The Effect And Mechanism Of Anti-CD47 Immunotherapy In Pancreatic Cancer

Part I:The expression of CD47 in tumor cells and the number of tumor-infiltrating macrophages are independent prognostic factors for pancreatic cancer patientsObjective:we are trying to investigating the relationship between CD47 expression,PD-L1 expression and the number of tumor-infiltrating macrophages in pancreatic ductal adenocarcinoma（PDAC）tumor tissues and determining the relationship between these indicators and the clinicopathological features and prognosis of PDAC patients.Methods:Immunohistochemistry（IHC）staining was performed on tumor sections of106 PDAC patients with anti-CD47/PD-L1/CD68/CD163 antibodies.Spearman’s rank correlation was used to analyze the correlation between these variables.Univariate and multivariate Cox analysis and Kaplan-Meier analysis were used for prognosis analysis.Results:IHC staining showed the high expression of CD47 and PD-L1 in tumor tissues which was accounted for 61.3%and 30.2%of all cases,respectively.High infiltration of CD68⁺macrophages（M）and CD163⁺M2 macrophages（M2）was accounted for 48.1%and 42.5%of all cases,respectively.Correlation analysis shown that the number of CD68⁺M was positively correlated with CD163⁺M2.Moreover,the number of CD68⁺M was significantly positively correlated with the expression of CD47,while there was no correlation between the expression of CD163⁺M2 and CD47.High infiltration of CD163⁺M2 was associated with higher T stage.However,the expression of CD68⁺M and CD47 had no significant correlation with the clinicopathological variables.Univariate and multivariate analyses indicated that the expression of CD47 in tumor was an independent prognostic factor for PDAC patients.We further examined the effect of CD47,PD-L1,CD68,and CD163 on the OS of PDAC patients,and we found that the combination of CD47 and CD68⁺M(CD47^high/CD68⁺M^high,CD47^high/CD163⁺M2^high,CD47^low/CD68⁺M^low,and CD47^low/CD163⁺M2^low)were independent prognostic factors for PDAC patients.Conclusion:In PDAC patients,the expression of CD47 of tumor cells is significantly correlated with the number of tumor-infiltrating macrophages.In addition,both CD47and tumor-infiltrating macrophages were independent prognostic factors for PDAC.Part II:Single-cell RNA sequencing revealed the role of anti-CD47immunotherapy in remodeling tumor-infiltrating immune cells in pancreatic cancerObjective:Investigate the antitumor effect and mechanism of anti-CD47 monoclonal antibody（m Ab）against pancreatic ductal adenocarcinoma（PDAC）in mouse models.Methods:The anti-tumor therapy effect of anti-CD47 m Ab was investigated in PDAC patients-derived xenograft（PDX）models.Further,to observing the anti-tumor effect of anti-CD47 m Ab,the mouse pancreatic cancer cell line Panc02 was used to construct a syngeneic mouse pancreatic cancer model by subcutaneously implanting the cell line into C57BL/6 mice.To further understand the mouse immune cell subpopulations associated with anti-tumor response following anti-CD47 treatment,we harvested the tumors after the 15 days anti-CD47 treatment and then analyzed the CD45-positive immune cells by single-cell RNA sequencing with 10x Genomics pipeline.Results:The tumor burden in PDX model did not reduced after anti-CD47 treatment,which may result of the fact that the PDX model was established by immunodeficient mice,which was lacked a complete immune system.We further established Panc02mouse model that has a complete immune system,and we found that tumor growth in Panc02 mouse model was significantly suppressed after anti-CD47 treatment.Furthermore,22608 tumor-infiltrating immune cells were further detected by single-cell sequencing and divided into the following clusters:eight lymphoid clusters,five monocyte/macrophage clusters,three neutrophil clusters,and three dendritic cell（DC）clusters.We found that the anti-CD47 treatment significantly increased the tumor-infiltrating CD4⁺T cells,remodeled the tumor-infiltrating Tregs clusters and induced a shift in the intratumoral CD8⁺T cell cluster toward activation.Moreover,we showed that anti-CD47 treatment significantly remodeled the macrophage compartments in Panc02 tumor-bearing mice by increasing the pro-inflammatory macrophages（M1 macrophages）that exhibit anti-tumor function,while reducing the anti-inflammatory macrophages（M2 macrophages）.Conclusion:Anti-CD47 m Ab treatment can inhibit the tumor growth of pancreatic cancer in mice,remodel the tumor-infiltrating immune cell clusters,and promote the M2 macrophages transfer into M1 macrophages,which play a role in improving the tumor immune microenvironment and inhibiting tumor cells.Part III:Anti-tumor effect and mechanism of anti-CD47 combined with anti-PD-L1 therapy in pancreatic cancer mouse modelsObjective:Investigate the anti-tumor effect and mechanism of combination immunotherapy targeting CD47 and PD-L1 in pancreatic cancer mouse models.Methods:To establish pancreatic cancer mouse models,Panc02 and MPC-83 cells were collected and subcutaneously injected into the right ventral region of C57BL/6 and KM mice,respectively.When the tumor volume reached 50～100 mm³,the mice were randomly divided into 4 groups（5 mice in each group）:control group,anti-CD47treatment group,anti-PD-L1 treatment group,and anti-CD47 combined anti-PD-L1treatment group.The control group was intraperitoneally injected with murine Ig G.The treatment group was treated with anti-CD47 or anti-PD-L1 antibody for 2 weeks.After the treatment,the mice were euthanized and the tumor were removed and measured.Flow cytometry and RNA sequencing were performed to detect the immune cells in each group.Results:We observed a synergistic anti-tumor effect of anti-CD47 combined with anti-PD-L1 in MPC-83 mouse model,but no synergistic effects were observed in Panc02 mouse model.In the MPC-83 tumor model,compared to either anti-CD47 or anti-PD-L1 alone,combination treatment increased the proportion of PD-1⁺CD8⁺T lymphocytes in spleens and tumors,and improved the expression of Cd8b1,Pdcd1,Arg1,Lag3,Nos2,Mrc1,Serpinb9 and Ifit3.However,the results was not observed in the Panc02 tumor-bearing mouse model.Conclusion:Our study showed that the combination therapy targeting of CD47 and PD-L1 had a synergistic anti-tumor effect in MPC-83 mouse model,but this effect was not observed in Panc02 mouse model,which may be caused by the different tumor microenvironment established by different pancreatic cancer cell lines.