The Mechanism Of Exogenous IL-33 In The Progression Of HCC And Its Clinical Significance

The global incidence of hepatocellular carcinoma(HCC)has been high in recent decades.Most patients are found to be at an advanced stage.In addition,both liver transplantation and radiofrequency ablation have strict indications,and only 5%to 10%of HCC patients are eligible for partial hepatectomy.Even advanced HCC patients are treated with multi-target kinase inhibitors such as sorafenib,their long-term prognosis is not unsatisfactory due to high rates of recurrence and metastasis.Therefore,it is urgent to explore key molecules in HCC progression and find valuable markers.Interleukin-33(IL-33)is believed to be an effective promoter of type II immunity.It has been widely studied as a pleiotropic cytokine since 2005.Endogenous IL-33 is released into the extracellular system,alerting the immune system after cell damage or necrosis.Activated IL-33 binds to a coreceptor,a heterodimer composed of ST2 and IL1RAP,and initiates the inflammatory pathway.ST2 is a specific receptor for IL-33,encoded by the gene IL1RL1 and mainly expressed by immune cells.Combined with ST2,IL-33 activates a variety of ST2+ immune cells,including innate lymphoid cells(ILC2s),regulatory T cells(Tregs),dendritic cells(DCs),natural killer cells(NK)and so on,inducing the secretion of a variety of chemokines and pro-inflammatory cytokines,and regulating local and systemic immunity.Further,IL-33 promotes inflammatory events in tumors and activates pro-or anti-tumor responses.Transgenic IL-33 activates NK cells and T cells,resulting in growth inhibition melanoma and lung cancer.The IL-33/ST2 pathway upregulates CD40L and suppresses the growth of murine colon cancer.However,IL-33 enhances type II immune response accelerating tumor progression in tumor-bearing animals.Colon cancer transfection with IL-33 promotes tumor metastasis by accumulating myeloid-derived suppressor cells(MDSCs)to regulate the tumor microenvironment(TME).Tumor-derived IL-33 activates mast cells(MCs)and macrophages,promoting the development of gastric cancer.Therefore,the role of IL-33 in cancer remains controversial.In several studies on HCC,the RS3821204 genotype of plasma ST2 is positively correlated with HCC risk in China.IL-33 in stromal cells regulated by the pDGF-BB-SOX7 axis promotes HCC metastasis through tumor-associated macrophages.However,tumor-infiltrating effector-memory CD8+T cells in surgically resected tissues producing IL-33 could prolong the survival of HCC patients.In addition,IL-33 released by the liver inhibits HCC growth by promoting T cell response.Therefore,the effect of IL-33 on HCC by the regulation the immune system should be further studied.Considering that the effect of exogenous IL-33 on HCC through immune cells has not been reported yet,therefore,we mainly study the mechanism of exogenous IL-33 in the progression of HCC and its clinical significanceHere,we explore the correlation between IL-33 expression and prognosis of HCC patients,and predict the related pathways of IL-33 gene sets,suggesting that IL-33 may be a marker of HCC poor prognosis.After that,we examine the effects of exogenous IL-33 on the biological characteristics of mouse hepatoma cells in vitro and in vivo,Then we explain the possible mechanisms from the remodeling of TME,the secretion of tumor proliferating factors,and the change of microvessel density.At last,we put forward the future development direction and possible research scope of IL-33.Part Ⅰ evaluating the prognostic value of IL-33 in HCC patients and predicting its functionObjective:In HCC clinical samples,the correlation between IL-33 and the clinicopathological characteristics of HCC was analyzed,and the relationship between IL-33 and the prognosis of HCC patients was clarified.In the TCGA database of Liver hepatocellular carcinoma(LIHC),several databases were used to predict the pathway most related to the IL-33 gene sets.Methods:IL-33 expression in HCC tissue array was detected by immunohistochemistry(IHC).A total of 69 HCC tissues and 64 paired para-carcinoma tissues were included in the analysis considering.Then,statistical software SPSS was used to analyze the correlation between IL-33 expression and clinicopathological characteristics of HCC,such as gender,tumor size,TNM stage,capsule integrity and so on.Moreover,Cox regression analysis showed the correlation between IL-33 expression and prognosis in HCC patients.In addition,we further analyzed the effect of IL-33 on the clinical follow-up time of 69 HCC patients.Overall survival(OS)and disease-free survival(DFS)curves were conducted with the log-rank test according to the Kaplan-Meier method.Moreover,LIHC TCGA database was used to find the top 300 most relevant genes of IL-33.gene ontology(GO),and kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were performed through the R package "clusterProfiler".Gene set enrichment analysis(GSEA)was performed with GO,KEGG and Reactome databases to explore the functions of IL-33 and its gene sets.Finally,gene set variation analysis(GSVA)was used to compare the difference in the pathway scores between the high and low IL-33 groups.Results:Immunohistochemical semi-quantitative analysis of IL-33 expression in HCC tissue array showed that it was higher in HCC tissues compared with that in the adjacent tissues.After excluding the influence of individual differences,we further analysed IL-33 expression in 64 paired HCC and adjacent para-cancer tissues in this dataset.It was found to be consistent with the previous trend in 44 pairs data.In addition,HCC patients with high expression of IL-33 showed significantly shortened OS and DFS.Multivariate Cox regression analysis also suggested that IL-33 expression in HCC patients could be an independent prognostic predictor.Then,in GO terms,biological processes(BP),such as endothelial cell migration and tissue migration,was significantly regulated by the IL-33 alterations.Cellular components(CC)and molecular functions(MF)including cell-cell junction and extracellular matrix structural constituent were significantly associated with these IL-33 alterations.KEGG pathway analysis showed that the most enriched pathways were PI3K-Akt pathway,cGMP-PKG pathway,calcium pathway and Apelin pathway,which were associated with tumorigenesis and HCC progression.In addition,GSEA indicated that many pathways involved in tumorigenesis and cell cycle were related to IL-33 alterations,especially PI3K-Akt and MAPK pathways.At last,GSVA showed that signature scores in multiple oncogenic pathways were higher in IL-33 overexpression patients,which may lead to a poor prognosis.Conclusion:It has been verified in clinical HCC samples that the high expression of IL-33 was closely related to the shortened OS and DFS,which was consistent with multivariate Cox regression analysis of HCC prognosis.Then,Go terms,KEGG pathway,GSEA and GSVA were performed to predict the pathways of IL-33 alteration,most of which are involved in tumor proliferation and invasion.This part of the study started from the clinic,and provided the clinical application prospect for the further development of IL-33 treatment strategy in HCC.Part Ⅱ Study on the biological function of exogenous IL-33 on mouse hepatoma cells in vitro and in vivoObjective:Based on the negative correlation between IL-33 expression level and the survival time of HCC patients,this part of the study will explore the effects of exogenous IL-33 on the biological characteristics of mouse hepatoma cells from the cellular and animal levels.Methods:We selected mouse hepatocellular carcinoma Hepa1-6 cells as the study object.CCK-8 proliferation experiment was used to study the effect of IL-33 on Hepa1-6 cells proliferation ability in vitro.In this experiment,multiple concentrations of IL-33 were selected as Ong/mL,10ng/mL,20ng/mL,50ng/mL,and 100ng/mL respectively,and then co-incubated with Hepa1-6 cells for 48 hours.In vivo experiments,female C57BL/6 mice aged 6-8 weeks were selected to establish subcutaneous tumor-bearing models with 5×106 and 8×106 Hepa1-6 cells to study the effect of IL-33 on hepatoma cells with different tumor-bearing capacities.From the third day after tumor bearing,mice in the experimental group were intraperitoneally injected with mouse recombinant IL-33 protein(0.4μg/mouse),and mice in the control group were injected with the same volume of PBS.The injection was given every other day for 5 consecutive times.Tumor volumes were measured and calculated on days 3,5,7,9 and 11.After euthanasia of mice,the tumor size,tumor weight and tumor volume at different time points in two groups were counted.Finally,Ki-67 expression level was semi-quantified by IHC to evaluate the proliferation ability of hepatoma cells in two groups mice.Results:Exogenous IL-33 had no direct effect on the proliferation of Hepa1-6 cells in vitro.Surprisingly,IL-33 promoted the mouse hepatoma cells proliferation with different tumor loads in vivo.As a result,tumor size in the IL-33 treatment group was larger than that in the PBS treatment group on days 7,9 and 11.After euthanasia,the tumor weight of experimental mice was higher than that of control mice.In addition,the staining intensity of proliferation marker Ki-67 in the IL-33 treatment group was stronger than that in the control group.All these suggested that IL-33 promoted the proliferation of mouse Hepa1-6 cells in vivo.However,the effects of exogenous IL-33 on the proliferation of Hepa1-6 in vivo and in vitro were inconsistent.Based on a large number of studies,it has been reported that IL-33 regulates a variety of immune cells during immune response and homeostasis.We speculated that the inconsistent effects of IL-33 on Hepa1-6 cells proliferation might be partly attributable to the immunogenicity of tumors,rather than the changes in their intrinsic characteristics.Conclusion:Systemic injection of IL-33 accelerated the growth of HCC cells in mice.However,it did not promote the proliferation of Hepa1-6 cells in vitro.Considering the reports of IL-33 regulating a variety of immune cells,we speculate that IL-33 may promote tumor proliferation through the TME.This lays a foundation for the subsequent exploration of the mechanism of IL-33 promoting HCC cells proliferation in vivo.These results suggest that IL-33 may be a key tumor promoter for HCC proliferation and tumorigenicity,providing a new idea for a comprehensive understanding of HCC occurrence and development.Part Ⅲ Study on the mechanism of exogenous IL-33 promoting the progression of hepatocellular carcinomaObjective:On the basis of clarifying the effects of IL-33 on the biological functions of mouse Hepa1-6 cells in vivo and in vitro,this part of the study aims to clarify the specific mechanism of IL-33 promoting HCC progression such as infiltration of various immune effector cells and immunosuppressive cells in mice,the secretion of chemokines recruiting myeloid cells and cytokines,and the changes of microvascular density.Methods:We established a mouse hepatocellular carcinoma subcutaneous tumor-bearing model.After IL-33 treatment,the infiltration of immunosuppressive cells,such as MDSCs and Tregs,and immune effector cells,such as NK cells and T cells,were detected by flow cytometry.To further explore the cytokines involved in IL-33 regulation,we examined the chemokines recruiting MDSCs into the TME and the expression of S100A9 secreted by myeloid cells.In addition,pro-inflammatory factors such as IL-6,IL-1β,and tumor necrosis factor(TNF)-α that stimulate tumor proliferation and angiogenesis were also examined.Finally,IHC was used to detect the micro vessel density in the tumors to fully reveal the mechanism of IL-33 in promoting mouse hepatoma cells proliferation in vivo from three aspects.Results:IL-33 administration markedly increased CD45+leukocyte immune cell infiltration.Among them,we observed a decrease in frequencies of intratumoral CD3NK1.1+NK cells in tumor-bearing mice with IL-33 treatment.The percentage of activated CD69+ CD8+ T cells was remarkably downregulated in the IL-33-treated group.While,there was no difference in the expression of IFN-γ between the two groups.These results demonstrated that IL-33 promoted tumor progression only in part due to a reduced ratio of CD8+ T cells and NK cells in vivo.Furthermore,MHC II expression in splenic DCs decreased in response to IL-33 administration,thus,blocking DC maturation and their cross-presentation ability.Meanwhile,IL-33 increased the splenic infltration of immunosuppressive cells in tumor-bearing mice.CD11c-CD11b+GR1+MDSCs and Tregs were significantly increased in the spleens of mice treated with IL-33,which may lead to the occurrence of tumor immune escape.Then,we detected the chemokines that recruited myeloid cells into the TME.The results showed that the mRNA levels of Csf2,Ccl2,Ccl5,and Cxcl1 chemokines signifcantly increased after IL-33 treatment.Then S100A9 secreted by myeloid cells was found to be up-regulated,inducing epithelial-mesenchymal transformation and participating in tumorigenesis.Therefore,IL-33 may induce tumor derived chemokines(e.g.Ccl5),which enhanced the recruitment of myeloid cells secreting S100A9 to promote tumor progression.In turn enlarged tumors secreted more chemokines,which recruited myeloid cells to form a closed loop promoting tumor growth.At last,we clarified the mechanism of IL-33 promoting HCC progression from the perspective of micro vascular changes.After IL-33 treatment,the expression of vascular endothelial growth factor(VEGF)as a key signal to stimulate angiogenesis was significantly increased.IL-33 also increased the IHC staining intensity of endothelial marker CD31,and the microvascular density was approximately higher in experimental mice than in untreated mice,suggesting that active angiogenesis and lymph angiogenesis occurred in IL-33-treated tumors.Conclusion:IL-33 not only inhibited the infiltration of immunoeffector cells and mobilized immunosuppressor cells,but also alters their subsets and functions.In addition,IL-33 regulated a variety of chemokines and cytokines,forming a closed loop that promotes tumor proliferation.At last,IL-33 promoted intratumoral neovascularization.Thus,IL-33 may act as an effective amplifier for signaling pathways that link the loops of inflammatory microenvironment to tumor amplification,thereby mediating tumor growth and proliferation.