M1-like polarization of microglia negatively correlation with GBM progression
To investigate the role of microglia polarization in GBM progression, we firstly assessed the M1-like and M2-like microglia related markers expression in GBM orthotopic tumor tissue. The expression level of CD80, M1-like microglia marker, was lower in the tumor tissues than the normal brain tissues, while the expression level of CD206, M2-like microglia marker was in verse as shown immunohistochemistry staining (Fig. 1A).
To connect this results to clinical application, using the GBM patient tissue specimens (patients information in Table 2), we performed the immunofluorescence staining and the results shown that CD80 was significantly downregulated in recurrence GBM tissues than the primary GBM tissues, while CD206 was in verse (Fig. 1B). Due to the limitations of the tissue samples, we evaluated the correlation of CD80 expression level and patients’ survival based on the information in TCGA database. The heat map revealed a linear reverse relationship between CD80 and tumor proliferation and metastasis indicators (BIRC5,MYC, and MMP9, Fig. 1C), which suggested the negatively relationship between with GBM progression and M1-like polarization of microglia.
Interestingly, we noticed a similar trend of expression level between CD80 and EphA2 in the TCGA database (Fig. 1C). Using the GBM patient tissue specimens, we found the expression level of EphA2 was also higher in the primary GBM tissues than the recurrence GBM tissues, indicating the potential correlation between EphA2 and M1-like polarization of microglia (Fig. 1D).
GBM cells prevented EphA2 expression and M1-like polarization of microglia
The polarization of microglia and protein expression is regulated by both instinct and external factors. We next assessed the role of GBM cells in determining EphA2 expression and polarization of microglia using the co-culture system (Fig. 2A). Co-cultured BV2 with the GBM cells (GL261 or C6) for the indicated time, qPCR shown that the expression level of CD80 in BV2 was downregulated in a time dependent manner in the presence of GL261 or C6, while the Arg-1 and CD206 were upregulated (Fig. 2B), which suggested GBM cells prevented M1-like polarization of microglia.
Moreover, co-cultured BV2 with GL261 or C6, the expression level of EphA2 in BV2 was significantly down-regulated in a time dependent manner with the peak inhibition at 24 h (Fig. 2C and 2D). The similar results can also be acquired by immunofluorescence staining (Fig. 2E), indicating GBM cells inhibited EphA2 in microglia.
Epha2 Promoted The M1-like Polarization Of Bv2
To investigate the role of EphA2 in microglia polarization, we first transfected EphA2 cDNA plasmid into BV2 cells and confirmed the overexpression of EphA2 proteins in these cells by western blot and qPCR (Fig. 3A and 3B). qPCR shown that iNOS significantly upregulated, while CD206 and Arg-1 downregulated in EphA2 overexpression BV2 cell (Fig. 3C). The similar results can also be acquired in the sorted primary microglia (Fig. 3D). Western blot shown the expression level of iNOS, TNF-α and IL-1β in EphA2 overexpression BV2 cell increased (Fig. 3E). These results indicated that EphA2 induce M1-like polarization of microglia.
To further elucidate the functional role EphA2 in BV2 polarization, two stable EphA2-knockdown cell clones (EphA2 shRNA1 and shRNA2) were established, and high silencing efficiency (90% silencing) was confirmed by western blot (Figure S1). Compared to the parental cancer cells, EphA2 knockdown cells showed the upregulation of CD206 and Arg-1, and downregulation of iNOS at the RNA level (Fig. 3F). Western blot shown the expression level of iNOS, TNF-α and IL-1β in EphA2 knockdown BV2 cell decreased (Fig. 3G). These results demonstrated knockdown EphA2 prevented M1-like polarization of microglia. Moreover, NLRP3 expression level increased in EphA2 overexpression BV2 cells, and decreased in EphA2-knockdown BV2 cells, which indicated that EphA2 was also involved in the inflammation response (Fig. 3E and G).
To further confirmed the role of EphA2 induced M1-like polarization of microglia in GBM cells metastasis, we performed Transwell migration and invasion assays. Co-cultured with EphA2 overexpression BV2 cells, the number of living C6 and GL261 GBM cells that passed through the membrane was much lower than the number of the cells for the control group (Fig. 3H-K). These results supported the hypothesis that EphA2 induced M1-like polarization of microglia prevents the invasion and metastasis potential of living GBM cells.
PI3K/Akt pathway is involved in EphA2-mediated M1-like polarization of BV2
To further understand the underlying mechanism of EphA2 regulating microglia polarization, iTRAQ labeling was firstly used to identify differentially expressed phosphorylated proteins. iTRAQ-LC-MS/MS analysis results demonstrated that a total of 2,232 phosphorylated proteins, 4,941 phosphorylated peptides and 7,136 phosphorylated sites were identified (Fig. 4A). A total of 443 quantified proteins with P < 0.05 and an expression change greater than 2-fold or less than 0.5-fold between the KO and control groups were manually selected. Compared with the control group, 370 proteins were upregulated and 73 were downregulated (Fig. 4B). To better understand the involvement of EphA2 in physiological functions in BV2 cells, Blast2 go software was used to determine all different phosphorylated peptides, and a GO functional enrichment analysis of proteins corresponding to the phosphorylated peptides was then performed by Fisher. The results demonstrated that EphA2 involved nucleic acid binding, intracellular organelles, nucleoplasm and organelles (data not shown). KEGG pathway enrichment analysis revealed that 21 phosphorylated peptides were related to PI3K-AKT pathway (Fig. 4C and 4D).
As the PI3K-AKT is an important regulator of macrophage polarization, we hypothesized that it may function downstream of EphA2 to mediate the M1-like polarization of BV2. To test this hypothesis, we assessed the PI3K-AKT pathway related proteins in the EphA2-knockdown, EphA2-overexpression and parental BV2 cells. Compared to the parental BV2 cells, the expression level of p-PI3K and p-AKT was significantly increased in EphA2-overexpression BV2 cells (Fig. 4E-F), while the significantly decreased expression of p-PI3K and p-AKT in EphA2-knockdown BV2 cells was observed (Fig. 4G-H).
To further confirm the role of PI3K-AKT pathway in EphA2 mediated the M1-like polarization of BV2, PI3K-AKT pathway activator, sc-79, was also used. As expected, the expression level of p-PI3K and p-AKT was increased in BV2 cells in the presence of sc-79 (Fig. S2). Treated EphA2-knockdown BV2 cells with sc-79, the expression level of CD80 and IL-6 was significantly upregulated, while Arg-1 was downregulated as assessed by RT-PCR (Fig. 4I). Moreover, co-cultured with EphA2-knockdown BV2 cells in the presence of sc-79, the ability of C6 and GL261 metastasis and invasion was significantly inhibited (Fig. 4J).
EphA2-mediated M1-like polarization of BV2 inhibited GBM cells proliferation and metastasis in vivo
To validate our in vitro observation EphA2-mediated M1-like polarization of microglia inhibiting GBM cells metastasis, we performed tumor growth and metastasis assays in vivo. We firstly tested examined the metastasis of GMB cells in nude mice by injecting 1 × 106 C6 cells co-cultured with BV2 cells (control group) or EphA2 overexpression BV2 cells into the mouse tail vein. From the HE staining, we observed that C6 cells exposed to EphA2 overexpression BV2 cells showed significantly more and larger metastatic foci than the control group (Fig. 5A). Moreover, we also assessed the proliferation ability of GMB cells in nude mice by injecting 1 × 106 C6 cells co-cultured with BV cells (control group) or EphA2 overexpression BV2 cells. As shown in Fig. 5B-D, tumors from EphA2 overexpression BV2 cells co-culture group grew slower and smaller than the control group, and larger necrotic area was also observed from the HE staining. Moreover, there was a significant reduction of proliferation (survivin and c-myc) and metastasis (ZEB1 and β-catenin) related markers in protein expression level (Fig. 5E). These in vivo results further supported the in vitro evidence that EphA2-mediated M1-like polarization of microglia prevented GBM cells metastasis.