MIF improves immune microenvironment of Lewis lung cancer brain metastases after radiotherapy via reducing M2 macrophages.

e21026 Background: Our previous studies showed that inhibition of MIF/CD74 signaling pathway can promote the transformation of intracranial macrophages from M2 to M1 type after radiotherapy in brain metastases(BMs). It is reported that M2 macrophages are the main components of tumor immunosuppression microenvironment, and the surface marker, ARG-1, can decompose the Arginine which is essential in the growth and development of T cells. Therefore, this paper will focus on the mechanism of MIF regulating tumor infiltrating lymphocytes (TILs) by inducing macrophage phenotype transformation. Methods: MIF-Knockdown in Lewis cells were constructed by lentiviral shRNA interference technique. The expression levels of MIF in Lewis and iNOS (M1 type marker), Arg-1 (M2 type marker) in BV2 cells were detected by WB. The RNA expression levels of M1 and M2 tags were detected by qPCR. The expression of iNOS, Arg-1 and Iba-1 in lung cancer BMs model mice was detected by IF. The content of arginine in tumor tissue and clinical patient specimens were detected by Arginine kit. The proportion of TILs in vivo was measured by flow cytometry. BMs tumor growth in mice was observed by Small Animals Imaging Technology. Results: In animal experiments, WB、PCR and IF results showed that MIF knockdown (sh-MIF) combined with whole brain irradiation(IR) can raise the expression of iNOS, while the Arg-1 lessend, suggesting that downregulation of MIF can promote M1 phenotype and reduce M2 phenotype. Flow cytometry results showed that sh-MIF combined with IR increased the proportion of CD8+T/CD4+T cells . After clearance of macrophage, Flow cytometry showed no difference between the sh-MIF group and the control group (NC). Small Animals Imaging results also showed that the inhibition of tumor growth by sh-MIF was less significant than macrophages presence. It is indicated that down-regulation of MIF expression combined with irradiation promote T cell infiltration and inhibit tumor may be mediated by macrophages. Arginine kit results showed that the content of arginine increased after IR or sh-MIF, while it increased overall after macrophage clearance, but the difference between each groups was not significant, suggesting that IR or sh-MIF could increase the content of arginine by reducing M2-type macrophages. Flow cytometry and animals imaging results showed that the CD8+/CD4+TILs ratio was increased and tumor growth was inhibited after the addition of exogenous arginine. In clinical samples, the content of arginine in CSF decreased after radiotherapy, which suggests that arginine supplementation has the potential to improve the effect of radiotherapy. Conclusions: Inhibitory MIF expression can reduce the expression of Arg-1 from M2 type macrophages in BMs, thus raise the content of arginine, promote the infiltration of lymphocytes, improve the immune microenvironment, achieve radiotherapy sensitization finally.