Preliminary Study Of IFNβ/ISG15/B7H4 In Cellular Immunosuppression Of Colorectal Cancer

Background and SignificanceColorectal cancer is the third most common form of cancer and the third most frequent cause of cancer deaths worldwide.An estimated 1.2 million new cases occur annually and expected death rate is more than 600,000 people.In the last few years,Although along with the constantly development of our country’s health care and medical technology,the treatment of patients with colorectal cancer have improved to a great extent,but the survival rate is still not improved greatly.The main reason is more than half of those patients have occurred micrometastases before doing radical operation and relapse and metastasis are the main reasons of post-operative death.The growth,invasion and metastasis of tumor cells rely on the accuracy and meticulous of microenvironment factors in dispatching invasive biological behavior of tumor.The dynamic balance of microenvironmental extracellular matrix,cellular component and the cytokines forming a unique space division and network regulating model has an influence on tumor survival,proliferation,differentiation and invasion,even metastasis.Among them,Cytokines,secreted by immune cells,such as macrophages and neutrophils derived of bone marrow have greatly negative effects on microenvironment,which,directly or indirectly,mediate the immunosuppression and inhibit the fuction of cytotoxic T lymphocyte and NK cells.State of cellular immune function can determine the survival of cancer cells.Therefore,clearing the state of immune function of tumor microenvironment and clarifying how to regulate the tumor cells survial,growth,differentiation and metastatic mechanism.And preventing the microenvironmental inhibiting factors in the process of tumor cells growth,can promote us to effectively explore anti-tumor strategy.Interferons,the one of cytokines are early applied to tumor immunotherapy.Type I interferons include IFNa and IFNβ,which mainly produced from macro-phages and plasmacytoid DCs and fibroblasts.Two kinds of interferons combine with the same receptor sharing with the same biological activities.Fibroblasts,tumor associated macrophages(TAM)derived of tumor microenvironment is the mainly source of IFNa and IFNβ.ISG15 encoded by ISG15 gene is one of hundreds of proteins expressed after IFN stimulated lymphocytes.The expression of ISG15 is under the stimulus of the IFNa,IFNβ and IFNy,but IFNβ has more apparent enhancemant to stimulating its expression.Lymphocytes express ISG15 and secrete to the extracellular matrix,but the secretion mechanism is not clear and the corresponding receptors and extracellular transport form have not been determined.B7H4 belongs to protein B7 family,one lymphocyte function inhibiting molecules.Its receptor and cellular immunosuppression way is also not certain and clear.The roles of ISG15 and B7H4 in tumor immunity are only reported in recent years remaining no making breakthrough progress.Abadi thought that the host mesenchymal cells expressing B7H4 to inhibit cellular immune through inducing g-MDSCs are worth attention when they found the gene B7H4 knock-out mice could suppress the pulmonary metastasis of breast cancer,which provided reference for research the microenvironmental cellular immunosuppression.But the immunological suppression induced by tumor cells highly expression of B7H4 are still unresolved.We find that compared with control group,IFNβstimulation group can increase subcutaneous tumor capacity,reduce tumor cell apoptosis,ihibit the ability of T cells lethality,urge highly expression of ISG15 and B7H4.We detect 649 cases of colorectal cancer samples through immunohistochemistry who have completely 11 years of follow-up data and results display that colorectal cancer patients with high ISG15 expression are poor prognosis.Screening of tumor cells antigen presenting and constimulatory molecules,we find that the expression of B7H4 is possibility improved.ICAM-1 is higher when the protein lysis buffer of high expression of ISG15 sensitizating DC cells,but the protein lysis buffer of high expression B7H4 or ISG15/B7H4 sensitizating DC cells can reduce ICAM-1.ICAM-1/LFA-1 is the mainly mechanism mediating antigen presenting of DC cells.Influencing the expression of ICAM-1 may lead to T cells failing to receive the antigen than DC cellls presented effectively.This topic intends to build colorectal cancer model by subcutaneous and intraperitoneal injection of mice colorectal cancer cell CT26.WT in Balb/c mice to establish the interaction between natural colorectal cancer occurrence and host microenvironment and discuss the influence of colorectal cancer cells on the antigen presentation of host cellular immunity,activation of immune cells,influence on immunological effect and the connection of ISG15 and B7H4.Thus,we speculate that IFNβ/ISG15/B7H4 may participate in the inhibition of cellular immunity and promote the growth and invasion of colorectal cancer cells.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.2.Cell culturing techniqueHuman colorectal cancer cells HT29,HCT116,LS174T,SW480,SW620,LoVo,Bladder cancer cell umuc,Lymphoma cell Ly10,mice colorectal cancer cell CT26.WT are cultured with the medium RPMI1640(containing 10%fetal bovine serum);Lung cancer cell Glc-2 is cultured with the medium DMEM;Cervical cancer cell HeLa is cultured with the medium MEM;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,appropriatly humidity condition of cultivation in the box.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/second antibody incubation,chemiluminescence detection.4.Cellular function experimentFACS was used to detect cell apoptosis and identify the surface of cells.5.Animal experimentThe 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,Appendix subserosal injection.Every group has seven Balb/c mice.The environment of SPF is used to raise mice.6.Immunohistochemistry(IHC)Tumor tissues obtained from above steps are used IHC operation method to detect the expression of ISG15 after formalin fixation,dehydration,paraffin embedding and sectioning.7.Statistical analysisExperimental datas use SPSS 17.0 software for statistical analysis.P values<0.05,the difference is statistically significant.Consequence1.The expression of ISG15 in the colorectal cancer tissues:(1)20 cases of colorectal cancer tissues and matched normal mucous epithelial tissues using IHC to detect the expression of ISG15,the results show that the expression of ISG15 is higher in cancer tissues than normal tissues.(2)649 cases with 11 years completely follow-up datas of colorectal cancer specimens which obtained from clinic are used to detect the expression of ISG15 in tumor tissues and combine with the characteristics of the patient’s clinical cases to do correlation analysis.IHC results show that ISG15 with higher expressing are closely related to poor prognosis(P<0.05,the difference statistically significant).2.The expression of ISG15 in colorectal cancer cells:We selecte colorectal cancer cells HT29,HCT116,LS174T,SW480,SW620,LoVo to extracting the cellular proteins,and Western Blot is used to detect the expression level of ISG15.Results displaying:the expression level of ISG15 is higher in SW620 and LoVo.3.The expression of ISG15 and B7H4 in colorectal cancer cells after stimulated by IFNβ:We add cytokines IFNβ to 50ml complete medium,the final using concentration is 1000 U/ml.The special medium is used to culture colorectal cancer cells HT29,HCT116,LS174T,SW480,SW620 and LoVo,CT26.WT,bladder cancer cell umuc,cervical cancer cell HeLa,lung cancer cell Glc,lymphoma cell Ly10.48 hours later,the protein is extracted from those tumor cells and Western Blot testing results showing:IFNβ stimulating those cells after 48 hours,the expression of ISG15 is significantly higher than control group(in addition to Ly10);the expression of B7H4 is higher in HT29,LoVo and CT26.WT.4.The impacts of IFNP on the biological characteristics in colorectal cancer cells:(1)Compared with 5-Fu induced control group,CT26.WT stimulated by IFNβcan transfer 5-Fu induced apoptosis.(2)After stimulated by IFNβ,SW620 obtains EMT thanslation.(3)The impacts of the host cellular immune after the colorectal cancer cell CT26.WT stimulated by IFNβ:The control group CT26.WT/Blank and experimental group CT26.WT/IFNβ do subcutaneous and intraperitoneal injection in Balb/c mice to establish animal models.Then we extract DC cells and T cells from mice to coculturing with CT26.WT for detecting cells apoptosis.Results displaying:CT26.WT stimulated by IFNβ can suppress the lethality of T cell and influence the host cellular immunity.(4)The influence of antigen presentation on DC cells of high expression of ISG15 and B7H4When the no-load and high expression plasmid of ISG15 and B7H4 transfect CT26.WT,we obtain the protein lysis buffer to sensitizate DC cell,then coculturing with normal T cells.Western Blot detects the expression of ICAM-1 on surface of DC cells.Results displaying:high expression of B7H4 and Isg15/B7H4 lead to down-regulation of ICAM-1 and lonely high expression of ISG15 lead to the high expression of ICAM-1.Conclusion1.Colorectal cancer cells stimulated by IFNβ can promote the expression level of ISG15 and B7H4 protein.2.ISG15 is high expression in colorectal cancer and association with poor prognosis in patients.3.SW620 stimulated by IFNβ obtain EMT conversion.4.IFNβ could improve the expression level of ISG15 and B7H4 to influence the expression of ICAM-1 and antigen presentation and suppress the host cellular immunity.