| ObjectiveSolid tumors are organic entities constructed by tumor cells and matrix components in a specific three-dimensional space.The distribution pattern of tumor cells within a tissue can be simply quantified using the mathematical indicator of tumor cell density.The changes in tumor cell density in response to tumor proliferation or treatment response will be combined with corresponding changes in secretome to induce a series of cascade reactions.Immune cells are the"double-edged sword"involved in tumor development.In the short-term effect,killer subpopulations such as CD8+T cells and M1 macrophages exert anti-tumor effects,while regulatory T cells(Tregs)and M2 macrophages subpopulations exert a"protective"effect by inhibiting anti-tumor immunity.In the long-term effect,the killing subgroup achieves immune editing effect by clearing the sensitive tumor subgroup,while the inhibitory immune cell subgroup has limited effect.Based on the influence of tumor secretome and metabolic products such as lactate on the functional differentiation of immune cell subpopulations,the response of immune cell subpopulations is both an upstream cause and a downstream result in the change of tumor cell density.Therefore,the purpose of this study is to explore the response patterns and mechanisms of key immune cell subpopulations,namely Tregs/CD8+T cells and M1/M2 macrophages,with changes in tumor cell density using hepatocellular carcinoma as a sample,and the potential significance of this response in tumor development.Methods1.Identify the response pattern of the ratio of Tregs/CD8+T cells to M1/M2macrophage subpopulations with changes in liver cancer cell density.Identify and count the tumor cell density and infiltration degree of Tregs,CD8+T cells,M1 and M2 macrophages in hepatocellular carcinoma(HCC)tissue samples through immunohistochemistry.Validate sample conclusions and screen potential mediators based on bioinformatics methods.2.Identify the potential role of interleukin 8(IL-8)derived from liver cancer cells in tumor density related immune fluctuations:establish a co culture model of gradient density liver cancer cells and human peripheral blood monocytes in vitro,and measure the polarization of Tregs using flow cytometry;Use lentivirus intervention and recombinant IL-8 to verify the effect of IL-8 expression on Tregs polarization.3.The promoting effect of IL-8 on lactate production in liver cancer cells.Quantify the correlation between IL-8 expression in liver cancer cells and lactate content in the culture medium using a lactate assay kit.The correlation between IL-8expression and the expression of three key enzymes and hypoxia inducible factor-1A(HIF-1A)in liver cancer cells was determined based on bioinformatics methods,q PCR,and western blot analysis.The correlation between IL-8/Death associated protein kinase 1(DAPK1)/PK activity/lactate expression in hepatocellular carcinoma was analyzed based on bioinformatics,Pyruvate kinase(PK)activity assay kit,immunohistochemistry,western blot and lentivirus intervention.4.Synergistic promotion of IL-8 and lactic acid in Tregs infiltration of liver cancer model.Lentivirus intervenes in mouse liver cancer cells to establish control group,high IL-8 group,high lactate group,high IL-8&high lactate group basic cell lines and mouse orthotopic liver cancer model.Verify the accuracy of the model through immunohistochemistry and lactate assay.Confirm the progression status of liver cancer in each group through live imaging and gross observation of samples.Identify target immune cells through immunohistochemical staining.5.The promoting effect of IL-8 on lymphocyte endothelial adhesion.Establish two-dimensional and three-dimensional co culture models of umbilical vein endothelial cells and liver cancer cells.The adhesion rate of lymphocytes was measured by flow cytometry after co incubation of lymphocytes and endothelial cells.The expression of adhesion molecules on the surface of umbilical vein endothelial cells was measured by q PCR,western blot,and immunofluorescence.6.The potential significance of immune fluctuations related to liver cancer cell density in the development of liver cancer.obtaining tumor subclone expression,ploidy data,and tumor mutation load data based on bioinformatics.Results1.The Tregs/CD8+T cell ratio in liver cancer infiltration fluctuates significantly with tumor cell density,mainly manifested as low value areas within the 5000 to 6000cell/mm~2 density range and high value areas on both sides of the density range(<5000cells/mm~2 and≥6000 cells/mm~2).The ratio of M2/M1 macrophages did not show significant changes with tumor cell density.2.The expression changes of IL-8 in liver cancer cells induce changes in the degree of Tregs polarization in a monocyte liver cancer cell co culture model,but IL-8alone did not alter the degree of Tregs polarization.3.IL-8 promotes lactate expression in liver cancer cells through the IL-8/DAPK1/PK axis,further promoting an increase in Tregs polarization in the monocyte liver cancer cell co culture model.4.High expression of IL-8 combined with high lactate secretion by liver cancer cells promotes a significant increase in the proportion of Tregs infiltration in liver cancer tissue and promotes tumor progression.Removing high lactate production will eliminate the promoting effect of IL-8 on Tregs infiltration.5.The degree of tumor ploidy and tumor mutation load dispersion fluctuate regularly with the increase of tumor cell density.Conclusion1.Tumor density dependent IL-8 secretion fluctuations are mediators of density dependent Tregs/CD8+T cell infiltration fluctuations.IL-8 promotes lactate secretion in liver cancer cells through the DAPK1/PK activity axis,thereby inducing increased Tregs enrichment in tumor tissue.2.The lymphocyte recruitment of IL-8 combined with the post-processing of high lactic acid environment provides another explanation for the establishment of the immunosuppressive microenvironment in liver cancer tissue,suggesting that inhibiting the infiltration of immune cells may not be the only or optimal solution for tumor to resist immune killing.3.The potential significance of immune fluctuations related to tumor cell density:Utilizing immune editing effects to eliminate the advantage of high mutation load subpopulations at density checkpoints,ensuring the overall stable development of tumor tissue. |
PDF Full Text Request