Blocking The CXCL1-CXCR2 To Remodel The Tumor Inflammatory Microenvironment And Inhibit The Progression Of Hepatocellular Carcinoma

Object Hepatocellular carcinoma cancer(HCC)is one of the most common malignant tumors of the digestive tract in the world.In the past few decades,TKI and immunotargeting drugs have made great progress in the treatment of liver cancer,but the therapeutic effect of advanced liver cancer varies greatly and the effect is not ideal.Among them,the differences in etiology and pathological mechanism of hepatocarcinoma are the important reasons for the heterogeneity of hepatocarcinoma.In our country,chronic hepatitis B cirrhosis is the main cause of hepatocarcinogenesis,and the inflammatory microenvironment of hepatocarcinoma is also the core mechanism of hepatocarcinogenesis.Chemokines play an important role as mediators in chronic inflammation and tumor inflammation.However,there is limited information on chemokines in hepatocellular carcinoma,and the disease is almost entirely due to chronic hepatitis.Therefore,we explored the mechanisms by which one of the chemokines,CXCL1,an inflammatory chemokine commonly found in cirrhosis that positively correlates with the extent of liver damage,influences tumorigenesis and progression in HCC.CXCL1 expression profiles can predict recurrence in HCC patients and promote tumor progression by increasing tumor cell metabolism and epithelial-mesenchymal transition(EMT).As a functional receptor of CXCL1,CXCR2 specifically binds to CXCL1 to drive multiple immune cell migration and other pro-cancer effects in liver cirrhosis have been fully explored.For example,CXCL1 is able to activate inflammasomes by stimulating the G protein-coupled receptor CXCR2 to release IL-1β,where the CXCL1-CXCR2 axis is thought to be closely associated with drug resistance in tumors.Therefore,we hypothesized that blocking the binding of CXCL1-CXCR2 in the inflammatory microenvironment would suppress the inflammatory response,thus further inhibiting the pro-oncogenic role of CXCL1 in HCC;The aim of this experiment is to verify our hypothesis through in vitro experiments.Method The GEPIA tumor database analyzed correlations between CXCL1 expression levels and Disease Free Survival(DFS)and Overall Survival(OS)in patients with HCC and performed immunohistochemistry(IHC)and immunofluorescence(IF)to further confirm in collected clinical samples.Type 0 macrophages(M0)were obtained from human monocyte THP-1 induced by a well-established phorbol ester(PMA)and tumorassociated macrophages(TAMs)were obtained by co-culture with the HCC cell line Huh7 in a simulated inflammatory microenvironment in vivo using a co-culture chamber.The expression of chemokine CXCL1 in the co-culture system was detected by PCR and Elisa.The cell viability,clonogenic ability and apoptosis rate of Huh7 cell line were detected by MTT assay,cell clonogenic assay after blocking the specific binding of CXCL1-CXCR2 axis by small molecule inhibitor(SB225002).The biological behavior changes of Huh7 cell line,including the ability of migration and invasion,were determined by cell scratch test and transwell test,expression levels of epithelial-tomesenchymal transition(EMT)-related representative indicators in Huh7 cells in different experimental subgroups we performed PCR and WB validation by extracting total RNA and total protein of the corresponding Huh7 cells.Result GEPIA tumor database analysis showed that patients with HCC with high CXCL1 expression had lower DFS and OS than patients with HCC with low CXCL1 expression.By immunohistochemistry and immunofluorescence in clinical specimens we obtained that tumor tissues had higher CXCL1 expression compared with adjacent non-cancerous tissues and were accompanied by more expression of the macrophage marker CD68 protein.Previous experimental studies have demonstrated that co-culture of macrophages with tumor cells can induce macrophages to differentiate into M2 cancer-promoting tams,and the growth activity,clonality,migration and invasion of tumor cells Huh7 are enhanced.Furthermore,the expression level of CXCL1 was significantly decreased within the macrophage-tumor cell co-culture system after we blocked specific binding of the CXCL1-CXCR2 axis using the CXCR2 small molecule inhibitor SB225002;The biological behavior of tumor cells was significantly inhibited,and the levels of E-cadherin and Vimentin were increased,while the levels of N-cadherin and Vimentin were decreased.Furthermore,we explored the mechanism underlying the changes in CXCL1 expression levels,and the analysis revealed that the upstream factor IL-1β,which acts directly on CXCL1,presented a corresponding expression trend.In addition,the EMT changes of Huh7 tumor cells were correlated with NFKB1,which was also verified at the protein level.Conclusion The expression of chemokine CXCL1 was positively correlated with the degree of inflammation and promoted the proliferation,migration and invasion of Huh7 cell line,the use of SB225002,a small-molecule inhibitor of CXCR2,blocks the specific binding of CXCL1-CXCR2 to reduce CXCL1 expression levels within the co-culture system.At the same time,the EMT level,proliferation,migration and invasion of Huh7 cell line were also inhibited,the mechanism of action may be that the use of SB225002,a small-molecule inhibitor,directly affects IL-1β and NFKB1,thereby acting downstream on CXCL1 and EMT.