To Investigate The Regulatory Roles Of The Cell Surface Adhesion Molecule CD58 Of Pancreatic Cancer Cells On The Tumor Immune Microenvironment

Background:Pancreatic ductal adenocarcinoma(PDAC)is one of the most aggressive and lethal cancers worldwide.Due to the high degree of malignancy,difficulty in early diagnosis,low chance of curative resection,and poor sensitivity to chemoradiotherapy,the prognosis of PDAC patients is extremely poor,with an overall survival rate of less than 10%at 5 years.The tumor microenvironment of PDAC is highly immunosuppressive,as shown by the lack of immune effector cells(CD4+T cells,CD8+T cells,dendritic cells)and the occupation of immunosuppressive cells(Tregs,MDSCs,M2 macrophages),which directly contribute to the drug resistance of immunotherapy.It was found that the immune adhesion molecule CD58 and soluble CD58(sCD58)had the significant regulatory effects on T/NK cell-mediated innate and cellular immune responses in the inflammation.However,their functions in the tumor immune microenvironment had not been reported.This study aims to investigate the roles of CD58 and sCD58 in the immune microenvironment of pancreatic cancer and the related molecular mechanisms.Methods:(1)To determine CD58 expression at the transcriptional and protein levels of PDAC tissues,its correlation with the demographic and clinicopathological characteristics,and the effect of its expression level on the survival prognosis by using different public databases and immunohistochemical(IHC)microarrays.Simultaneously,the correlation and potential functional role of CD58 with immune cell infiltration in cancer foci were preliminarily explored.(2)By IHC and immunofluorescence(IF)staining,PDAC tissues and cell lines were utilized to clarify the subcellular localization of CD58;qRT-PCR and Western blot were used to investigate CD58 expression at the mRNA and protein levels in different PDAC cell lines;and then CD58 was stably knockdown to investigate its effects on the survival activity,proliferation,migration,and invasion capacities of PDAC cells using colonyforming assay,CCK-8 proliferation assay,wound healing assay,and transwell invasion assay,respectively.(3)Indirect co-culture of macrophages and PDAC cells,using qRT-PCR and flow cytometry,to clarify the interaction between PDAC cells and macrophages;based on stimulated/inhibited intervention and rescue experiments,using bioinformatics analysis,Western blot,flow cytometry,and ELISA,to explore and verify the relationship among TGF-β/smad2/3 signaling pathway,mCD58 on the surface of PDAC cell membrane and sCD58 in the supernatant;simultaneously,to elucidate the molecular mechanism of TGFβ1 induced CD58 "expressional separation";then,using active human recombinant CD58 protein,to observe whether it could induce the polarization of immature macrophage M0 to immunosuppressive M2 type.(4)To preliminarily investigate the correlation among ADAM 17,TGF-β1,and CD58 through bioinformatics analysis and qRT-PCR validation in different PDAC cell lines;to explore ADAM 17 expression in pancreatic cancer tissues,correlation with clinicopathological characteristics,influence on survival prognosis,IF staining for localization,and immune cell infiltrations using different public databases and online analysis platforms;and to propose the mechanistic hypothesis of ADAM17-mediated"expressional separation" of CD58.(5)Using the T/NK cell isolation kit,mouse splenic T/NK cells were extracted to clarify CD2 expression on the surface of T/NK cells;lentiviral transfection of mouse pancreatic cancer cells was used to establish a stable CD58-overexpressing cell line,which was successfully verified using GFP green fluorescence microscopy,IF staining,flow cytometry,and Western blot;by direct co-culture of T cells and mouse pancreatic cancer cells in vitro,the positive ratio of CD 107a and perforin in T cells among the groups was detected using flow cytometry to clarify the effect of CD58 overexpression and sCD58 on T cell activation;moreover,the effect of overexpressed CD58 and sCD58 on T/NK cell activation and cytotoxicity was confirmed by establishing an immunocompetent murine subcutaneous tumor bearing model and intraperitoneal xenograft model in vivo.(6)Through collecting serum samples from 537 patients including PDAC,low-grade pancreatic malignant tumors,benign pancreatic tumors,benign pancreatic diseases,other malignant tumors,and healthy controls,the differences in the contents of sCD58 and TGF-β1 among the groups were compared,the correlation between TGF-β1 and sCD58 in serum samples was investigated,the diagnostic value of TGF-β1 and sCD58,as well as their combination with CA199,was assessed.Results:(1)The expression of CD58 was significantly upregulated in PDAC tissues and cell lines,which was correlated with worse histological grade and larger tumor diameter in PDAC patients.Its high expression often predicted shorter survival time and faster relapse,indicating a poor prognosis.(2)In addition to its localization on the cell membrane surface of PDAC,some of CD58 molecules accumulate in the cytoplasm and even form a "cytoplasmic pool".Furthermore,the cytoplasmic membrane distribution of CD58 was varying in different PDAC cells.The CD58 expression was elevated in multiple cell lines of PDAC at both transcriptional and protein levels.However,CD58 knockdown did not significantly affect survival activity,proliferation,migration and invasion capabilities of PDAC cells.(3)PDAC cells were able to induce the polarization of immature macrophage M0 to M2 type;TGF-β1 secreted by TAMs could promote the CD58 "expressional separation" of PDAC cells through the Smad2/3 signaling pathway,that is,the decrease of mCD58 on the surface of tumor cell membranes,accompanied by an increase of sCD58 content in the extracellular supernatant.Besides,sCD58 failed to induce the polarization of immature macrophage M0 into M2 type.(4)There were significant correlations among ADAM 17,TGF-β1,and CD58.ADAM 17 was upregulated at both transcriptional and translational levels in PDAC tissues;the more advanced the PDAC stage and the poorer degree of differentiation,the higher transcriptional level of ADAM 17.ADAM 17 and multiple immune cell infiltration in PDAC tumors were correlated,suggesting that it might play a role in the tumor immune microenvironment.Upregulated ADAM 17 often predicted shorter overall survival and faster recurrence,hinting a poor prognosis.More importantly,ADAM 17 was likely to act as an exoenzyme resulting in proteolytic shedding of CD58 on the surface of PDAC cells to the ECM and formation of sCD58,that is,mediating CD58 "expressional separation".(5)The positive rate of CD2 in T/NK cells was nearly 100%.Overexpression of CD58 could promote the activation of T/NK cells,enhance their cytotoxicity,and induce the secretion of anti-tumoral cytokines including IFN-γ and TNF-α via CD2-CD58 interaction.Local high concentration of sCD58 accumulation in PDAC tissues could interfere with the CD2-CD58 axis by competitively binding CD2,inhibit the activation of T/NK cells,attenuate their cytotoxicity,and reduce the secretion of anti-tumoral cytokines including IFN-y and TNF-α.(6)The contents of sCD58 and TGF-β1 in the serum of PDAC patients were increased,but decreased in the serum of patients with low-grade pancreatic malignant tumors,benign pancreatic tumors,and benign pancreatic diseases,while the expression in the serum of patients with other malignant tumors was elevated,even higher than that in PDAC patients.TGF-β1 and sCD58 were positively correlated in human serum,including PDAC.The Triple Model of sCD58+TGF-β1+CA199 had clinical diagnostic value for differentiating patients with PDAC from non-PDAC.Conclusions:(1)The expression of CD58 is strikingly elevated in PDAC tissues,and strongly correlated with the cell differentiation and tumor size of PDAC patients,and its high expression often indicates shorter survival and faster relapse.Therefore,CD58 can be used as a potential marker to predict the survival prognosis of PDAC patients.(2)TAM-mediated TGF-β1/smad2/3 signaling pathway can induce "expressional separation" of CD58 in PDAC cells.Of note,ADAM 17 is likely to be involved in the generation of sCD58 as a membrane sheddase."Expressional separation" of CD58 inhibits the activation of T/NK cells,cytotoxicity and secretion of anti-tumoral factors,mediates the inhibitory reprogramming of PDAC microenvironment,and promotes the immune escape of PDAC cells.Therefore,the development of targeted drugs or related immunotherapeutic means against the "expressional separation" of CD58 has broad prospects,which provides novel ideas for immunochemotherapy of PDAC.(3)The contents of sCD58 and TGF-β1 are increased in the serum of PDAC patients,and the Triple Model of sCD58+TGF-β1+CA199 has clinical diagnostic value for PDAC.