槐耳多糖通过调控JAK1/STAT3/CCL2轴减少瘤内髓源性抑制细胞募集以抑制三阴性乳腺癌进展OA

Objective Myeloid-derived suppressor cells(MDSCs)are frequently over-recruited in the immune microenvironment of triple-negative breast cancer(TNBC),forming an immunosuppressive barrier that contributes to the poor response rates to existing immunotherapies.This study aims to investigate the effects of polysaccharide of Trametes robiniophila Murr(PS-T)on the immune microenvironment in TNBC and clarifying the underlying molecular mechanism by which PS-T inhibits MDSC recruitment through suppression of the JAK1/STAT3/CCL2 axis.Methods TNBC xenograft model was established in 36 female BALB/c mice(6 to 8 weeks,weighing 18 to 20 g).Mice were randomly divided into 2 groups(n=6)for intragastric administration:PS-T group(100 µL solution containing 3 mg of PS-T)and control group(100 µL isotonic sodium chloride solution).Tumor volume was measured on days 5,7,9,11,13,15,17,19,and 21 post-inoculation.On day 21,tumor tissues were harvested and photographed,and then processed into single-cell suspensions and tissue sections for flow cytometry and immunohistochemical staining to evaluate the infiltration of MDSCs and T cells.RNA sequencing data were analyzed using GO and KEGG enrichment analyses to identify signaling pathways involved in the PS-T-mediated MDSC regulation in TNBC.RT-qPCR,Western Blot,and immunohistochemical staining were used to detect JAK1,STAT3,and CCL2 expression.Transwell co-culture assay was performed to verify that PS-T inhibits MDSCs recruitment by suppressing CCL2 secretion.JAK1-knockdown 4T-1 cell line was constructed,and Western blot and ELISA were used to assess the effects of JAK1 knockdown on PS-T-mediated inhibition of phosphorylated STAT3 and CCL2.Results ① Compared with the control group,PS-T significantly inhibited the growth of TNBC(P=0.003)and remodeled the immune microenvironment.Flow cytometry showed that the proportion of intratumoral CD8⁺ T cells was increased in the PS-T group(P=0.002),which was confirmed by immunohistochemistry demonstrating elevated numbers of intratumoral CD8⁺ T cells(P=0.003).② Single-cell RNA sequencing of the immune cell composition in tumor tissues from TNBC-bearing mice revealed that PS-T treatment increased CD8⁺ T cell infiltration from 3.6%to 9.33%,while markedly reducing the proportion of MDSCs from 15.64%to 0.17%.Immunohistochemical staining further showed a significant decrease in the number of CD11b⁺ cells in the PS-T group(P=0.008).Flow cytometric analysis of MDSC subsets indicated that PS-T significantly reduced the proportion of PMN-MDSCs(gated as CD45⁺CD11b⁺Ly6G⁺Ly6Clow cells)(P=0.012).③ GO and KEGG enrichment analyses of differentially expressed genes between the control and PS-T groups revealed significant enrichment in cytokine-related pathways and the JAK/STAT signaling pathway.RT-qPCR and Western blot assays demonstrated that PS-T significantly downregulated the mRNA and protein levels of CCL2 in 4T-1 cells(P<0.001),which was further confirmed by immunohistochemistry showing reduced CCL2 expression in vivo(P<0.001).Co-culture of flow cytometry-sorted MDSCs with 4T-1 cells in Transwell chambers in the presence of MK-0821,an antagonist of the CCR2 receptor,attenuated the PS-T-mediated inhibition of MDSC recruitment to the lower chamber(P<0.01).④ Both immunohistochemistry and Western blot analyses demonstrated that PS-T inhibited the phosphorylation of STAT3(p-STAT3)without altering the total STAT3 protein level.⑤ Western blot analysis showed that PS-T reduced the protein level of JAK1,the kinase responsible for STAT3 phosphorylation(P=0.003).Knockdown of JAK1 in 4T-1 cells abrogated the inhibitory effects of PS-T on p-STAT3 and CCL2 expression,as verified by both Western blot and ELISA assays.Conclusion PS-T suppresses intratumoral MDSC recruitment by inhibiting the JAK1/STAT3/CCL2 signaling pathway in TNBC tumor cells,thereby remodeling the immunosuppressive microenvironment and exerting anti-tumor effects.These findings provide novel theoretical support for the application of PS-T in combination immunotherapy for TNBC.