The Mechanism Of Squamous Cell Carcinoma-associated Fibroblasts Facilitating The Generation Of Monocytic Myeloid-Derived Suppressor Cells Via Secreting Exosomes

Background and objective:Esophageal cancer is a common type of tumor in digestive system and is the leading cause of cancer-related death.Currently,the traditional treatment of esophageal cancer is mainly surgery,radiotherapy and chemotherapy,but still can not significantly improve the prognosis of patients.In the recent years,the rise of immunotherapy has brought new treatment strategies for the cancer patient.However,in esophageal cancer,immunotherapy has not achieved significant effect,so it is important to explore the reasons for failure of immunotherapy.Numerous studies have reported that the tumor immunosuppressive microenvironment is the major cause of the failure of immunotherapy,which consists of immunosuppressive cells and cytokines.In the esophageal cancer microenvironment,tumor-associated fibroblasts(CAFs)and myeloid-derived suppressor cells(MDSCs)are two important populations of immunosuppressive cells.CAFs occupy the main components of esophageal cancer tissues,and differs greatly in morphology and function from normal fibroblasts(NFs).CAFs express high levels of smooth muscle protein-?(?-SMA)and vimentin,which can remodel extracellular matrix,promote tumor angiogenesis as well as secret various cytokines to form an immune barrier to inhibit the infiltration and function of immune cells.MDSCs are a group of heterogeneous cells derived from bone marrow,which are mainly divided into two subtypes:monocytic MDSCs(M-MDSCs)and polymorphonuclear MDSCs(PMN-MDSCs).Both subtypes of MDSCs can inhibit T cell function and promote tumor cell proliferation.At present,most studies focus on the interaction of tumor cells with CAFs and tumor cells with MDSCs.However,the interaction of these two immunosuppressive cells in the tumor microenvironment remains unclear,therefore,exploring the interaction between CAFs and MDSCs can help to better understand the formation of immunosuppressive microenvironment in esophageal cancer.The aim of this study was to investigate the mechanism of CAFs-induced M-MDSCs generation and clinical significance of CAFs and M-MDSCs accumulation in esophageal cancer and provided theoretical evidence and new ideas for immunotherapy of esophageal cancer.Methods:TCGA database was used to analyze the correlation between CAFs and MDSCs in esophageal cancer and further verified by immunohistochemistry of clinical specimens;immunofluorescence was used to detect the expression of?-SMA and vimentin in primary cultured fibroblasts of squamous cell carcinoma(CAFs)and normal human skin-derived fibroblasts(NFs);flow cytometry and GEO database were used to analyze the differences of surface makers and functional molecules of monocytes from healthy donors and patients peripheral blood and tumor tissues;monocytes were incubated with supernatant of CAFs and NFs,flow cytometry and immunofluorescence were used to detect the changes of surface makers and functional molecules;qRT-PCR was used to detect the expression of immunosuppressive genes after co-incubation;CD8~+T cells were cultured alone or co-cultured with co-incubated monocytes,and the flow cytometry was used to detect the cytokine secretion,proliferation and apoptosis of CD8~+T cells;ultracentrifuge was used to isolate exosomes from supernatant of NFs and CAFs;transmission electron microscop was used to detect morphology of exosomes;nanoparticle tracking technique was used to detect the size of exosomes;western blotting and flow cytometry were used to detect the surface makers of exosomes;immunofluorescence was used to detect the location of exosomes in monocytes;monocytes were cultured alone or co-incubated with exosomes secreted by NFs and CAFs,and flow cytometry was used to detect the phenotype of monocytes;CD8~+T cells were cultured along or co-cultured with co-incubated monocytes,flow cytometry was used to detect the secretion of cytokines,proliferation and apoptosis of CD8~+T cells;TCGA and GEO databases were used to screen miRNAs in exosomes and verified by qRT-PCR;GEO database combined with STRING website were used to predict signaling pathway activated by miR-21;qRT-PCR and western blotting were used to detect PTEN,total and phosphorylated STAT3 expression in monocytes after treated with miR-21 mimic or control;after adding STAT3 inhibitor,Sattic,flow cytometry was used to detect phenotypic changes of monocytes;qRT-PCR was used to detect the expression of miR-21 exosomes isolated from peripheral blood of healthy donors and esophageal cancer patients;clinical specimens and TCGA database was used to analyze the relationship between CAFs,M-MDSCs accumulation and miR-21 expression with clinical parameters and prognosis.Results:1)TCGA database and squamous cell carcinoma specimens proved that the accumulation of CAFs and M-MDSCs has a strong correlation.2)Flow cytometry and GEO database showed that monocytes in peripheral blood and tumor tissues of patients have M-MDSCs phenotype.3)Immunofluorescence confirmed that primary cultured esophageal carcinoma fibroblasts had CAFs-like characteristics with high expression of?-SMA and vimentin.4)Flow cytometry,qRT-PCR and immunofluorescence results showed that CAFs supernatant could induce the phenotypic and functional transformation of monocytes into M-MDSCs.5)CAFs-induced M-MDSCs could inhibit the function and proliferation of CD8~+T cells and promote CD8~+T cell apoptosis.6)CAFs secreted amounts of exosomes and could be engulfed by monocytes,promoting the transformation of monocytes into M-MDSCs.7)TCGA,GEO and in vitro experiments confirmed that exosomes secreted by CAFs contained a large amount of miR-21.8)The GEO,STRING database and in vitro experiments confirmed that miR-21can induce the transformation of monocytes to M-MDSCs by inhibiting the expression of PTEN and activating p-STAT3 in monocytes.9)The levels of miR-21 in exosomes had a strong correlation with the ratio of M-MDSCs in esophageal cancer patients peripheral blood.10)Immunohistochemistry of esophageal squamous cell carcinoma specimens have confirmed that miR-21 expression is strongly correlated with aggregation of CAFs and M-MDSCs.11)Immunohistochemistry of esophageal squamous cell carcinoma specimens and TCGA databases indicated that the expression of miR-21 and the accumulation of CAFs and M-MDSCs were associated with lymph node metastasis,tumor stage and clinical prognosis in patients with esophageal cancer.Conclusion:1)Exosomes secreted by CAFs can be phagocytosed by monocytes and inhibited the expression of PTEN and activated p-STAT3 by releasing miR-21 encapsulated in exosomes,inducing the transformation of monocytes into M-MDSCs.2)CAFs-induced M-MDSCs have immunosuppressive function.3)The expression of miR-21 is associated with accumulation of CAFs and M-MDSCs in esophageal cancer tissues,and high expression of miR-21 and accumulation of CAFs and M-MDSCs predict poor prognosis of esophageal patients.