Salvianic acid A enhances anti-PD-1 therapy by promoting HEV-mediated stem-like CD8 T cells infiltration in TNBC.

Abstract

Objective: This study aims to investigate the potential of Salvianic acid A (SAA) to enhance the efficacy of anti-PD-1 immunotherapy in triple-negative breast cancer (TNBC), with a focus on elucidating the mechanisms.

Methods: To explore the effects of SAA on anti-PD-1 therapy efficacy, we established a mouse tumor model using 4T1 breast cancer cells and treated groups with SAA, anti-PD-1 (αPD-1), or their combination. Tumor growth, weight, and survival were monitored. A melanoma mouse model using B16 melanoma cells was also used to validate the efficacy of SAA enhanced immunotherapy. Tumor tissues were analyzed histologically and by flow cytometry to assess immune cell infiltration and function. The expression of immune markers and cytokines was evaluated using immunohistochemistry, Western blot, and quantitative RT-PCR. In vitro experiments were conducted on 4T1, MDA-MB-231, and MDA-MB-453 breast cancer cell lines, as well as CD8 T cells and endothelial cells, to investigate the direct effects of SAA on cell viability, activation, and phenotype maintenance. Additionally, the impact of SAA on high endothelial venules (HEVs) was assessed using immunofluorescence and flow cytometry.

Results: The combination of SAA and anti-PD-1 therapy significantly inhibited tumor growth and prolonged survival in the 4T1 mouse model and B16 mouse model respectively, compared to controls (P < 0.001). Tumor volumes and weights were consistently lower in the combination group, with no significant weight loss or toxicity observed. Histological analysis revealed increased stromal content and reduced tumor cell density in the SAA + αPD-1 group, indicating enhanced immune cell infiltration and tumor cell death. Flow cytometry showed that SAA significantly increased the infiltration of CD8 T cells and stem-like CD8 T cells (TCF1 and SLAMF6) into the tumor microenvironment when combined with αPD-1 (P < 0.001). The combination also enhanced the expression of IFN-γ and Ki-67 in CD8 T cells, indicating improved functional capacity. Additionally, SAA promoted the formation of HEVs in tumor tissues, as evidenced by increased CD31 and MECA-79 staining (P < 0.001). In vitro, SAA did not directly inhibit breast cancer cell viability or activate CD8 T cells but maintained the high endothelial phenotype in endothelial cells by upregulating key markers such as ACKR1 and CDH5. These findings demonstrate that SAA enhances anti-PD-1 efficacy by modulating the tumor immune microenvironment and promoting HEV formation, without direct cytotoxic effects on cancer cells or immune cells.

Conclusion: SAA significantly enhances the efficacy of anti-PD-1 therapy by promoting HEV-mediated stem-like CD8 T cells infiltration in TNBC. The combination of SAA and αPD-1 represents a promising therapeutic strategy that warrants further exploration in preclinical and clinical settings.