Shuangshen Granule Regulates Tumor Cell Exosomes Through MIF-miR-34a-KLF4 Pathway and Affects Macrophage Polarization...

The incidence and mortality of lung cancer are among the highest in the world, involving a variety of complex pathological mechanisms and processes. Previous studies have demonstrated that the tumor immunosuppressive microenvironment (TIME) plays a key role in tumor progression, promoting tumor invasion and metastasis and leading to resistance to therapy. Tumor-associated macrophages (TAMs) are important cell components in TIME, and their phenotypes affect the prognosis of tumors. Tumor-derived exosomes carrying specific information as a medium of crosstalk between macrophages and tumor cells are considered to be an underexplored information transfer medium affecting tumor progression and tumor microenvironment remodeling. In the previous study, we found that Shuangshen granule (SSG) can reduce the formation of lung tumors in mouse models of spontaneous lung cancer, but the specific mechanism of SSG inhibiting lung cancer has not been solved. Starting from the exosomes of tumor cells, we established the M1-like TAMs overexpression mouse lung cancer transplantation tumor model and constructed MIF^+/+ Lewis stable transfection cell line and shMIF Lewis stable transfection cell line to analyze the specific molecular biological mechanism of SSG inhibiting lung cancer. The experimental results showed that SSG significantly reduced the volume and mass of Lewis lung cancer xenografts in mice, and its inhibitory effect on Lewis lung cancer was related to the increase in the proportion of M1-like TAMs. Flow cytometry and western blot revealed that SSG can increase the proportion of CD8⁺ central memory T cells, reduce the proportion of CD8⁺ effector memory T cells, reduce the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells, and play a role in regulating the tumor microenvironment. In vitro experiments further showed that SSG drug-containing serum could inhibit the expression of MIF protein and RNA in MIF^+/+ Lewis cells and increase the expression of miR-34a in MIF^+/+ Lewis cell exosomes. Interestingly, we also found that MIF^+/+ Lewis cell-derived exosomes after SSG-containing serum intervention can reverse the increase in the proportion of M2-like TAMs and increase the proportion of M1-like TAMs. Subsequent gene modification experiments showed that MIF^+/+ Lewis cell exosomes can increase the expression of the KLF4 gene in macrophages in vitro. In conclusion, our study revealed that SSG can regulate the polarization of TAMs through lung cancer cell-derived exosomes, and its mechanism is related to the MIF-miR-34a-KLF4 pathway.

•SSG can inhibit the proliferation of lung cancer, which is related to the antitumor mechanism involving macrophages.•The poor prognosis of lung cancer caused by macrophage migration inhibitory factor (MIF) is related to the TAM phenotype, and SSG can inhibit lung cancer by inhibiting MIF.•SSG regulates the tumor cell MIF-MiR-34a exosome pathway and regulates the polarization and function of TAMs.