Extended Data Fig. 4: TFPI2 promotes microglia migration via activating STAT6 signaling.
a, Amplification pattern of TFPI2 and CLOCK in GBM and low-grade glioma (LGG) patient tumors. b, GSEA analysis shows the top 10 enriched hallmark pathways in TFPI2-high GBM patient tumors compared to TFPI2-low tumors. Blue highlighted pathways relate to immune response. c, Representative of transwell migration of PrhMG following stimulation with conditioned media (CM) from GSC262 or GSC23 expressing control and TFPI2 overexpression (OE) plasmid. Scale bar, 400 µm. d, Representative of transwell migration of PrhMG, HMC3 and SIM-A9 microglia following stimulation with CM from GSC272 (for PrhMG and HMC3) or QPP7 cells (for SIM-A9) expressing control shRNA (shC) and TFPI2 shRNAs (shTFPI2). Scale bars, 400 µm for PrhMG; 200 µm for HMC3 and SIM-A9. e, f, Representative (e) and quantification (f) of flow cytometry for the percentage of CD45lowCD11b+ and CD45highCD11b+ cells in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. n = 4 and 3 biological replicates for shC and shTfpi2, respectively. g, h, Representative (g) and quantification (h) of flow cytometry for the percentage of CD45highCD11b+CD68+ macrophages in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. n = 4 and 3 biological replicates for shC and shTfpi2, respectively. i, Correlation between microglia signature and STAT6, AKT1, AKT2 or AKT3 in TCGA GBM dataset. R and P values were determined by Pearson correlation. j, Representative of transwell migration of mouse primary microglia isolated from wild-type (WT) and STAT6 knockout (KO) mice treated with or without TFPI2 recombinant protein (20 ng/ml). Scale bar, 200 µm. k, Representative of transwell migration of PrhMG, HMC3 and SIM-A9 microglia following stimulation with TFPI2 protein (20 ng/ml) in the presence or absence of STAT6 inhibitor AS1517499 at indicated concentrations. Scale bars, 400 µm for PrhMG; 200 µm for HMC3 and SIM-A9. Error bars indicate mean ± SEM. One-way ANOVA test.
