Study On The Expression And Function Of Programmed Cell Death 1 Ligand 2(PD-L2)in Colorectal Cancer

Objective In the first part of this study,we performed the pan-cancer analysis to explore the expression,function,prognostic significance,and predictive value in immunotherapy cohort of programmed cell death 1 ligand 2(PD-L2).Methods In this study,R language commands such as "gsub","avereps" and "sapply"were used to extract the expression level of pdcd1lg2 mRNA(PD-L2 encoding gene)in 33 types of human malignant tumor and 8 types of malignant tumors internationally from The Cancer Genome Atlas(TCGA)database and International Cancer Genome Consortium(ICGC)database.The immunohistochemical staining results of 19 kinds of malignant tumors in the Human Protein Atlas(HPA)database were analyzed to determine the expression of PD-L2 protein in tumor tissues.The expression level of pdcd1lg2 mRNA was sorted from low to high by using the "quantile" command in R language,and divided into the lowest 30%group and the highest 30%group.Then Gene Set Enrichment Analysis(GSEA)software was used for functional enrichment analysis with Kyoto Encyclopedia of Genes and Genomes(KEGG)gene set and Reactome gene set as reference.The R language"estimateScore" and other commands were used to calculate the correlation between the expression of pdcd1lg2 mRNA and tumor stromal score and tumor immune score in pan-cancer.Spearman correlation test was used to determine the expression of pdcdllg2 mRNA and the mRNA expression of immunomodulatory molecules(immunostimulatory molecules,immunosuppressor molecules,chemokines and chemokine receptors)and Tumor Mutation Burden(TMB)and MicroSatellite Instability(MSI).Set the "minimum required interaction score" to 0.700 in the Search Tool for the Retrieval of Interacting Genes(STRING)database to identify molecules that interact with PD-L2.The correlation between the infiltration abundance of 21 different types of immune cell and the expression level of pdcd1lg2 mRNA was calculated by the "Gene" module in the TIMER database.Kaplan-Meier Survival analysis and Cox regression analysis were performed using R language commands such as "survfit" and "coxph" respectively to determine the relationship between pdcd1lg2 mRNA and Overall Survival(OS)or Progression-Free Survival(PFS).Expression difference of pdcd1lg2 mRNA output by selecting "Gene"module in Tumor Immune Syngeneic MOuse(TISMO)database in different treatment response groups in multiple mouse malignant tumor immunotherapy cohorts.The accuracy of 7 common immunotherapy efficacy markers and pdcd1lg2 mRNA expression in the prediction of immunotherapy efficacy in human malignant tumors was compared by the"Biomarker Evaluation" module in Tumor Immune Dysfunction and Exclusion(TIDE)database.Results Pdcd1lg2 mRNA expressed in 33 types of common human cancer types in TCGA database and in 8 types of tumor tissues from 12 human cancer queues in ICGC though it exhibited inconsistent mRNA expression.Moreover,the expression of pdcd1lg2 mRNA was not completely consistent among different pathological subtypes of malignant tumors in the same site and the expression of pdcd1lg2 mRNA in different sites of the same pathological type of malignant tumors may also be different.Similarly,PD-L2 protein was widely expressed in 19 kinds of malignant tumor tissues in HPA,with great expression differences among different tumor tissues.The results of GSEA suggested that the expression of pdcdllg2 mRNA in pan-cancer were associated with multiple immune function related pathways,including ANTIGEN PROCESSING AND PRESENTATION and TCR SIGNALING.etc.The expression of pdcdllg2 mRNA in 32 types of cancer,except thymoma,in the TCGA database was positively correlated with immune score and stromal score(R>0.25,p<0.05).In addition,Spearman correlation analysis showed that the expression of pdcdllg2 mRNA was negatively correlated with the expression of multiple immunostimulatory molecules(Cor<0,p<0.05),such as ULBP1.TNFSF9 and TNFSF15,was positively correlated with the expression of several immunosuppressive molecules(Cor>0,p<0.05),such as TIGIT,PDCD1 and LAG3.The protein-protein interaction in STING database showed that PD-L2 protein were significantly correlated with the expression of multiple immune-related molecules,such as PD-1,CD3 and CD28.Based on the above results,we concluded that the function of PD-L2 was closely related to tumor immunity process in pan-cancer.Therefore,we further studied the relationship between PD-L2 and immune cells.The multiple algorithms in the TIMER showed that the expression of pdcd1lg2 mRNA was correlated with the infiltration of various immune cells(such as CD8+T.B cells,macrophages and dendritic cells.etc).It should be noted that the abundance of tumor-associated macrophages was the most positively associated with pdcd1lg2 mRNA in colon cancer(EPIC algorithm:Rho=0.917:MCP-COUNTER algorithm:Rho=0.913;XCELL algorithm:Rho=0.818:TIMER algorithm:Rho=0.496).In the study of clinical application of PD-L2,Kaplan-Meier survival curves indicated that high pdcd1lg2 expression was significantly associated with the deteriorated outcomes in 10 types of malignant tumors,such as colon cancer(p=0.032).uroepithelial carcinoma of bladder(p=0.011),and papillary cell carcinoma of kidney(p=0.01),and better prognosis in the other 10 kinds of malignant tumors,such as isoinvasive breast carcinoma(p=0.021),cervical cancer(p=0.011)and esophageal cancer(p=0.044).The results of Cox regression analysis demonstrated that high expression of pdcdllg2 mRNA was a risk factor for the prognosis of three malignant tumors.including renal papillary cell carcinoma(p=0.009),low-grade glioma(p<0.001)and thymoma(p=0.010),and a protective factor for the prognosis of patients with cutaneous melanoma(p<0.001).In the part of evaluation of the predictive value of PD-L2 in immunotherapy,the correlations were weak between the pdcdllg2 expression and TMB and MSI(R<0.4).Among 65 immunotherapy cohorts of tumor-bearing mice,the expression of pdcd1lg2 mRNA in tumor changed in 21 cohorts,Compared with baseline level.the expression levels of pdcd1lg2 mRNA in tumor tissues was increased in 19 cohorts.The predictive accurate of pdcdllg2 was more than 50%(AUC>0.5)in 15 immunotherapy cohorts of tumor patients,which was better than that of MSI score and B clone.Conclusion In pan-cancer tumor tissues.PD-L2 expression can be enriched in multiple immune-related functional pathways and is associated with the expression of multiple immune-related molecules,so it plays an important role in the tumor immune microenvironment.It is worth noting that PD-L2 may be highly expressed in tumor-associated macrophages in colon cancer.In addition,PD-L2 has prognostic value for various tumor patients and predictive value of immunotherapy efficacy.Objective According to the analysis of TIMER database in the first part of this study,compared with the other immune cells in other tumor tissues,the expression of pdcdllg2 mRNA was the strongest associated with the abundance of tumor-associated macrophages(TAMs)in colon cancer tumor tissue(EPIC algorithm:Rho=0.917;MCP-COUNTER:Rho = 0.913;XCELL:Rho=0.818;TIMER:Rho=0.496).Therefore,this part of the study will verify the distribution of PD-L2 expression in colon cancer tissues and further explore the effect of PD-L2 expression in TAMs on tumor progression and its mechanism.Methods Tumor Immune Single-cell Hub(TISCH)database was used to analyze the single-cell sequencing data sets of 8 colorectal cancer tissues to understand the expression of pdcd1lg2 mRNA in tumor cells,immune cells and stromal cells.Paraffin sections of tumor tissues of colon cancer patients who had undergone surgical treatment were taken,and PD-L2 and CD68 multiple immunofluorescence staining were performed to analyze the expression of PD-L2 on TAMs.In addition,fresh tumor tissues of colon cancer patients undergoing surgical treatment were extracted into single-cell suspension and then analyzed by flow cytometry to detect the expression of PD-L2 on other immune cells and tumor cells.After murine subcutaneous transplanted tumor model was constructed by MC38 colon cancer cell,PD-L2 expression in immune cells and tumor cells were detected by flow cytometry at different time points after modeling(day 7,day 14,day 21 and day 28).Moreover,PD-L2+TAMs and PD-L2-TAMs in tumors were sorted by fluorescence activated cell sorting(FACS)on the 14th day for follow-up experiments.The RNA sequencing data of human colon cancer cell lines were analyzed in the "Expression&CN" module of Cancer Cell Line Encyclopedia(CCLE)database,and the expression level of pdcdllg2 mRNA in human tumor cell lines was output by using R language "ggplot" and other commands.The expression of PD-L2 in 6 human colon cancer cell lines and 3 murine colon cancer cell lines was determined by Western Blot analysis.Mouse primary bone marrow derived macrophages were extracted and induced to be polarized into M1 and M2 macrophages.Subsequently.Ml and/or M2 markers on PD-L2+TAMs.PD-L2-TAMs,M0 macrophages,M1 macrophages and M2 macrophages were detected by qRT-PCR and flow cytometry to determine the direction of polarization of PD-L2+TAMs.After mixing PD-L2+TAMs or PD-L2-TAMs with Escherichia coli,flow cytometry was used to detect the number of phagocytic bacteria to understand their phagocytic ability.Flow cytometry was used to determine the expression of lysosome-associated membrane protein 1(LAMP1),LAMP2 and Ki-67 in PD-L2+TAMs and PD-L2-TAMs to understand the lysosome status and proliferation capacity of the TAMs.Transwell culture mode was used to co-culture PD-L2+TAMs/PD-L2-TAMs and MC38 which is the colon cancer cell for 24 h or 48 h,and the number of migrating and invading colon cancer cells was counted.Transwell culture mode was also used to co-culture two types of TAMs and colon cancer cells for 7 days,and the number of tumor cell clones was counted.Mouse primary T cells were isolated,activated by CD3 and CD28,and cocultured with PD-L2+TAMs/PD-L2-TAMs.The proportion of CD8+T cells expressing Ki-67 and producing interferon γ(IFN-γ)was determined by flow cytometry.After obtained by the FASC,PD-L2+TAMs and PD-L2-TAMs were sequenced to search for specific expression genes and differential expression genes(|log2FC|≥1,Qvalue≤0.05),and GO and KEGG enrichment analysis were performed.The culture supernatant of PD-L2+TAMs and PD-L2-TAMs was collected after 48h culture,and cytokine protein array was used to detect the differences in cytokine secretion between the two types of TAMs.Results Through analysis of 8 single cell sequencing data sets of colorectal cancer tissue in TISCH database,it was found that PD-L2 encoding gene pdcdllg2 mRNA was mainly expressed in immune cells and stromal cells of tumor microenvironment(TME),especially in monocyte/macrophage cell population and not expressed in tumor cells.By analyzing the tumor tissues of colon cancer patients undergoing surgical treatment in Colorectal Surgery Department,we found that PD-L2 was expressed on TAMs in 4 of the 7 enrolled colon cancer patients,accounting for about 20%of the number of TAMs.The double positive cell count was rare in 3 patients.Flow cytometry analysis showed that CD8+T cells in the tumor tissues of colon cancer patients could be divided into PD-L2HighCD8+T cells(group I)and PD-L2LowCD8+T cells(group II),the proportions of CD8+T cells in the two groups were 51.11±7.26%and 8.23±5.55%,respectively.About 34.8±2.55%of B cells expressed PD-L2.Dendritic cells and tumor cells did not express PD-L2.Flow cytometry analysis found that the number of PD-L2+TAMs cells.PD-L2+CD4+T cells and PD-L2+CD8+T cells were not constant,but changed with the tumor volume.The number of PD-L2+TAMs peaked on the 14th day(30.35±5.45%).while the number of PD-L2+CD4+T cells and PD-L2+CD8+T cells peaked on the day7.In order to further confirm whether PD-L2 can be expressed on tumor cells.the transcriptome data of 73 human colorectal cancer cell lines in the CCLE database was analyzed in this study.and it was found that 24 of them did not express pdcdllg2 mRNA,and 41 cell lines had very low expression level.Western Blot analysis of 6 human and 3 mouse colon cancer cell lines showed that the expression level of PD-L2 protein was consistent with that of pdcdllg2 mRNA in CCLE database.In conclusion.since PD-L2 is mainly expressed in TAMs in colon cancer tissues,subsequent experiments are mainly to explore the nature and function of PD-L2+TAMs.qRT-PCR and flow cytometry analysis showed that the expression levels of M2 markers(CD206,Arg-1,IL-10,TGF-β and Ym1)on PD-L2+TAMs were higher than those on PD-L2+TAMs(p<0.05),and similar with those on M2-type macrophages from mouse bone marrow,therefore it is believed that PD-L2+TAMs was more likely to be M2-type.Flow cytometry showed that although the number of E.coli engulfed by PD-L2+TAMs increased compared with PD-L2-TAMs,the difference was not statistically significant(p>0.05),and both types of TAMs had strong lysosome activity.Additionally.flow cytometry analysis also showed that almost all TAMs(including PD-L2+TAMs and PD-L2-TAMs)had strong proliferative ability.In the study of tumor progression,it was found that PD-L2+TAMs increased the migration(p<0.001).invasion(p<0.05)and proliferation(p<0.05)of tumor cells compared with PD-L2-TAMs.Meanwhile,it could inhibit the activation and proliferation of T cells but the difference was not statistically significant(p>0.05).Finally,transcriptome sequencing of the two types of TAMs showed that a total of 14,834 genes were shared by PD-L2+TAMs and PD-L2-TAMs,while 576 genes were specific to PD-L2+TAMs and 670 genes were specific to PD-L2-TAMs.What’s more,308 genes of PD-L2+TAMs were up-regulated and 113 genes were down-regulated compared with PD-L2-TAMs.Enrichment analysis of up-regulated genes in KEGG pathway found that it was mainly concentrated in the "cytokine-receptor interaction" pathways.After FASC,the cell culture supernatant of the two kinds of TAMs were collected to detect the differences in secretion of 308 cytokines.The results showed that PD-L2+TAMs showed higher expression of Resistin and VEGFR1 and other pro-tumor cytokines,while lower expression of MIP-1α(tumor suppressor cytokines).Conclusion The expression of PD-L2 on TAMs can affect the biological behavior of tumor cells and CD8+T cells,thus promoting the progression of colon cancer by secreting high level of Resistin and VEGFR1,and low level of MIP-1α.