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. 2025 Jun 30;74(8):256.
doi: 10.1007/s00262-025-04116-x.

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

Xiaoming Ding  1 , Gai Liang  2 , Yan Luo  2 , Xiaomei Zhou  2 , Qu Zhang  3   4   5 , Bo Luo  6   7   8   9
Affiliations

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

Xiaoming Ding et al. Cancer Immunol Immunother. .

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.

Keywords: CD8 T cell; High endothelial venules; Immunotherapy; PD-1; Salvianic acid A; Triple-negative breast cancer.

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Conflict of interest statement

Declarations. Conflict of interest: The authors declare no competing interests. Ethics approval and consent to participate: The study protocol was approved by the Ethics Committee of Hubei cancer hospital (LLHBCH2023YN-069). All the participants provided written informed consent for participation. The study was conducted in accordance with the principles of the Declaration of Helsinki. Consent for publication: Not applicable.

Figures

Fig. 1
Fig. 1
Salvianolic Acid A (SAA) increases the efficacy of anti-PD-1 therapy in in vivo. A Tumor growth curves for four treatment groups: Control (Ctrl), SAA alone, Anti-PD-1 (αPD-1) alone, and the combination of SAA + αPD-1. Tumor volumes were measured at indicated time points, and data are presented as mean ± SD. B Tumor weights at the conclusion of the experiment (day 20) for each treatment group. Each dot represents an individual tumor, and the horizontal line indicates the mean. C Body weights of mice in each treatment group throughout the 20-day study period, indicating the tolerability of the treatments. D Kaplan–Meier survival curves for each group, showing the proportion of mice surviving post-treatment initiation. E Percentage increase in tumor volume from baseline for each treatment group, calculated at the end of the study. F Representative hematoxylin and eosin (H&E) stained sections of tumor tissues from each treatment group, illustrating histological differences. G Tumors dissected in each group. The tumors were harvested 20 days after the inoculation. ***P < 0.001, **P < 0.01, *P < 0.05, ns statistically nonsignificant
Fig. 2
Fig. 2
SAA combined with anti-PD-1 therapy enhances the anti-tumor immune microenvironment. A Flow cytometric analysis of CD45 immune cell subsets in tumor infiltrates from different treatment groups: Control (Ctrl), Salvianolic Acid A (SAA), Anti-PD-1 (αPD-1), and the combination of SAA + αPD-1. The subsets analyzed include CD8 T cells, T regulatory cells (Tregs), M1 macrophages, and M2 macrophages. B Immunohistochemical analysis of CD8 T cell infiltration in tumor tissues from each treatment group. (scale bars = 100 μm). C Flow cytometric analysis of relative mean fluorescence intensity (MFI) of CD45 cells, PD-L1, and Caspase 3, as well as functional CD8 T cell subsets within the tumor microenvironment. The percentages of IFN-γ CD8 T cells and Ki-67 CD8 T cells are presented, along with the relative mRNA expression of VEGF. D Western blot analysis of PD-L1, VEGF, and GAPDH protein expressions in tumor lysates from each treatment group. The blots demonstrate differential expression levels of PD-L1 and VEGF in response to the treatments. ***P < 0.001, **P < 0.01, *P < 0.05, ns statistically nonsignificant
Fig. 3
Fig. 3
SAA Increases Stem-like CD8 T Cell Infiltration in Tumors. A Immunofluorescence images of tumor sections from Control (Ctrl), SAA-treated, αPD-1-treated, and SAA + αPD-1-treated mice. Sections are stained for TCF1 (yellow), CD8 (purple), and DAPI (blue). Co-localization of TCF1 and CD8indicates the presence of stem-like CD8 T cells. The SAA + αPD-1 group exhibits increased co-localization, suggesting enhanced infiltration of stem-like CD8 T cells (Scale bar: 100 μm). The tumors were harvested 20 days after the inoculation. B Quantification of CD8T cells (left) and TCF1 CD8 T cells (right) per mg of tumor tissue. The SAA + αPD-1 group shows a significant increase in both populations compared to other groups (**P < 0.001 for CD8 T cells, **P < 0.01 for TCF1 CD8T cells; ns = not significant). C Flow cytometry histograms showing SLAMF6 and TCF1 expression within CD8T cells. The SAA + αPD-1 group demonstrates a rightward shift, indicating increased expression of these stem-like markers. D Quantification of mean fluorescence intensity (MFI) for SLAMF6 and TCF1. The SAA + αPD-1 group has significantly higher MFI for SLAMF6 (*) and TCF1 (**), compared to control (*P < 0.05, **P < 0.01). E Ratio and number of different CD8 T cell subsets per mg of tumor: SLAMF6PD-1TIM3−, TCF1PD-1TIM3−. The SAA αPD-1 group significantly increases all subsets (**P < 0.01, *P < 0.05). F Percentage of peripheral blood cells that are CD8CD3, SLAMF6PD-1TIM3−, and TCF1PD-1TIM3− across treatment groups. The SAA + αPD-1 group shows no significant increase in these subsets. ***P < 0.001, **P < 0.01, *P < 0.05, ns statistically nonsignificant
Fig. 4
Fig. 4
Effects of SAA and αPD-1 treatments on endothelial cell and high endothelial venule in vivo A CD31 Immunostaining and Endothelial Cell Density (EV/area). Representative histological sections from control, SAA-treated, αPD-1-treated, and αPD-1 + SAA-treated mice show CD31-positive endothelial cells in tumor tissues. The tumors were harvested 20 days after the inoculation. Quantitative analysis reveals that the combination treatment of αPD-1 + SAA significantly increases endothelial cell density compared to control and αPD-1 alone (P < 0.001). B MECA-79 Immunostaining and High Endothelial Venule Density (HEV/area). Sections stained with MECA-79 highlight HEVs in the same treatment groups. Quantification indicates that both αPD-1 and αPD-1 + SAA treatments significantly increase HEV density compared to control and SAA alone (P < 0.001). Scale bars represent 100 μm. C Multiplex Immunofluorescence Analysis of Endothelial Cells and HEV in Different Treatment Groups. Representative images show the expression of DAPI (nuclear stain), CD31 (endothelial cells), MECA-79 (HEV marker), TCF1 (T cell differentiation), CD8 (cytotoxic T cells), and a merged image combining all stains. The SAA + αPD-1 combination treatment significantly increases the intensity and distribution of CD31, MECA-79, TCF1, and CD8 staining compared to individual treatments and control, suggesting a synergistic effect on HEV formation and stem-like CD8 T cell infiltration. D Quantitative Analysis of MECA-79⁺ Tumor-Associated High Endothelial Cells (TA-HECs) Across Treatment Groups. Flow cytometry data show that the combination treatment significantly increases the percentage of CD31⁺CD45⁻ cells among total cells (P < 0.01), the percentage of MECA-79⁺ TA-HECs among CD31⁺CD45⁻ cells (P < 0.001), the absolute number of MECA-79⁺ TA-HECs per mg tumor tissue (P < 0.05 and P < 0.01) and the mean fluorescence intensity (MFI) of MECA-79⁺ TA-HECs (P < 0.01). ***P < 0.001, **P < 0.01, *P < 0.05, ns statistically nonsignificant
Fig. 5
Fig. 5
In vitro effects of SAA on breast cancer cell viability and growth (A–C) Relative cell viability of 4T1 (A), MDA-MB-231 (B), and MDA-MB-453 (C) cells after 24-h treatment with increasing concentrations of Salvianic acid A (SAA; 0, 10, 20, 40, 80 μmol/L). Cell viability was determined using the CCK8 assay and is expressed as a percentage of the untreated control. No significant differences were observed across all concentrations tested (ns, non-significant). (D-F) Growth kinetics of 4T1 (D), MDA-MB-231 (E), and MDA-MB-453 (F) cells treated with 80 μmol/L SAA over 72 h. Optical density at 450 nm (OD450 nm) was measured at 0, 24, 48, and 72 h to assess cell growth. SAA treatment did not significantly affect cell growth compared to the control (ns, non-significant). (G, H) Survival analysis of 4T1 (G) and MDA-MB-231 (H) cells treated with varying concentrations of IFN-γ (0, 5, 10, 20 ng/mL) alone or in combination with 80 μmol/L SAA for 48 h. Cell survival was quantified using the CCK8 assay and is presented as a percentage of the untreated control. The addition of SAA did not significantly enhance the IFN-γ-mediated inhibition of cell survival (ns, non-significant)
Fig. 6
Fig. 6
Effects of Salvianolic Acid A (SAA) on CD8 T Cells. A Time- and concentration-dependent cell viability using the CCK-8 assay. CD8 T cells were treated with 20 μmol/L SAA for 0, 24, 48, and 72 h. Data are mean ± SD from three experiments. B Cell viability at 72 h with varying SAA concentrations. CCK-8 assay results are shown as mean ± SD (n = 3). Then, purified CD8 T cells from the spleen stimulated with anti-CD3/anti-CD28 and IL-15 with or without presence of SAA. C Expression of activation markers CD69, CD25, and CD44 over time with 20 μmol/L SAA treatment. D TCF1 expression at 72 h with different SAA concentrations. E TCF1 expression at 24 and 48 h with 20 μmol/L SAA. Flow cytometry data are mean ± SD (n = 3); ns = not significant. F Tcf7 gene expression at 48 h via RT-PCR. Data are normalized to β-actin and shown as mean ± SD (n = 3). G SLAMF6 expression at 72 h with varying SAA concentrations. Flow cytometry data are mean ± SD (n = 3); ns = not significant
Fig. 7
Fig. 7
In vitro, SAA maintains high endothelial phenotype in endothelial cells. A Real-time PCR analysis of Ackr1, Nr2f2, Cdh5, and Pecam1 gene expression in flow-sorted high endothelial cells from mouse tumors treated with SAA (20 μmol/L) for 48 h. Data are presented as fold change relative to control, normalized to β-actin. **P < 0.01, *P < 0.05. B Real-time PCR analysis of Cd34, St3gal6, Emcn, and B3gnt3 gene expression in flow-sorted high endothelial cells from mouse tumors treated with SAA (20 μmol/L) for 48 h. Data are presented as fold change relative to control, normalized to β-actin. ***P < 0.001, **P < 0.01, *P < 0.05. C Flow cytometric analysis of ACKR1 and MECA-79 expression in CD31 mouse tumor endothelial cells treated with SAA (20 μmol/L) for 24 h and 48 h. D Quadrant analysis shows the percentage of cells in each quadrant. E Real-time PCR analysis of ACKR1 and CDH5 gene expression in HUVECs treated with SAA (20 μmol/L) for 48 h. Data are presented as fold change relative to control, normalized to β-actin. **P < 0.01, *P < 0.05. F Western blot analysis of ACKR1 and CDH5 protein expression in HUVECs treated with varying concentrations of SAA (0, 10, 20, 40 μmol/L) for 48 h. GAPDH is used as a loading control
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