Study Of Glioma Malignant Biological Behavior Mediated By Astrocytes In Tumor Microenvironment

Part I The mechanism of astrocytes promoting glioma chemoresistanceIntroductionGlioblastoma multiforme (GBM) is the most common and deadiest of primary malignant tumor in the central nervous system. Its invasive characteristics make complete resection difficult. Although surgery combined with radiotherapy plus temozolomide-based chemotherapy improve survival, the prognosis remains poor with high recurrence rates and a median survival of only approximate 14.6 months. Resistance to chemotherapy is one of the major causes of relapse as well as reduced survival in glioma patients. Temozolomide (TMZ) and vincristine (VCR) are two clinically widely used chemotherapeutic drugs, which have distinct anticancer mechanism. Although these anticancer agents show effective cytotoxicity on glioma cells in vitro, they fail to show satisfactory clinical outcomes in vivo because of drug resistance. Therefore, developing novel strategies to improve the efficacy of chemotherapy or overcoming chemoresistance is significant for glioma treatment and the focus of clinical research at present.The conventional research on resistance of tumors only focused on tumor itself. On the other hand, the tumor microenvironment, especially the tumor stromal compartments, has a major influence on tumors. Recently, accumulating researches have demonstrated that tumor stromal cells can influence the malignant biological behavior of tumor. Astrocytes are a unique group of stromal cells in the glioma microenvironment, with the widest distribution and the most abundant glial cell population. In addition, astrocytes are the first cell type in the brain to react with gliomas and surround them. Under pathological conditions, such as ischemia and trauma, astrocytes become activated, and can protect neurons from injury-induced apoptosis. Studies have revealed that astrocytes play an important role in glioma growth, invasion and angiogenesis. But few studies have investigated the role of astrocytes in glioma chemoresistance.ObjectiveThe main aims of our study are to explore the function and mechanism of astrocytes in glioma chemoresistance and to provide both new therapeutic target and theoretical basis for management of glioma, especially in avoiding resistance, enhancing sensitivity in chemotherapy.Methods and results1. Human astrocytes protect glioma cells from apoptosis induced by TMZ and VCR in vitro through direct contactWe established a co-culture system of CFSE-glioma cells and astrocytes to simulate the tumor microenvironment and to detect the effect of astrocytes on glioma chemoresistance. We found co-culture with human astrocytes significantly reduced the chemotherapy-induced apoptosis in human glioma cell A172 by approximate 50%. These data indicated that astrocytes could protect human glioma cells from apoptosis induced by TMZ or VCR.To determine whether protection by astrocytes required direct contact or cytokines secreted by astrocytes, we established both transwell and direct contact co-culture experiments to explore this issue. The apoptosis of glioma cells in the transwell co-culture system decreased slightly but not significantly. These results showed that the protection conferred by astrocytes required direct contact between astrocytes and glioma cells.2. Gap junctional communication (GJC) between astrocytes and glioma cells is required for protection from apoptosisThe gap junction (GJ) is a vital structure formed by direct cell-to-cell contact and has been reported to play a crucial role in apoptosis and death of tumor cells. To determine whether there was a functional gap junction between astrocytes and glioma cells, the function of the GJs was evaluated using the dye transfer assay. Transfer of calcein through GJ between astrocytes and glioma cells was observed when these cells were co-cultured in direct contact. Moreover, the potent GJC inhibitor carbenoxolone (CBX) significantly inhibited dye transfer between glioma cells and astrocytes.Then, we investigated the role of GJC between astrocytes and glioma cells in glioma cell chemoresistance. CBX treatment significantly promoted the glioma cells apoptosis in direct contact co-culture, as the apoptosis of glioma cells in co-culture with CBX was significantly higher than that without CBX, when incubation with TMZ or VCR. The data showed that GJC inhibition could reverse astrocytes’ protection of glioma cells from TMZ- and VCR-induced apoptosis, suggesting that GJC participated in astrocyte-mediated protection of glioma cells.3. The GJC-mediated protection of astrocytes is connexin 43 (Cx43) dependent and can be reversed by Cx43 knockdownAs connexin 43 (Cx43) is the major protein of GJ in both central nervous system and gliomas, we further investigated whether the protection by astrocytes mediated by GJC was dependent on Cx43. As immunofluorescence showed, both astrocytes and glioma cells expressed Cx43 protein, and GJ channels can be formed between them. Then we knocked down Cx43 protein in glioma cells using siRNAs to identify the function of Cx43 in GJC. Compared with the negative control group, the dye transfer was significantly decreased in gliomas transfected with siCx43 by 81.77% and showed no significant difference from the CBX group. These results suggested that small molecules transferred through GJC depended on Cx43, and Cx43 may be the main functional component of GJ between astrocytes and glioma cells.To further confirm Cx43 function in glioma chemoresistance to TMZ and VCR, we measured the apoptosis of siCx43-transfected glioma cells. When A172 cells were transfected with siCx43, the protection of glioma cells in co-culture from chemotherapeutic drugs was abolished, and neither did GJC inhibition by CBX treatment affect glioma cell apoptosis. These data above underscored the importance of Cx43 dependent GJC between astrocytes and glioma cells to glioma chemoresistance, and there may be certain molecules transmitted between astrocytes and glioma cells through GJ that regulate the sensitivity of glioma cells to chemotherapeutic drugs.4. Astrocytes control Ca2+ concentration in glioma cells through GJC to prevent calcium overload and reduce apoptosisCalcium ions are an important second messenger that transmit through GJC, and play a key role in the activation and execution of apoptosis and death. Our results showed that cytoplasmic Ca2+ concentrations in glioma cells positively correlated with glioma cell apoptosis. In addition, the Ca2+ chelator BAPTA-AM inhibited TMZ-induced apoptosis of glioma cells and this inhibition effect was dose-dependent. After TMZ treatment, astrocytes had a substantially lower cytoplasmic Ca2+ concentration than A172 cells cultured alone. In addition, the cytoplasmic Ca2+ concentration of the co-cultured A172 cells was also lower. However, after GJC inhibition by CBX, the cytoplasmic Ca2+ concentration of the co-cultured A172 cells showed no significant difference from that of A172 cells cultured alone. The above data indicated there may be calcium ions flowing through GJ because of the different Ca2+ concentrations between glioma cells and astrocytes. As a consequence, Ca2+ concentrations in glioma cells were controlled through GJC by astrocytes, thus preventing calcium overload and reducing apoptosis, likely at least in part by blocking the Ca2+ -dependent apoptosis process.5. The interaction between human astrocytes and glioma cells in vivoTo confirm the interaction between astrocytes and glioma cells in vivo, we verified the distribution and functional state of astrocytes in human glioma specimens by immunohistochemistry of GFAP staining. GFAP-positive astrocytes surrounded and infiltrated into the tumor. Astrocytes in the peri-tumor zone became activated, as indicated by the upregulation of GFAP and a morphological transformation to multipolar stellate form. This phenomenon was also confirmed in co-culture experiments in vitro. All data above showed that there was direct contact between infiltrated human astrocytes and glioma cells in vivo and in vitro.Conclusion1. Human astrocytes, in tumor microenvironment, can protect glioma cells from cytotoxicity of chemotherapeutic drugs in vitro.2. Astrocytes enhanced chemoresistance in glioma cells by controlling cytoplasmic Ca2+ through GJC between astrocytes and glioma cells.3. Chemoresistance associated with GJC between astrocytes and glioma cells was dependent on Cx43.4. There was interation between astrocytes and glioma cells in tumor microenvironment. Therefore, successful clinical chemotherapy treatment of gliomas would require combination with GJC inhibitor, considering effects of astrocytes.5. Cx43-GJC between astrocytes and glioma cells may be a potential target for overcoming chemoresistance in gliomas clinically.Conclusion1. Human astrocytes, in tumor microenvironment, can protect glioma cells from cytotoxicity of chemotherapeutic drugs in vitro.2. Astrocytes enhanced chemoresistance in glioma cells by controlling cytoplasmic Ca2+ through GJC between astrocytes and glioma cells.3. Chemoresistance associated with GJC between astrocytes and glioma cells was dependent on Cx43.4. There was interation between astrocytes and glioma cells in tumor microenvironment. Therefore, successful clinical chemotherapy treatment of gliomas would require combination with GJC inhibitor, considering effects of astrocytes.5. Cx43-GJC between astrocytes and glioma cells may be a potential target for overcoming chemoresistance in gliomas clinically.Part Ⅱ Effects of human astrocytes on migration and invasion property of glioma cellsIntroductionThe invasive phenotype of GBM makes tumor impossible complete resection and resistance to current therapeutic intervention, which is the major cause of poor prognosis in glioma patients. Therefore, developing novel strategies to restrain their invasive capacity as well as understanding the mechanisms that regulate glioma invasion are critical to improve poor prognosis. Recently, accumulating research has demonstrated that the tumor microenvironment especially the tumor stromal cells contribute to the glioma malignant behavior. Astrocytes are a unique group of stromal cells in the glioma microenvironment, with the most abundant glial cell population. More importantly, studies have revealed that astrocytes enhance the invasion potential of glioblastoma stem-like cells and play an important role in glioma growth and angiogenesis. Additionally, astrocytes in glioma microenvironment became functionally reactive and constantly secreted a variety of cytokines to regulate glioma proliferation and invasion. The impact of microglia, another type of stromal cells in the glioma microenvironment, on glioma expansion was well elaborated. However, the function of astrocyte in glioma and its mechanism associated with tumor invasion have not been fully elucidated.Membrane type 1 matrix metalloproteinase (MT1-MMP; MMP14), a transmembrane proteinase of MMP family, is critical for degration of the extracellular matrix. MMP 14 can enhance tumor invasion and dissemination by cleavage of CD44, a cell surface glycoprotein or by activation of MMP-2 and MMP9 via a combination of TIMP-2 with MMP 14. A growing body of evidence indicates that MMP 14 is upregulated in diverse aggressive tumor cells and tumor associated stromal cells, and MMP 14 of microglia or glioma cells has become an emerging intervention target for glioma therapy. Interleukin-6 (IL-6), one of growth and survival cytokines involved in the modulation immune and inflammatory responses, was recently revealed to promote invasion and migration of glioma cells. IL-6 was also proved to increase the activation of MMP2 and MMP9 to enhance the cancer cells’ invasion. Since MMP 14 is an activator of MMP2 and MMP9, it is decisive to provide a hypothesis that IL-6 regulates MMP 14 involving the activation of MMP2 and MMP9, thereby influencing tumor migration and invasion in glioma.ObjectiveOur study was to explore the mechanism of glioma migration and invasion mediated by astrocytes in tumor microenvironment and to provide potential target for glioma treatment of migration and invasion propensity.Methods and results1. Human astrocytes significantly increased the migration and invasion of glioma cells by releasing soluble factors in vitroWe co-cultured glioma cells with human astrocytes in vitro, and found that astrocytes significantly increased glioma migration and invasion propensity via transwell migration and invasion assays.2. The activated signaling pathways and upregulation of gene and protein associated with invasion induced by astrocytesWe used astrocytes conditional medium (ACM) to stimulate glioma cells. After analysis of migration and invasion associated genes and proteins such as MMP and Rho family by qRT-PCR and western blot assays, we found membrane type 1 matrix metalloproteinase (MMP 14) of glioma cells was the most obviously upregulated in both glioma cells. In addition, ERK1/2, AKT and p38MAPK pathways were activated, which was also reported to be associated with migration and invasion.3. Astrocytes upregulated cytomembrane MMP 14 expression of glioma cells that promoted migration and invasion via activation of MMP2 but not cleavage of CD44Via flow cytometry, we found the cytomembrane MMP 14 expression of glioma cells was upregulated by ACM stimulation, but the cytomembrane CD44 of glioma cells was not decreased at the same time. Downregulation of MMP 14 by siRNA decreased the activity of MMP2, whereas upregulation induced by astrocytes increased the activity of MMP2. In addition, knockdown of MMP 14 by siRNA resulted in decreasing glioma migration and invasion propensity. These data indicated that upregulation of MMP 14 by astrcoytes contributed to promote glioma migration and invasion via activation of MMP2 but not cleavage of CD44.4. IL-6 secreted from astrocytes was responsible for cytokine that upregulated cytomembrane MMP 14 expression on glioma cellsCytokine Arrays analysis of 507 cytokines indicated that interleukin-6 (IL-6) secreted by astrocytes was profoundly increased, especially in co-culture medium, which was confirmed by ELISA assay. In addition, incubation with IL-6 could increase cytomembrane MMP14 expression of glioma cells while adding IL-6 neutralizing antibodies almost abolished MMP14 upregulation induced by ACM. These data indicated that IL-6 was the key cytokine that induced the cytomembrane MMP 14 expression on glioma cells.5. Both IL-6 and MMP14 expression were associated with the advanced WHO grade and the poor overall survival of gliomas, and MMP14 expression was correlated with IL-6Via immunohistochemistry of IL-6 and MMP14 on human tumor specimens, we found both MMP14 and IL-6 expression were associated with the advanced WHO grade, which predicted the poor overall survival. In addition, MMP14 expression was correlated with IL-6, and normal brain with edema had also high IL-6 expression, which confirmed our hypothesis of IL-6—MMP14 axis between astrocytes and glioma cells.Conclusion1. Human astrocytes released cytokines to induce cytomembrane MMP14 expression of glioma cells, thereby enhancing the migration and invasion of glioma cells by activation of MMP2.2. IL-6 secreted from astrocytes was responsible for cytokine that upregulated cytomembrane MMP14 expression of glioma cells.3. MMP14 expression was correlated with IL-6 expression, and both of them were associated with the advanced WHO grade in glioma specimens and demonstrated a poor overall survival.4. IL-6 and MMP14 axis between astrocytes and glioma cells may become a potential target for glioma treatment.