Enhanced Therapeutic Efficacy of Combining Losartan and Chemo-Immunotherapy for Triple Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer, which is relatively resistant to anti-programmed cell death-1 (α-PD1) therapy, characterized as non-immunogenic, dense stroma and accumulation of M2 tumor-associated macrophages (TAMs). Despite progress in strategies to deplete extracellular matrix (ECM) and enhance tumor-cell immunogenicity, the combinatorial anti-cancer effects with α-PD1 need to be explored. Here, we applied doxorubicin hydrochloride liposome (Dox-L) as immunogenic cell death (ICD)-inducing nano-chemotherapy and used losartan as stroma-depleting agent to improve α-PD1 efficacy (Losartan + Dox-L + α-PD1). The results showed that losartan could cause ECM reduction, facilitating enhanced delivery of Dox-L and further dendritic cell (DC) maturation. Additionally, losartan could also alleviate hypoxia for TNBC, thus reprogramming pro-cancer M2 TAMs to anti-cancer M1 TAMs, successfully overcoming immune-suppressive microenvironment. These modifications led to a significant increase in T cells' infiltration and augmented anti-tumor immunity as exemplified by the notable reduction in tumor size and lung metastases. In summary, our findings support that combined treatment of losartan with Dox-L normalizes immunological-cold microenvironment, improves immuno-stimulation and optimizes the efficacy of TNBC immunotherapy. A novel combinational strategy with FDA-approved compounds proposed by the study may potentially be useful in TNBC clinical treatment.

Keywords: chemotherapy; extracellular matrix; immunotherapy; positron emission tomography (PET); triple-negative breast cancer.

Figure 1

Losartan significantly inhibits the growth of tumors treated with Dox-L. (A) Schematic illustration of losartan and Dox-L combination therapy to tumor models (n = 5). (B) Average tumor growth curves of 4T1-tumor-bearing mice from different groups of mice. (C) Average weights of tumors at the end of treatments. (D) Photographs of excised tumors at the end of treatments. (E) Weight of mice after different treatments. Data are expressed as the mean ± SEM. Data are expressed as the mean ± SEM. Statistical significances were calculated via Student’s t-test, ***p < 0.001 and ****p < 0.0001.

Figure 2

Antitumor effect of losartan, Dox-L plus anti-PD1 immunotherapy in orthotopic 4T1 tumor models. (A) Schematic illustration of combined treatment to tumors. (n = 5) (B) Tumor growth curves of different groups of orthotopic tumor-bearing mice after various treatments as indicated in the figure. (C) Average weights of tumors at the end of treatments. (D) Photographs of excised tumors at the end of treatments. (E) Weight of mice after different treatments. (F) The numbers of lung nodules were counted under an anatomy microscope (n = 5). (G) Representative Lung photographs of 4T1 murine breast tumors treated as indicated. Red arrows indicate the metastatic nodules on the lungs. (H) Representative H&E histopathological images of the lung metastasis. Scale bar = 200μm. Data are expressed as the mean ± SEM. Statistical significances were calculated via Student’s t-test and one-way analysis of variance (ANOVA), *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3

Enhanced tumor normalization by losartan relieves dense tumor stroma, increasing perfusion and alleviating tumor hypoxia. (A) Representative images of CD31 (endothelial marker, green), Collagen I (stroma content, green), α-SMA (pericyte marker, red), HIF-1α (hypoxia marker, green) immunofluorescence staining and DAPI (blue) nuclear staining after various treatments as indicated. Quantification of CD31 (B), Collagen I (C), α-SMA (D), HIF-1α (E) positive staining signals from the images shown in (A). Scale bar = 100μm. Data are expressed as the mean ± SEM. Statistical significances were calculated via one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 9). ****p < 0.0001. ns, no significance.

Figure 4

Losartan combined with chemo-immunotherapy promotes immune-stimulation in orthotopic 4T1 breast tumors. (A) Representative fluorescence images of 4T1 tumor slices immunostained for (effector T cell marker CD8, green), CD86 (M1-like TAM marker, green), CD206 (M2-like TAM marker, red), CRT (ICD effect marker, red) and DAPI (cell nuclei, blue). (B–D) Representative flow cytometry plots showing the tumor immune cells, including CD8+ T cells (CD45+, CD3+, CD8+), M1-like TAMs (CD11b+, F4/80+, CD86+) and M2-like TAMs (CD11b+, F4/80+, CD206+) in tumors after different treatments. (E) Heat map plot of ELISA results. (F) Quantification of the level of CD8+ T cells by flow cytometry analysis (n = 5). (G) Ratio of M2-like TAM to M1-like TAM by flow cytometry analysis (n = 5). (H) Quantification of CRT positive staining signals from the images shown in (A) (n = 9). Cytokine quantification of the secretion of TNF-α (I), IFN-γ (J), IL-6 (K) in sera from mice after various treatments as indicated in the figure (n = 3). Scale bar = 100μm. Data are expressed as the mean ± SEM. Statistical significances were calculated via one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ns, no significance.

Figure 5

Investigation of the splenic DCs infiltrate and splenic ¹⁸F-FDG uptake in mice bearing 4T1 tumors after combination therapy. (A, B) The percentage of mature DCs (CD11c+, CD80+, CD86+) was analyzed by flow cytometry (n = 5). (C) Representative PET/CT images showing the ¹⁸F-FDG uptake in the spleens of 4T1 tumor-bearing mice at PET scans (n = 5). White dashed line = spleen. (D) Splenic ¹⁸F-FDG uptake was determined by scanning in 4T1 tumor-bearing mice. (E) The ratio (spleen/muscle) is expressed as decay-corrected mean SUV of ROI of spleen divided by decay-corrected mean SUV of ROI of muscle. (F, G) Correlation of the ¹⁸F-FDG uptake and the ratio (spleen/muscle) respectively with the number of splenic DC cells present. Data are expressed as the mean ± SEM. Statistical significances were calculated via one-way ANOVA using the Tukey post-test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 6

The scheme illustrates the mechanism of losartan-based chemo-immunotherapy to achieve systemic antitumor immune responses.