The Role Of STAT3 In The Development Of Hepatocellular Carcinoma In Myeloid Cells

Hepatocellular carcinoma（HCC）is the fourth most common cause of cancer-related deaths worldwide.Risk factors for hepatocellular carcinoma include chronic hepatitis B and C,alcohol addiction,metabolic liver disease（especially non-alcoholic fatty liver disease）,and exposure to dietary toxins such as aflatoxin and aristolochic acid.Available treatment options include chemotherapy,radiation therapy,and immunotherapy in addition to surgical treatment methods.The signal transducer and activator of transcription（STAT）plays an important role in signaling in hepatocellular carcinoma and is a member of seven families of classical cancer pathways.STAT3 was originally identified in 1996 as an acute phase response factor（APRF）in IL-6 signaling during the study of interferon-induced gene transcription and is involved in cancer cell proliferation,apoptosis,invasion,metastasis,etc.Our group has demonstrated that STAT3 is expressed in liver parenchymal cells,tumor cells,vascular endothelial cells,and various immune cells in the tumor microenvironment,and the activation of STAT3 in various cells is closely related to cancer development,but the mechanism of how STAT3 activation in various cells promotes cancer development in the liver cancer environment is still unclear.Myeloid cells,as opposed to germline cells,are the general term for common myeloid progenitor cells and the various types of precursor cells and mature cells formed by their differentiation.They include myeloid common progenitor cells,granulocyte-monocyte progenitor cells,macrophage-dendritic progenitor cells,dendritic common progenitor cells,mononuclear common progenitor cells,granulocytes,monocytes,dendritic cells,macrophages,NK cells,etc.They circulate through the blood and lymphatic systems and are rapidly recruited to tissue injury and infection sites by various chemokine receptors.Within tissues,they are activated for phagocytosis as well as for inflammatory cytokine secretion,thus playing a major role in protective immunity.Bone marrow cells can also be present in tissues under homeostatic conditions,and they control development,homeostasis and tissue repair.Previous studies have shown that in the tumor microenvironment,activation of STAT3 in myeloid cells promotes tumorigenesis development.In the present study,our group similarly demonstrated that STAT3 in myeloid cells promotes hepatocellular carcinoma development in a diethylnitrosamine（DEN）-induced hepatocellular carcinoma model,and that knockdown of STAT3 in myeloid cells resulted in more pronounced changes in the expression of pro-inflammatory factors such as IL12,IFNγ,and TNFαin tumor tissues compared with adjacent non-tumor tissues.However,the reasons for these changes in STAT3 in myeloid cells are still unclear,and further investigation of this phenomenon is beneficial for target mining and precise treatment.Various immune cells in the tumor microenvironment interact to maintain the growth of HCC.Hepatocellular carcinoma is an inflammation-driven disease.Chronic inflammatory factor-related immunosuppression hasised to growth factors,cytokines an,d chemokines produced by stromal cells and tumor cells.Among them,activation of tumor cell STAT3 followed by cytokine production can weaken the tumor surveillance effect of other immune cells in the tumor microenvironment and continue to activate the expression of STAT3 in myeloid cells and other stromal cells such as vascular endothelial cells to enhance the immunosuppressive effect in the tumor microenvironment,but how myeloid STAT3 interacts with other cells in the tumor microenvironment is not clear.In this study,STAT3^{F/F Lys+/-Cre}mice in the experimental group and STAT3^F/F mice in the control group were obtained by mouse genotype identification and mouse mating and breeding methods.And DEN/CCl4-induced mouse liver cancer model was used to detect the number of tumors,maximum tumor volume,liver mass as a percentage of body mass,maximum tumor length diameter,and maximum tumor width on the liver.The levels of ALT and AST in hepatocellular carcinoma and acute liver injury conditions and the levels of IFNγand TNFαin the hepatocellular carcinoma environment were detected by ELISA kits.The effect of myeloid STAT3 on the recruitment numbers of CD8⁺T cells,CD4⁺T cells,NKT cells,Ly6C^hi cells,neutrophils,and myeloid-derived suppressor cells in the immune microenvironment of mouse hepatocellular carcinoma was detected by flow cytometric analysis.The expression of Th1,Th2,and Th17 intracellular factors was also examined to determine the changes in the proportion of each CD4⁺T cell subtype,and Liver tissue injury and hepatocyte proliferation were observed by HE staining and Ki67 staining.1.Effect of myeloid STAT3 in DEN/CCl4-induced hepatocellular carcinoma model in miceOur group obtained experimental STAT3^{F/F Lys+/-Cre} mice and control mice STAT3^F/Fby genetic identification and mating breeding.The mice were injected intraperitoneally with 25 mg/kg of DEN every two weeks at 2 w,4 w,and 6 w after birth,and switched to 0.5 ml/kg of CCl4 solution once a week until 22 w after 8 w.The mice were sacrificed and liver tissues were isolated to observe liver tumor growth and found that the number of liver tumors in STAT3^{F/F Lys+/-Cre} mice increased significantly,suggesting that knockdown of STAT3 expression in myeloid cells significantly promoted the growth of tumor number.The maximum volume of liver tumors was also found to be significantly increased in STAT3^{F/F Lys+/-Cre} mice compared to the control group,suggesting that knockdown of STAT3 expression in myeloid cells promoted the maximum volume of tumors.liver mass as a percentage of body weight,maximum length diameter,and maximum width of tumors in STAT3^{F/F Lys+/-Cre} mice treated with DEN/CCl4 also significantly increased.All these results suggest that STAT3 expression in myeloid cells has an inhibitory effect on tumor growth in hepatocellular carcinoma model mice.2.Effect of knockdown of STAT3 in myeloid cells on the levels of ALT,AST,IFNγand TNFαin the liver tumor environmentSerum alanine transaminase（ALT）and serum aspartate transaminase（AST）in STAT3^{F/F Lys+/-Cre} and STAT3^F/F mice were examined in the DEN/CCl4-induced hepatocellular carcinoma immune microenvironment under chronic inflammatory conditions.Aspartate aminotransferase（AST）levels and serum IFNγand TNFαexpression,the results showed that in the DEN/CCl4-induced hepatocellular carcinoma model,compared with STAT3^F/Fmice,serum ALT and AST levels in STAT3^{F/F Lys+/-Cre}mice did not significantly change.However,knockdown of STAT3 expression in myeloid cells was found to elevate serum IFNγand TNFαlevels in DEN/CCl4-treated mice,and these suggest that myeloid-specific deletion of STAT3 in the DEN/CCl4 hepatocellular carcinoma model leads to an increased inflammatory response in the immune microenvironment of hepatocellular carcinoma and thus increases hepatocarcinogenesis.3.Effect of myeloid STAT3 on immune cells in liver tumor tissuesThe effect of STAT3 on the recruitment of CD8⁺T cells,CD4⁺T cells,NKT cells,Ly6C^hi cells,neutrophils,s and myeloid-derived suppressor cells（MDSC）to tumor tissues as a proportion of immune cells in tumor tissues was examined by flow cytometric analysis in the immune microenvironment.The ratio of CD8⁺T cells in the immune microenvironment of STAT3^{F/F Lys+/-Cre} mice with hepatocellular carcinoma did not show significant differences,but knockdown of myeloid-derived cells STAT3 in the tumor immune microenvironment significantly increased the recruitment of CD4⁺T cells.The difference in the proportion of NKT cells recruited to tumor tissues in STAT3^F/F mice compared to STAT3^{F/F Lys+/-Cre} mice was not significant.The difference in the proportion of Ly6C^hi monocytes in tumor tissues in STAT3^F/F and STAT3^{F/F Lys+/-Cre} mice The difference in the percentage of immune cells was also not significant,indicating that the lack of STAT3 in myeloid cells did not alter the extent of Ly6C^hi monocyte infiltration in liver tumors.Meanwhile,neither the proportion of neutrophils nor the infiltration of myeloid-derived suppressor cells in tumor tissues was affected by myeloid STAT3.4.Effect of myeloid STAT3 on the differentiation of CD4⁺T cells in liver tumor tissuesThe frequency of IL17-A,IL-4,and IFNγexpression in CD4⁺T by myeloid STAT3was detected using flow cytometry and the percentage of Th17,Th1,and Th2 cells in CD4⁺T was counted.The proportion of CD4⁺T cells differentiated toward Th17 was significantly higher in the immune microenvironment of STAT3^{F/F Lys+/-Cre} mice with hepatocellular carcinoma.In contrast,there was no statistically significant difference in the number of Th1 and Th2 cells as a proportion of CD4⁺T cell population in liver tumor tissues of STAT3^{F/F Lys+/-Cre} mice relative to STAT3^F/F mice.The expression of IL-17A and RORγt in CD4⁺T cells was significantly higher in the tumor immune microenvironment of STAT3^{F/F Lys+/-Cre} mice,suggesting that knockdown of STAT3 in myeloid cells promoted the differentiation of CD4⁺T cells toward Th17 in the liver tumor immune microenvironment.5 STAT3 in myeloid cells significantly ameliorates DEN-induced early liver injury.STAT3 in myeloid cells showed a significant protective effect against acute liver injury caused by DEN.After 24 h,STAT3 in myeloid cells significantly reduced serum AST and ALT levels as detected by ELISA kit.Conclusions:1.Myeloid STAT3 inhibited tumorigenesis in DEN/CCl4 mouse liver cancer model.2.Knockdown of myeloid STAT3 increased the levels of IFNγand TNFαin the immune microenvironment of liver tumors.3.Knockdown of myeloid STAT3 increased the recruitment of CD4⁺T cells in liver tumor tissues.4..Myeloid cells STAT3 inhibit the differentiation of CD4+T cells to Th17 in the liver tumor immune microenvironment.5.Myeloid STAT3 significantly ameliorates DEN-induced early liver injury.