Single-cell Analysis Reveals Potential Mechanisms Of Immune Checkpoint Inhibitors Resistance In Advanced Triple-negative Breast Cancer

Objective: To analyze the mechanism of drug resistance of immune checkpoint inhibitors in advanced triple-negative breast cancer through bioinformatic technology.Methods: In this study,a dataset from Gene Expression Omnibus database with single-cell RNA sequencing data of tumors and peripheral blood immune cells and corresponding clinical information from 11 patients treated with paclitaxel combined with atezolizumab after being diagnosed as advanced triple-negative breast cancer was reanalyzed.There were 4 cases of 11 patients with partial response,and 7 cases of stable disease with a total of 89,951 cells.The partial response group included 3 PD-L1-positive patients,and the stable disease group included 2 PD-L1-positive patients.Through bioinformatics technology,the sequencing results were reduced,clustered and annotated,and the changes of tumor-infiltrating immune cells under different treatment results were compared,and the in-depth analysis of the immune cell population with significant changes was obtained.This includes fine clustering of immune cell populations to discover key cell populations affecting immune checkpoint inhibitors resistance.Through pseudotime analysis,important pathways and genes affecting cell differentiation were discovered.Enrichment analysis and a protein-protein interactive network was made to uncover key genes influencing immune checkpoint inhibitors resistance.Results: B cells,NK cells and monocytes/macrophages had the most significant impact on immune checkpoint inhibitors resistance.In the B cell subpopulation,germinal center B cells were significantly associated with remission.Germinal center B cells inhibit drug resistance by promoting the anti-tumor immune effect of CXCL13+T cells.The results of pseudotime analysis showed that the upregulation of GADD45β reduced the formation of Cyclin B1-CDK1 complexes,stagnated the cell cycle in the G2/M phase,and made it more difficult to differentiate into germinal center B cells.Patients who are not sensitive to immunotherapy are infiltrated with a large number of low-cytotoxic GNLY-NK cells around the tumor,competitively inhibiting the anti-tumor immune effect of GNLY+NK cells and T cells.Monocytes/macrophages in patients with partial response upregulated IL1 B,upregulated chemokines CCL20 and CCL4L2 and downregulated CCL8 and CCL2,increased chemotaxis on dendritic cells,Th cells and NK cells,and decreased chemotaxis on monocytes and granulocytes.Based on the analysis results of three immune cells,it was found that tumor-infiltrating immune cells had abnormal activation of NF-κB pathway,increased transport of P50 homodimer to the core,and inhibition of heterodimer,which affected the resistance of immune checkpoint inhibitors.Conclusion: Abnormal activation of NF-κB pathway in triple-negative breast cancer tumor-infiltrating immune cells,upregulating GADD45β to inhibit the G2/M phase by forming P50 homodimer and binding to BCL3,hindering the differentiation of B cells to germinal center B cells,inhibiting the transcription of cytotoxicity-related genes in NK cells,and regulating the expression of chemokines in monocytes/macrophages,thereby affecting the resistance of immune checkpoint inhibitors.