Experimental study on the macrophage polarization characteristics of glioma-macrophage fusion cells and their impact on the proliferation and invasion capabilities of glioma cells
Objective To establish a fusion cell model between glioma cells and macrophages to explore the polarization characteristics of glioma-macrophage fusion cells and their effects on the proliferation and invasive capabilities of glioma cells.Methods Mouse-derived glioma cells GL261 and macrophages RAW264.7 were used to construct and screen the GL261xRAW264.7 fusion cells.GL261,RAW264.7,and GL261xRAW264.7 fusion cells were cultured separately to collect their supernatants,and the respective conditioned media were obtained(CM,namely GL261-CM,RAW264.7-CM,GL261xRAW264.7-CM).Flow cytometry was employed to detect the chromosomal content of GL261xRAW264.7 fusion cells and their expression levels of macrophage markers CD86 and CD206.The differences in proliferation and migration abilities between GL261xRAW264.7 fusion cells and their parent cells(GL261 and RAW264.7 cells)were evaluated using CCK-8,Transwell,and scratch assays.Additionally,the effects of GL261xRAW264.7-CM,compared with parent cell CMs,on the proliferation,migration,and invasion capabilities of cultured GL261 cells were assessed.Immunomagnetic bead flow cytometry was utilized to detect differences in the secretion of macrophage M1-type cytokines TNF-α,IL-1β,IL-6,and IL-23 and M2-type cytokines IL-10,TGF-βbetween RAW264.7 and GL261xRAW264.7 fusion cells.Results The GL261xRAW264.7 fusion cells were successfully constructed,and flow cytometry confirmed a significant increase in their chromosomal content(right shifts in G1 and G2/M peaks compared to parent cells).CCK-8 assays showed that the proliferation ability of GL261xRAW264.7 fusion cells was stronger than that of GL261 and weaker than that of RAW264.7 with significant differences(all P＜0.05).Transwell assays demonstrated that the migration ability of GL261xRAW264.7 fusion cells was enhanced compared with the parent cells with significant differences(all P＜0.001).Flow cytometry revealed statistically significant differences in the expression rate of CD206 between GL261xRAW264.7 fusion cells and RAW264.7 cells[(75.80±2.31)％vs.(4.72±0.47)％,P＜0.001].However,there were no significant differences in CD86 expression[(4.27±0.40)％vs.(4.18± 0.41)％,P＞0.05].Immunomagnetic bead flow cytometry showed that compared with RAW264.7,GL261xRAW264.7 fusion cells exhibited increased expression of IL-10 and TGF-β,decreased expression of TNF-α and IL-1β with significant differences(all P＜0.01).However,there were no significant differences in the expression of IL-6 and IL-23(all P＞0.05).CCK-8,Transwell and scratch assays all indicated that the proliferation,migration and invasion of GL261 cells cultured in GL261 xRAW264.7-CM were enhanced compared with those cultured in parent cell CM with significant differences(all P＜0.01).Conclusion The in vitro constructed GL261xRAW264.7 fusion cells possess M2-type macrophage immune characteristics and can promote the proliferation,migration and invasion capabilities of glioma cells.
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