ISG15 Suppresses The Aggregation Of CD8 Positive Cells In The Microenvironment Of Colorectal Cancer In Association With Unfavorable Prognosis

Background and SignificanceThe homeostasis of extracellular matrix,cells and cytokines inmicroenvironment may form unique regulation networksand spatial distribution patterns,resulting in impacting the survival,proliferation,differentiation,invasion and metastasis of tumor cells.Avoiding or preventing the immunitywithin the microenvironment is essential for tumor cells to survive.Therefore,tumor cells may actively or passively participate in and influence the microenvironment,actively suppress cellular immune functions,induce other cells to secrete cytokines,inhibit the immune toxicityof cytotoxic Tlymphocytes(cytotoxic T lymphocyte,CTL)and natural killer cells(natural killer cell,NK).The state of cellular immune functions may determine the survival of tumor cells,in which the CTL cells plays a leading role and directly affects the adaptive immune of tumor cells in microenvironment.Proliferation of tumor cells is the result of cellular immunity failure.In addition to the immune evasion,negative antigen expression of tumor cells and weak capacity of antigenpresentation of immune cells,immune cytokines regulation concessions,the supression of cellular immune function,etc are also important reasons why tumor cells can not be killed.Interferon(interferons,IFN)is one of the early used cytokinesin cancer immunotherapy while it did not achieve the anticipated effects.Interferonstype Ⅰinclude IFNa and IFNβ,which are mainly generated by macrophages,plasmacytoid DCs and fibroblasts.Encoding by ISG15gene,ISG15 is one of hundreds of proteins expressed bylymphocytesafter IFN stimulation.It can be expressed by IFNa,IFNβ or IFNγ stimulation,but bythe stimulation of IFNβ the expression is the most obviously enhanced.B7H4 is a recently discovered negative co-stimulatory factorof cellular immune.B7H4 can suppress the activation of CD4+T and CD8+T cells and their release of cytokines.Abadi,etc.found that in B7H4 gene knockout mice,the lung metastasis of breast cancer was significantly inhibited,considering that the host stromal cells expressed B7H4 to inhibit cellular immune functions by inducing the tranformation of MDSCs(bone marrow-derived suppressor cells),which provides important evidences for the study of the suppression of cellular immune in microenvironment.Studies have shown that the high expression of B7H4 in prostate cancer tumors is also relevant to tumor overgrowth.However,the reason why tumor cells with high expression of B7H4 can induced immunosuppression is still unknown.The incidence and mortality rates of colorectal cancer(CRC)are the third highest in the world,new cases each year are about 120 million people,and the death toll is expected to exceed 60 million.Thus,we expect to build research programscalled "Tumor cell-tumor microenvironment","tumor microenvironment-tumor immunity interactive"in colorectal cancer.On one hand,we intend to show the initiative of the discipline to microenvironment by tumor cells,on the other hand,we attempt to show microenvironmental factors that passively preventing tumor cells from being killed and promoting tumor growth.We analysed colorectal cancer tissues with 11 years of follow-up data by immunohistochemistry,found that patients with high expression of ISG15(total 649 cases)or low expression of CD8(249 cases)had poor prognosis.In order to resolve the model of information exchangebetween the tumor cell sand metastasismicroenvironment,we inoculated murine colon carcinoma cell line CT26.WT with or without IFNβ-stimulusin to Balb/c mice to build the adaptive immune model of tumor and immunity to study the effect oncellular immune responses in vivo by tumor cells after IFNβ-stimulus.In vivo,we found that compared to the control group CT26.WT/Blank(non-IFNβ-stimulus group),the capacity of subcutaneous tumor formation in miceof the experimental group CT26.WT/IFNβ(IFNβ-stimulus group)was enhanced;the tumor cells are with high expressionsof ISG15 and Ki-67;CD8 cells within the tumor tissue were significantly reduced;the FADD expression of tumor cellswas also significantly reduced.The co-cultivation of tumor cells,T cells and DCs showed that the T cells and DCsextracted from experimental group CT26.WT/IFNβ mice can cause reduction of the apoptosis of tumor cells.In our previous work,we screened tumor antigen presenting and costimulatory signal molecules and found that after IFNβ-stimulus,the expression of B7H4 protein in the colon cancer cells was upregulated.We analysed 249 clinical tissue samples of colorectal cancer with 11-year follow-up data,concluding that tumor cells expressing high level of B7H4 were associated with poor prognosis in patients.After transplanting CT26.WT cells with IFNp-stimulusto Balb/c mice subcutaneously,B7H4 was also highly expressed in the tumors.These results suggest that ISG15 may inhibit the raise of CTL cells in tumor microenvironment and affect the expression of B7H4 to exert immunosuppressive effects,thereby inhibiting the immune destruction,collabratedly maintaining the survival microenvironment of tumor cells and promoting tumor progress.In this study,we constructed the BALB/c mice tumor model,which were injected into the mouse colon cancer cell CT26.WT through the following three ways:subcutaneous injection,intraperitoneal injection,the spleens being injected under the film,to simulate the natural occurrence of colorectal cancer,and the interaction between the development and the body microenvironment,and investigate the effect of ISG15 on the immune response of colorectal cancer and its correlation with the prognosis of the tumor.Materials and methods1.Clinical specimens collected649 cases of colorectal cancer who had completely 11 years of follow-up data and confirmed by experienced pathology doctors.Specimens obtained from patients were not received any chemotherapy or any treatment.Patients and/or their families signed a consent form for the use of cancer tissue specimens for scientific research.2.Cell culturing techniqueHuman colorectal cancer cells HT29,HCT116,LS174T,SW480,SW620,LoVo,mice colorectal cancer cell CT26.WT are cultured with the medium RPMI1640(containing 10%fetal bovine serum);Dendritic cells(DCs)derived from mice are cultured with the medium RPMI-1640(containing 10%fetal bovine serum),which containing granulocyte macrophage colony stimulating factor(GM-CSF,50 ng/ml),interleukin 4(IL-4,25 ng/ml).The conditions are 37℃ and 5%CO2.3.Western Blot(WB)Cells are cultured with proper concentration and after the pancreatic enzyme digestioning,we collect cellular precipitation supernatant and extracting protein lysates.BCA protein content detection kits were used to detect protein concentration.Then the protein lysates were separated by SDS-PAGE,electrophoretically transferred to membrane,incubation with 5%milk,primary/secondary antibody incubation,chemiluminescence detection.We use Western blot(WB)to verify the ISG15 expression in different cells.In HT29,HCT116,LS174T,SW480,SW620,LoVo and mice colorectal cancer cell CT26.WT,before and after application of IFN beta stimulationin cellculture,we carryed out a comparative analysis c to evaluate the stability of ISG15 expression.At the same time,in the cells before and after culture of more than IFN beta stimulation,Western blot was used to screening related immune molecules.4.Immunohistochemistry(Immunohistochemistry,IHC)stainingFormalin fixed human colorectal cancer tissues,mice subcutaneous tumor and liver metastasis tumor tissue specimens,after dehydration,paraffin embedded,sliced,according to the operation method of immunohistochemistry,microscope observation and collection of images,we detected ISG15,B7H4,CD8,FADD,and Ki-67 expression levels,and analyzed their intrinsic relationships.5.Immunofluorescence(immunofluorescence,IF)stainingWe selected tissues from patients with colorectal cancer to carry immunofluorescence analysis.The steps are as follows:dewaxing;hydration;autoclave;goat serum blocking,4℃ overnight incubation fertility anti,fluorescently labeled second antibody,at room temperature,close incubation;DAPI counterstained nucleus,Containing anti fluorescence quenching quenching agent mounting mounting medium and under confocal laser scanning microscope observation,the acquisition image.IF experiments verify that B7H4 has no co localization with CD3 and other molecules in human colorectal cancer tissues.6.Cellular function experimentWe used CCK-8 proliferation assay,scratch test,and Transwell invasion assay to detect the effect of IFN on the functional aspects of colorectal cancer cell line CT26.WT;cell surface markers of T cells(DC cells)were identified by flow cytometry;the apoptosis of tumor cells was detected by flow cytometry in tumor cells and T cells and DC cells.7.Animal experimentWe used 4-6 weeks Balb/c mice,which raised on the environment of SPF.The two cells of CT26.WT/Blank,CT26.WT/IFNβ do the following three types processing and plant in Balb/c mice:subcutaneous injection,intraperitoneal injection,appendical subserosal injection.The growth of tumor tissue,the expression of the tumor and the survival period of the mice in the two groups were observed respectively.8.Statistical analysisExperimental datas use SPSS21.0 software for statistical analysis.P values<0.05,the difference is statistically significant.Consequence1.The expression of ISG15,CD8 and B7H4 in colorectal cancer tissues:We detected the expression level of ISG15 in tumor tissue in 649 patients with colorectal cancer with complete follow-up data for 11 years.The results showed that the high expression of ISG15 was closely related to the poor prognosis of the clinical cases(P<0.05,the difference was statistically significant).In 249 clinical samples,the results showed that the high expression of B7H4 was closely related to the poor prognosis of the patients(P<0.05,the difference was statistically significant);We evaluated the correlation between ISG15 and CD8 expression in tumor tissue,and the relationship between the two groups showed negative correlation(P<0.05,the difference was statistically significant).2.Expression of IFNβ/ISG15 signaling pathway in tumor cells before and after IFNβ stimulationWe selected colorectal cancer cell lines,HT29,HCT116,LS174T,SW480,SW620,LoVo and CT26.WT,to extract lysates of these tumor cell.At the same time,the cytokine IFNβwas added to 50 ml complete culture medium,and the cell lines were cultured for 48 hours,then the protein of the tumor cells was extracted;We used WB to detect IFNAR2,Jakl,Tyk2,STAT1,STAT2,IRF9,and ISG15 expression levels.The results showed that the expression of IFNβ/ISG15 protein was significantly higher than that of the control group after 48 hours of IFNβ.3.Effects of ISG15 on the biological behavior of colorectal cancer cells in mice(1)Compared with 5-Fu induced apoptosis,CT26.WT cells could reverse 5-Fu induced apoptosis by IFNβ stimulation.(2)CCK-8 proliferation assay,transwell invasion assay,scratch test results showed that there was no significant difference between the CT26.WT/Blank group and the CT26.WT/IFN group.4.Identification of surface markers of immune cells:We used flow cytometry to identify the surface molecular markers of mouse DC cells:CD11c,CD40,MHC-Ⅱ,CD80,and their positive rates were considerable;For mouse T cells obtained by magnetic cell double sorting of CD3 and CD8,we used flow cytometry to detect CD3+T cells and CD8+T cells proportion.5.The effect of ISG15 on immune response of host cells(1)We extracted the mouse bone marrow DC cells,spleen sorted T cells,and CT26.WT cells,then the three cells were co-cultured to detect tumor cell apoptosis.The results showed that the experimental group CT26.WT/IFNβ mouse source extracted T cells,DC cells,could reduce the apoptotic rate of CT26.WT cells.(2)We inoculated the murine colon cancer cell line CT26.WT with or without IFNβ stimulationinto Balb/c mice.Subcutaneous inoculation results showed that compared with the control group CT26.WT/Blank(culture group without IFNβstimulation),the experimental group CT26.WT/IFNP(IFNβ stimulation group)Subcutaneous tumor developedin Balb/c mice was enhanced,and the expression rate of ISG15,B7H4 and Ki-67 in tumor cells were increased,and CD8+cells in tumor tissue were significantly reduced,and the expression of FADD in tumor cells was also significantly decreased.6.High expression of immune molecule B7H4 in colorectal cancer cells stimulated by IFNβCytokine IFNβ was added to 50 ml complete medium,and the final concentration was 1000 U/ml.We used the medium to culture colonic cancer cells,HT29,HCT116,LS174T,SW480,SW620,LoVo,CT26.WT,and thenthe proteins were extracted from the tumor cellsafter 48-hour stimulation.WB test results showed that the expression of B7H4 was significantly increased in HT29,LoVo and CT26.WT after IFNβ stimulating for 48 hours.7.Localization of B7H4 in colorectal cancer tissuesIn 20 cases of colorectal carcinoma tissues,we used immunofluorescence experiments to verify whether there was a colocation betweenB7H4 and CD3,or CD20,CD34,CD68,CD 163,and Actin.The results showed that B7H4 was expressed specilfically in human colorectal cancer tissues,and there was no colocalization with these molecules.Conclusion1.In human colorectal cancer tissue,high expression of ISG15 and B7H4,low expression of CD8were corporatelyassociated with unfavorable prognosis tothe patients.ISG15 expression in the cancer tissues was negatively correlatedto CD8+cells in the interstitial tissues.2.IFNβactively promotes colorectal cancer cells to enhance expression of ISG15 and B7H4.3.ISG15 promotes tumor cells to express B7H4 might be importance of inhibiting the recruitment of CTL cells in tumor microenvironment,thus inhibiting the immune killing of the host cells.The innovation of this research1.This study provides evidences for "seed" and "soil" hypothesis that a crosstalking mechanism between the tumor cells and microenvironment.2.The balance of "attacking" and "defensef"between tumor cells and cytotoxic cells in the microenvironmentshowedthat mutual impacting mechanism of immunosuppressive effects and cancer progression.