YAP/STAT3 inhibited CD8+ T cells activity in the breast cancer immune microenvironment by inducing M2 polarization of tumor-associated macrophages

Abstract

Background: Breast cancer (BC) is the leading cause of cancer-related death among women. One of the hallmarks of cancer is sustained angiogenesis. YAP/STAT3 may promote angiogenesis and driving BC progression. This study aimed to investigate how YAP/STAT3 affects the immune microenvironment in BC and understand the underlying mechanism.

Methods: To establish a tumor-associated macrophages (TAMs) model, macrophages were cultured in the 4T1 cell culture medium. A BC mouse model was created by injecting 4T1 cells. The expression of YAP, STAT3, p-STAT3, VEGF, VEGFR-2, and PD-L1 was analyzed using immunofluorescence, western blotting, and quantitative real-time PCR. Flow cytometry was used to identify M1 and M2 macrophages, CD4⁺ T, CD8⁺ T, and Treg cells. Levels of iNOS, IL-12, IL-10, TGF-β, Arg-1, and CCL-22 were measured using enzyme-linked immunosorbent assay. Co-IP was used to verify whether YAP binds to STAT3. Hematoxylin-eosin staining was used to observe tumor morphology. Cell counting kit-8 was selected to detect T-cell proliferation.

Results: YAP, STAT3, P-STAT3, VEGF, VEGFR-2, and PD-L1 were highly expressed in BC tissues. The M2/M1 macrophages ratio increased in the TAMs group compared with the control group. Inhibiting of YAP and STAT3 decreased the M2/M1 macrophages ratio. YAP was found to bind with STAT3. T-cell proliferation was enhanced after YAP inhibition, and overexpression of STAT3 reversed the regulation of YAP on T-cell proliferation. In animal studies, inhibiting YAP inhibited tumor weight and volume development. After YAP inhibition, inflammatory infiltration, M2/M1 macrophage ratio, and Treg cell ratio declined, while CD8⁺ and CD4⁺ T-cell ratio increased.

Conclusion: In conclusion, this study suggested inhibition of YAP/STAT3 reversed M2 polarization of TAMs and suppressed CD8⁺ T-cell activity in the BC immune microenvironment. These findings open up new avenues for the development of innovative therapies in the treatment of BC.

Keywords: CD8+ T; TAMs; YAP/STAT3; breast cancer.