The Research Of Apolipoprotein A-I Mimetic Peptide L-4F In Regulation Of Bone Marrow-derived Suppressor Cells And Pancreatic Cancer Progression

Background and objective:L-4F is an amphipathic apolipoprotein A-I?ApoA-I?peptidomimetic composed of 18 amino acids that has been engineered to mimic the anti-inflammatory and antioxidant functions of ApoA-I.Studies have shown that L-4F has the effect on inhibiting tumor growth,and inhibiting inflammatory by down-regulating the secretion of IL-17A,IFN-?,IL-1?,IL-6 in tumor tissues or immune cells,and reducing the action of endotoxin.As the king of cancer,pancreatic cancer has a high mortality rate and a high transfer capacity.In the process of tumor progression,tumor cells can secrete a large number of cytokines into the tumor microenvironment,which promotes the immunosuppressive function of immune cells such as myeloid-derived suppressor cells?MDSCs?,resulting in tumor-to-host immune response.Ultimately,the tolerance further contributes to the growth of the tumor.In this study,we analyzed whether L-4F can affect the proportion of MDSCs in pancreatic cancer tumor tissues and it can inhibit the immunosuppressive function of MDSCs on T cells in vitro,including reducing the production of ROS and H₂O₂ in MDSCs,increasing T cell proliferation and secretion levels of IFN-?and increasing the infiltration of CD4⁺T and CD8⁺T cells in mouse spleen and pancreatic cancer tissues,and made studies about p-STAT3 signaling pathway in PMN-MDSC treated with L-4F.Method:This study is divided into two parts in vivo and vitro respectively.In vivo studies,we first cultured mouse high-metastatic pancreatic cancer cell line H7,and implanted H7 cells into the pancreas of C57BL/6 mice by pancreatic pancreatic tumor in situ to establish a pancreatic cancer model.From the third day after modeling,the tumor-bearing mice were randomly divided into two groups,and the mice were injected with Sc-4F control peptide or L-4F peptide daily by intraperitoneal injection.At the end of the treatment,the mice were sacrificed and their spleens and tumors were harvested.The inhibition degree of L-4F on pancreatic cancer tumor tissues was evaluated by directly measuring tumors size.At the same time,the infiltrated immune cells were isolated in mouse spleens and tumors,and the ratio of MDSCs,CD4⁺T and CD8⁺T cells in spleens and tumors were detected by flow cytometry,which were used to preliminarily measure the impact of L-4F on tumorigenesis and immune microenvironment during the tumor progression.In vitro studies,we first isolated myeloid cells from the hind limbs of wild-type C57BL/6 mice and induced differentiation into MDSCs by GM-CSF and IL-6.Using Sc-4F control peptide or L-4F to stimulate MDSCs,and using Sc-4F control peptide or L-4F stimulated MDSCs in co-culture with mouse normal spleen cells,to detect whether L-4F has a direct effect on MDSCs by apoptosis assay and proliferation experiment after T cell co-culture,the immunosuppressive effect of MDSCs on T cell proliferation after L-4F treatment was evaluated.The effect of L-4F on MDSCs and ratio of CD4⁺T and CD8⁺T differentiation in the cell population was detected by flow cytometry.The effect of L-4F on the release of reactive oxygen species?ROS?from MDSCs was further examined.Finally,we examined the changes of STAT3 phosphorylation in MDSCs after treated with L-4F by flow cytometry in vitro.Result:The results of in vivo studies indicate that:1.After treatment with L-4F,the tumor volume of mice in the tumor-bearing group was significantly smaller than control group treated with Sc-4F;2.By separating and detecting the infiltration of immune cells in mouse spleens and tumors,we found that there was no significant change in the proportion of MO-MDSC in spleens and tumors as compared with the control group treated with Sc-4F.And the proportion of PMN-MDSC decreased significantly by L-4F treatment;3.We found that the proportion of CD8⁺T cells in the spleens of the L-4F treatment group was significantly higher than the Sc-4F control group,while there was no change in the proportion of CD4⁺T cells as compared with the Sc-4F control group,the proportion of CD4⁺T cells and CD8⁺T cells in the tumor tissues treated with L-4F was significantly increased by separating and detecting the infiltration of immune cells in the mouse spleens and tumors;The results of in vitro studies indicate that:1.We found that the number of PMN-MDSC was significantly lower than the Sc-4F control group in the group treated with L-4F.However,there was no significant change in the proportion of MO-MDSC populations;2.Compared with the cells treated with Sc-4F,the number of apoptotic cells in L-4F-treated MDSCs did not change significantly;at the same time,compared with untreated or low-dose treated cells,there was no significant change in the proliferative capacity of MDSCs treated with L-4F group;3.Compared with the proliferation ability of cells treated with the Sc-4F,L-4F effectively blocked the immunosuppressive effect of MDSCs on T cell proliferation;T cells were co-cultured with the MDSCs streated with Sc-4F.After culture,the ratio of CD4⁺T cells to CD8⁺T cells was significantly reduced in the Sc-4F treated MDSCs group;when T cells were co-cultured with L-4F-treated MDSCs,the inhibition of proliferation of CD4⁺T cells and CD8⁺T cells were alleviated;4.Compared with the non-MDSCs group,MDSCs can inhibit the secretion of IFN-?in the Sc-4F control treatment group,and L-4F can attenuate this immunosuppressive function of MDSCs;5.Compared with the Sc-4F control group,the concentrations of ROS and H₂O₂ were significantly reduced in the L-4F treatment group;6.L-4F significantly inhibited the phosphorylation level of STAT3 in PMN-MDSCin a dose-dependent manner.Conclusion:Based on the above results,it can be concluded that L-4F has an inhibitory effect on mouse pancreatic cancer.At the same time,it can reduce the proportion of PMN-MDSCs in mouse spleens and tumors,and can promote the infiltration of CD8⁺T cells in spleens and tumors,and promote the infiltration of CD4⁺T cells in tumor.Therefore,L-4F may play a dual role by inhibiting immunosuppression and activating immune responses.In addition,L-4F can inhibit the differentiation of MDSCs in vitro,and can also attenuate the inhibitory effect of MDSCs on spleen T cells and increase the ability of T cells secreting IFN-?.Moreover,L-4F inhibits the immunosuppressive effect of MDSCs by reducing the phosphorylation level of STAT3 in MDSCs,and finally inhibits the occurrence and development of pancreatic cancer.