| 【Aim】The tumor microenvironment plays an extremely important role in the occurrence and development of tumors.Fibroblasts and macrophages are two key cells in the tumor microenvironment.They jointly promote tumor cell proliferation,invasion and metastasis,and at the same time promote tumor angiogenesis and tumor immune escape.However,it is unclear what the mechanism of synergism between fibroblasts and macrophages.Endosialin is a single transmembrane glycoprotein that is specifically overexpressed in tumor fibroblasts and pericytes,and promotes tumor proliferation and metastasis,but the mechanism is unclear.Therefore,this subject intends to conduct research from the following four aspects:1.To detect the expression of endosialin in bladder cancer,liver cancer and other tumors,and to detect the relationship between the expression of endosialin and the prognosis of patients.2.To detect the effect of endosialin-positive fibroblasts on tumor growth and the effect on macrophage infiltration and polarization in tumors.3.Explore the relationship between endosialin-positive fibroblasts and macrophages through a variety of high-throughput methods.4.Construct a fully human anti-endosialin antibody IgG78,detect the inhibitory effect of IgG78 on endosialin positive tumor model,and explore the mechanism of action.【Methods】1.The double immunofluorescence staining and immunohistochemical(IHC)staining of endosialin and other markers were performed on tumor tissues of bladder,liver,ovarian,breast,prostate and adjacent cancer specimens.The results of IHC were scored and a survival analysis was performed based on the score results and the survival information of patients.2.Fibroblasts from fresh tumor and adjacent tissues were isolated and identified by immunofluorescence and western blot,and the expression of endosialin was detected in isolated fibroblasts.3.Endosialin-positive fibroblasts and the tumor cell line Huh7 was mixed and inoculated to form subcutaneous tumors in nude mice to detect the effect of endosialin on tumor growth and macrophage infiltration and polarization in tumors.4.By co-immunoprecipitating(co-IP)of endosialin-positive fibroblasts and mass spectrometric analysis of the precipitated product,the possible interaction protein of endosialin was found,and then verified by ELSIA,Pull-Down,and endogenous or exogenous co-IP assay.Truncated bodies of extracellular domains(ECDs)of endosialin was constructed based on the domain structure of endosialin to detect the interaction between ECDs and candidate CD68.5.Chemotactic and adhesion effects of endosialin-positive fibroblasts on macrophages were detected through adhesion experiments and chemotaxis experiments.6.Endosialin-positive fibroblasts were co-cultured with the monocyte cell line THP-1 to detect the effects of endosialin-positive fibroblasts on differentiation and polarization of THP-1 cells.To find out factors secreted by endosialin-regulated fibroblasts which can promote macrophage polarization,transcriptome sequencing(RNA-seq)on endosialin-positive fibroblasts was performed.Western blot,q-PCR,and si RNA transfection were used to detect the effect of GAS6 on macrophage polarization.7.Anti-endosialin antibody IgG78 was constructed.SDS-Page and mass spectrometry were used to detect the purity and status of IgG78.Flow cytometry and cellular ELISA were used to detect the specific binding ability of IgG78 to endosialin.Subcutaneous tumor xenograft model and orthotopic liver tumor model were used to detect the ability of IgG78 to target tumors in vivo by in vivo imaging.8.IP assay was used to detect the binding domain of endosialin by IgG78 and the binding of deglycosylated endosialin by IgG78.The internalization and degradation of endosialin caused by IgG78 was detected by flow cytometry,immunofluorescence staining and western blot.The neutralizing effect of antibodies on the polarization of macrophages by endosialin-positive fibroblasts were tested by polarization experiments.9.Subcutaneous tumor xenograft model and orthotopic liver tumor model were developed in nude mice,and then were treated with IgG8 and control antibodies to test the influence of IgG78 on tumor growth and macrophage infiltration and polarization in the tumor.【Results】1.Endosialin is expressed in fibroblasts and perivascular cells of bladder cancer,ovarian cancer,breast cancer and prostate cancer,and is expressed in activated fibroblasts of liver cancer.The expression of endosialin is inversely related to the prognosis of patients with HCC and bladder cancer.The expression intensity of endosialin in tumors is higher than that in adjacent tissues and benign disease tissues.The expression of endosialin in fibroblasts isolated from tumors is higher than in fibroblasts isolated from adjacent tissues.2.Endosialin can promote the growth of mixed subcutaneous tumors.After knocking down of endosialin,fibroblasts’ role in promoting tumor growth is weakened.Co-IP and mass spectrometry assay revealed that endosialin interacted with CD68.CD68 binds with the ECD1 of endosialin,including a C-type lectin-like domain,a sushi domain,and an EGF-like domain.3.Endosialin positive fibroblasts adhere to CD68-positive macrophages mediated by the interaction of endosialin and CD68.In addition to the transmembrane form of endosialin,free endosialin also exists.Free endosialin is secreted after the C-terminus of endosialin being cut by the matrix metalloproteinase(MMP).However,the secreted endosialin cannot chemo CD68 positive macrophages.4.Endosialin-positive fibroblasts can promote the polarization of macrophages to M2 phenotype.RNA-seq found that endosialin regulates the expression and secretion of GAS6 in fibroblasts.Fibroblasts are the main source of GAS6.GAS6 can promote the polarization of macrophages to M2 phenotype.5.The anti-endosialin antibody IgG78 was successfully expressed and purified.IgG78 specifically bind endosialin-positive fibroblasts,and can specifically target tumors in vivo.6.IgG78 binds with the ECD2 of endosialin,which contains the musin domain.Therefore,IgG78 cannot directly block the interaction between endosialin and CD68.However,IgG78 can neutralize endosialin-mediated adhesion and polarization of macrophages.IgG78 can promote endosialin degradation by mediating endosialin internalization into cells and reduce GAS6 expression,thereby neutralizing endosialin-mediated adhesion and polarization of macrophages.7.IgG78 can inhibit tumor growth in vivo.Infiltration of M2 macrophages and proliferation of tumor cells decreased in the IgG78 treated group.【Conclusion】The results of this study showed that endosialin is specifically and highly expressed in tumor fibroblasts and regulates the tumor-promoting effect of fibroblasts.This tumor-promoting effect relies on endosialin-mediated adhesion and M2 polarization of macrophages by fibroblasts.The above results indicated that in addition to the interaction and influence of the tumor microenvironment on tumor cells,the cells in the tumor microenvironment also interact and affect each other,and this mutual influence further promotes tumor progression.Endosialin’s tumor-specific expression and important functions indicate that endosialin is an ideal target for tumor treatment.The anti-endosialin antibody IgG78 can neutralize endosialin-mediated adhesion and polarization of macrophages,and can inhibit tumor growth in vivo.Therefore,IgG78 provided a promising therapeutic candidate for tumor treatment. |
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