The Role Of SIRPα-CD47 Signaling Pathway In Crosstalk Between Macrophages And Oral Leukoplakia And Oral Squamous Carcinoma Cells

In recent years,the tumour microenvironment has been highlighted as an important hallmark of cancer.In tumour microenvironments,macrophages represent a major inflammatory component of the stroma and affect many aspects of the neoplastic tissue.Based on their role in linking innate with adaptive immunity and their immunoregulatory capabilities,macrophages are important cells when investigating tumour immunology.Macrophages in tumors are often called tumor-associated macrophages(TAMs)and are regarded as an important component of the tumor microenvironment,featuring remarkable diversity and plasticity.Signal regulatory protein α(SIRPα)is a cell-surface protein mainly expressed on myeloid cells,including macrophages and dendritic cells.SIRPα can bind to either widely expressed transmembrane ligand CD47 or soluble ligands.SIRPα and CD47 are involved in the pathogenesis and promote malignancies such as lymphoma,leukaemia,melanoma,lung cancer and multiple myeloma,and their expression varies during infection and malignancies.However,the profile of SIRPa expression in oral leukoplakia(OLK)and oral squamous cell carcinoma(OSCC)and the mechanism by SIRPa regulating macrophages or oral cancer cells remain unclear.Thus,the aim of the present study was to investigate the distribution and the incidence of SIRPα on macrophages and the relation to clinicopathological factors.Moreover,this study explored the regulation of SIRP and CD47 on cell phagocytosis,phenotype,proliferation,invasion,and migration of macrophages.We further explored the relationship between SIRPα and NF-κB,and studied the role of SIRPα at regulating the phenotype of macrophages.Also,the relationship between CD47 and STAT3/JAK2 signling was analysised.At the last,folic acid was conjugated to chitosan and used to formulate siRNA-CD47 into nanoparticles capable of anti-tumor therapy.Part ⅠThe expression of SIRPα in oral leukoplakia and oral squamous cell carcinomaObjection:SIRPα is a cell-surface protein expressed on macrophages that are regarded as an important component of the tumor microenvironment.The present study investigated the expression of SIRPα in OLK and OSCC.Methods:Immunohistochemical and double-labelling immunofluorescent analysis were carried out to detect the expression of SIRPa on twenty-five tissue samples.For subgroup analysis,OLK samples were divided into OLK with low-risk dysplasia(LR-OLK)and OLK with high-risk dysplasia(HR-OLK).Results:Both the expression of CD68 and CD 163 were detected different significantly by pathological grade(p<0.05).The expression of SIRPa in LR-OLK was higher than in HR-OLK(p=0.01)and OSCC(p=0.04).Compared with OSCC,the expression of SIRPa in HR-OLK was lower than OSCC,but there was no significant difference(p=0,15).The expression of SIRPa positively correlated with the expression of CD68 and negatively correlated with CD 163 on macrophages.In OLK,the percentages of CD163+SIRPa+ and CD68+SIRPa+ macrophages were higher than normal oral mucosa and OSCC(p<0.01).Conclusions:SIRPa could be applied as a prognostic marker to predict OLK and oral cancer progression.Part II The role of SIRPa in interaction between macrophages and oral squamous cell carcinomaObjection:The tumour microenvironment has been highlighted as an important hallmark of cancer.In tumour microenvironments,macrophages represent a major inflammatory component of the stroma and affect many aspects of the neoplastic tissue.The present study further explored the mechanism of SIRPa on the phenotype,phagocytosis ability,migration,and invasion of macrophages in OSCC.Methods:After stimulated by phorbol-l2-myristate-13-acetate(PMA),human acute monocytic leukemiacell line(THP-1)was co-cultured with Cal-27 cell lines.The subcellular localization of SIRPa was determined by immunofluorescence.Co-cultured with Cal-27 and SCC-9 cell lines for 9 days,the expression of SIRPa on macrophages were detected by flow cytometry.The effect of LPS,IL-4,and hypoxia on macrophages were explored.PDTC,as an inhibitor of NF-κB,were used to inspect the relationship between SIRPa and the phenotype of macrophages.The influence of SIRPa(knockdown by siRNA)on macrophages phagocytosis,proliferation,invasion,and migration were evaluated.In addition,the influence of SIRPa on Cal-27 cells invasion,and migration were detected.The expression of SIRPa on macrophages were assessed using western blotting.Alterations in messenger RNA(mRNA)expression of M1-related and M2-related genes were analyzed using quantitative reverse transcription polymerase chain reaction.Results:The expression of SIRPa on THP-1 cells was decreased gradually after co-cultured with Cal-27 and SCC-9 cell lines for 1,3,5,7,and 9 days.After co-cultured with Cal-27 cell lines,the expression of CD 163,IL-10,and TGF-β mRNA on macrophages were elevated,while the expression of SIRPα,IL-6,and TNF-a mRNA were decreased,indicating that macrophages were induced to M2 phenotype.Knockdown of SIRP promoted macrophages switching to M2 phenotype,along with inhibition of phagocytosis ability and IL-6,TNF-a productions,upregulated proliferation,migration,and IL-10,TGF-β productions of macrophages.Inhibited by PDTC,macrophages upregulated the expression of SIRPa and phagocytosis ability,inhibited the migration and invasion.Conclusions:SIRPa might be an important modulator of tumor-polarized macrophages in OSCC by targeting NF-κB.Part III CD47 as a therapeutic target of oral leukoplakia and oral squamous carcinomaObjection:CD47 is an antiphagocytic molecule that combines with signal regulatory protein alpha on macrophages,involving several tumor immunologic escape.However,the mechanism of CD47 on oral squamous cell carcinoma is not well documented.The present study is aim to investigate the mechanism of CD47 on the development of oral squamous cell carcinoma.Methods:CCK8 was used to detect the proliferation of Cal-27 cells after interfered with siRNA-CD47.Cal-27 cells were transfected by shRNA-CD47 to knockdown the expression of CD47.The effect of CD47 on the apoptosis of Cal-27 cells was detected by flow cytometry.Transwell assay was used to analysis the effect of CD47 on the migration and invasion ability of Cal-27 cells.In vitro phagocytosis of Cal-27 cells by macrophages and in vivo tumorigenicity were measured when knockdown the expression of CD47.The related protein in STAT3/JAK2 signal pathway was measured by western blot.Results:The proliferation of Cal-27 cells was inhibited by siRNA-CD47.But the effect of CD47 on the apoptosis of Cal-27 cells had no significant change.Knockdown of CD47 inhibited the migration and invasion ability of Cal-27 cells.After nine weeks after injection of Cal-27 cells,knockdown of CD47 cells showed reduce of the tumor volume in nude mice tumorigenicity.Knockdown of CD47 significantly enhanced in vitro phagocytosis of Cal-27 cells by macrophages and positively correlated with the STAT3 and JAK2 protein.Conclusions:CD47 is a promising therapeutic target in oral squamous cells carcinoma.Part Ⅳ Folic acid conjugated chitosan for targeted delivery of siRNA-CD47 for oral squamous cells carcinomaObjection:The aim of this study is to develop a delivery system that targets siRNA to silencing the gene CD47 in oral cancer cells and resisting the phagocytosis of macrophages.Exploiting the presence of folate receptors on the surface of Cal-27 cells,folic acid was conjugated to chitosan(FA-CS)and used to formulate siRNA-CD47 into nanoparticles capable of cell specific delivery.Methods:The physiochemical properties of the nanoparticles,including size,zeta-potential and encapsulation efficiency,were characterized and the intracellular uptake and tumoricidal efficiency were studied in vitro.Results:The structure was identified by infrared spectrum,1HNMR and elementary analysis.FA-CS-siRNA-CD47 significantly inhibited the proliferation of Cal-27 cells compared with CS-siRNA-CD47 and siRNA-CD47.FA-CS-siRNA-CD47 enhanced cellular uptake in Cal-27 cells and oral normal keratinocyte compared with CS-siRNA-CD47 and siRNA-CD47.Conclusions:FA-CS might be a potential siRNA-CD47 carrier for anti-inflammatory therapy in oral squamous cells carcinoma.