Effect Of Hyperthermic Intraperitoneal Chemotherapy On Immune Microenvironment In Patients With Peritoneal Carcinomatosis

Background:Peritoneal Carcinomatosis(PC)includes primary peritoneal cancer and metastatic peritoneal cancer.Patients with PC have a short survival time and poor prognosis.Hyperthermic Intraperitoneal Chemotherapy(HIPEC)is the main treatment for PC.However,the effect of HIPEC as a chemotherapy method on the tumor immune microenvironment(TIME)of PC patients has not been reported.Studying the effect of HIPEC on the immune function of PC patients and further explaining the mechanism of HIPEC can provide a basis for patients to better choose HIPEC treatment,and may provide a theoretical basis for HIPEC combined with immunotherapy.Objective:The effect of HIPEC on TIME in PC patients was explored by comparing the changes of TIME in tumor tissue samples and cytokines and T lymphocyte subsets in blood samples before and after HIPEC.Methods:In this study,patients with PC who underwent HIPEC in the Cancer Center of the Second People’s Hospital of Changzhou from December 2020 to December 2022 and were willing to be followed up for a long time were selected.Each patient received 4 times of HIPEC.Blood samples were collected before and after HIPEC treatment,and flow cytometry was used to detect changes in serum T lymphocyte subsets and 12 cytokines related to immune status:IL-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12P70,IL-17,IFN-α,IFN-γ,TNF-α.Blood samples were collected from 18 patients,and tumor tissue samples were collected from 6 patients before and after HIPEC and analyzed by Nano String n Counter Analysis.In addition,the tumor tissue samples of 2 patients were collected and analyzed by single-cell RNA sequencing(Sc RNA-seq)to verify the Nano String analysis results.At the same time,all patients were followed up,and the Progression-Free Survival(PFS)and overall Survival(OS)of patients with ascites were recorded.Kaplan-Meier method was used for survival analysis.Results:(1)Four serum cytokines were significantly increased after HIPEC in 18patients:IL-2(P=0.022,IL-6(P=0.002),IL-8(P=0.005),and IFN-γ(P=0.028)were significantly increased in 18 patients,and the other 8 cytokines had no significant change before and after treatment.The number of CD4~+,CD8~+and CD3~+T lymphocytes in peripheral blood T lymphocyte subsets increased significantly after HIPEC(420.67±137.41 vs 589.67±178.29,385.22±139.07 vs 477.17±141.11,842.44±219.86 vs 1055.78±248.48,P<0.01),but the CD4~+/CD8~+ratio did not change significantly.(2)Among them,Nano String RNA sequencing before and after HIPEC in 6patients showed a total of 13 differential gene expressions,including CXCL5(P<0.001),S100A8(P=0.001),S100A9(P=0.001),CXCL8(P=0.004),CCL18(P=0.014),CCL4(P=0.014),CCL2(P=0.021),IFIT1(P=0.021),IL1B(P=0.021),CXCL2(P=0.027),CCL20(P=0.049),CXCL1(P=0.049),and S100A12(P=0.049).KEGG and GSEA enrichment analysis showed that the gene sets significantly enriched after treatment were interleukin-17(IL-17)signaling pathway and chemokine signaling pathway.In the analysis of TIME cell type differences,a total of 14 cell types were analyzed before and after HIPEC treatment,and there were no significant differences,only regulatory T cells(Tregs)had a downward trend in the treated samples(P=0.094).TIME-related gene signatures changed before and after HIPEC did not change significantly,only T cell-inflamed gene expression profile(GEP)score showed an upward trend in the treated samples(P=0.063).(3)A total of 4 samples from 2 patients were analyzed by Sc RNA-seq,and the cell profiles of 4 samples were a total of 33858 cells and 12 cell types.The T cell-inflamed GEP scores of these 12 cell types were analyzed.Endothelial cells(ECs),B cells,T and NK cells and mononuclear phagocytes(MPs)were found to have higher scores than other cell types.The scores of these four cell types before and after HIPEC treatment were analyzed.It was found that the scores of T and NK cells and MPs T cell-inflamed GEP in both patients were significantly increased after HIPEC(P<0.001).T and NK cells were further selected for secondary subtype subdivision clustering and annotation,and 10 different subtypes were obtained by subdivision annotation.The results showed that among NK cells,the score of NK＿XCL1 subgroup increased significantly after HIPEC(P<0.05);In T cells,the scores of CD8Teff＿GZMH,CD8Teff＿GZMK,CD4Naive T＿CCR7 and CD4Treg＿FOXP3 subsets were significantly increased after HIPEC(P<0.05).(4)After HIPEC,10(83.3%)of the 12 patients with ascites at baseline achieved CR and 2(16.7%)achieved PR,with an ORR rate of 100%.The median PFS of ascites was 10.3 months in 12 patients with ascites,and the median OS in all patients was 18.4 months.Conclusions:HIPEC can significantly increase the serum levels of IL-2,IL-6,IL-8and IFN-γand the number of immune cells in patients with PC,thereby enhancing the anti-tumor immune function of PC patients.HIPEC can significantly increase the T cell-inflamed GEP score of immune cells,thereby improving the immunosuppressive state in PC patients and producing anti-tumor immune effects.