Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

Here we describe a new method of glioma cell visualization in living brain slices that can be used for evaluation of tumor size or visualization of internal tumor structures. Glial cells, as well as glioma cells of glial origin, express high levels of organic cation transporters. We demonstrate that application of a fluorescent substrate for these transporters 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+) to the incubation medium leads to quick accumulation of fluorescence in glioma cells during early developmental stages and in astrocytes, but not in neurons. Stained brain slices can be immediately investigated using confocal or fluorescence microscopy. Glioma and glial cells can be discriminated from each other due to their different morphology. The method described has an advantage of staining living tissue and is simple to perform.

Organic cation transporters (OCTs) were first found and then isolated from cultured glioma cells. When glioma cells are implanted into brain the fate of OCTs varies with time after implantation and transporter type. Here we report that OCT1, OCT2 and OCT3 immunofluorescence is significantly reduced over time in implanted GL261 glioma cells, during tumor development in the brain. By day 21 after glioma implantation, OCT1, OCT2 and OCT3 immunofluorescence was reduced more than 10-fold in the cytoplasm of glioma cells, while OCT3 immunofluorescence became concentrated in the nucleus. The well-known fluorescent substrate for OCT transporters, NIH-PA Author Manuscript, previously shown to accumulate in glioma-cell cytoplasm in in vivo slices, began to accumulate in the nucleus of these cells, but not in cytoplasm, after 21 days post-implantation. Considering this mislocalization phenomenon, and other literature on similar nuclear mislocalization of different transporters, receptors and channels in glioma cells, we suggest that it is one of the "omens" preceding the motility and aggressivity changes in glioma behavior.

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Glioblastomas, the most malignant form of gliomas, harbor multiple cell types. In particular, microglial cells can contribute up to 30% of a brain tumor mass, and can promote glioma cell growth and dispersal. The purpose of the present study was to test the hypothesis that glioma cells recruit nearby microglia through an MCP-1-mediated mechanism and enhance their production of MCP-1 in the tumor microenvironment. We evaluated the role of MCP-1 on glioma cell proliferation and invasion. Consistent with previous studies in rat models (Platten et al., 2003), we found that U-87 and A-172 human glioma cells promote microglial migration towards the glioma environment using a modified Boyden chamber migration assay. Furthermore, this effect was decreased after immunoneutralization of MCP-1 released from glioma cells. Additionally, using an antibody array we found that U-87 or A-172 glioma cell conditioned medium stimulates a 7-10 fold increase of MCP-1 release by microglia by 24 hours. We next analyzed the consequences of elevated MCP-1 on the invasiveness and proliferation of glioma cells. Using standard invasion assays with or without microglia in a lower compartment, we demonstrated that microglia significantly increased invasion of glioma cells, but this effect was not blocked by MCP-1 immunoneutralization. This suggests that the stimulatory effect of microglia on glioma invasion was due to some other factor such as release of matrix metalloproteinases (MMPs). In contrast, microglia increased U-87 and A-172 glioma cell proliferation within 72 hours and MCP-1 immunoneutralization reversed this proliferative effect of microglia in both cell lines. Based on these results, we propose that glioma cells release low levels of MCP-1 to recruit nearby microglia and enhance the production of MCP-1. This increased secretion of MCP-1 from microglial cells recruits more microglial cells into the tumor and stimulate glioma progression. A variety of substances released by microglia in tumor microenvironment, including cytokines, growth factors, proteases and extracellular matrix components could lead to increased proliferation and invasiveness of glioma cells. Supported by G11 HD052352, G12 RR03035, 8G12MD007583-27, U54 NS039408 and by the UCC Pilot Project Program Citation Format: Lilia Y. Kucheryavykh, Aixa F. Rivera-Pagán, Kimberleve Rolón-Reyes, Serguey N. Skatchkov, Misty J. Eaton. Role of monocyte chemotactic protein-1 (MCP-1) in the tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2013-1429