Identify Functional Molecules Of Tumor-associated Macrophages And Their Mechanism Based On Single-cell Sequencing

Tumor-associated macrophages(TAM)are the main immune cells in tumor tissue.TAM is a special macrophage population with significant heterogeneity and has dual functions on tumor cells.TAM can not only play the killing role of anti-tumor immunity,but also promote the immune escape,proliferation and metastasis of tumor.A large number of studies have shown that TAM has prominent immunosuppressive activity and promoting tissue growth,but lacks cytotoxic activity and pro-inflammatory effect.The immunosuppressive effect of TAM is a major obstacle to tumor immunotherapy.Taking TAM as the target of tumor therapy,regulating the number of TAM or remodeling the phenotype of TAM is expected to find a new direction for tumor immunotherapy.Single-cell RNA sequencing(sc RNA-seq)can analyze the gene expression with high resolution at the single-cell level,avoiding the error caused by the average signal of traditional genome technology.By studying the difference of transcriptional expression between different cell subsets,we can identify cell subsets and abnormally expressed genes that play a key role in the process of tumor development,so as to lay a research foundation for tumor treatment.Previous studies have used sc RNA-seq to map the composition and function of myeloid cells,including TAM,in multiple cancer species,proving that sc RNAseq is a powerful tool for studying the phenotype and function of TAM.In order to explore the role of TAM in the tumor microenvironment,we plan to use the public database and our research team’s published data to analyze and screen the TAM.The single-cell sequencing dataset PRJEB45598 is from tumor biopsies of patients with advanced gastric cancer before and after treatment of standard first-line chemotherapy.We analyzed PRJEB45598 and found that galactose-specific lectin 3(LGALS3),or galectin-3,was highly expressed in M2-like TAM,and the expression level was further improved after chemotherapy.In order to verify this result,we used C57BL/6J mice and B16-F10 cells to construct a tumor model(cell-derived tumor craft)and obtain tumor tissue.Flow cytometry was used to obtain TAM cells.RT-q PCR and WB experiments showed that LGALS3 in M2-like TAM was significantly higher than M1-like TAM in both m RNA and protein levels.In addition,LGALS3 recombinant protein can protect tumor cells from the killing of chemotherapy drugs.In conclusion,the M2-like TAM after chemotherapy can promote the chemoresistance of tumor cells through the synthesis and secretion of LGALS3,which provides a potential target for tumor treatment.In view of the expected data analysis results from the public database,we further used the dataset GSE210177 from published work of our research team to analysis.We found that chromobox 7(CBX7)was highly expressed specifically in TAM.CBX7 is a subunit of polycomb repressive complex(PRC1),which has the effect of catalysing histone modifications and regulating gene expression.Using si RNA to interfere the expression of CBX7,we found that m RNA level of IL-1β was down-regulated.ELISA confirmed that CBX7 can promote IL-1β secretion by macrophage.Ch IP and CUT&Tag indicated that CBX7 can combine with Il1 b promoter region,but its role in regulating gene transcription was not associate to PRC1-related histone modification.It has been reported that CBX2,a member of the CBX family,can undergo phase separation.By experiments we found that CBX7 has the same capability in vivo,which may play the role in transcriptional regulation.Given that the tumorigenic effect of IL-1β has been found in a variety of mouse and human tumors,we speculate that TAM may influence tumor microenvironment and promote tumor progression by releasing IL-1β.Generally,by using single-cell sequencing data,we analyzed the different expression genes from different TAM subgroups and searching for functional molecules in tumor progression and chemotherapy resistance.LGALS3 and CBX7 was screened and verified by experiments,and the specific molecular mechanism of their effect was further explored,which provided new ideas and methods for TAM-targeting tumor treatment and coping with tumor chemotherapy resistance.