosure: All authors have declared no conflicts of interest.