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.

FIGURE 1

YAP and STAT3 were highly expressed in the BC tissues. (A) IF staining was utilized to identify the YAP, STAT3, and p‐STAT3 expression in tissues. The red signal represented positive staining for YAP, p‐STAT3, and STAT3, while the blue signal indicated nuclear staining. (B). WB was selected to examine the levels of the YAP, STAT3, p‐STAT3, VEGF, VEGFR‐2, and PD‐L1 proteins in tissues. (C) The co‐localization of YAP and M2 macrophage (CD163) in tissues were detected using IF. (D) The co‐localization of YAP and CD8 (CD68/CD163) in tissues was analyzed using double IF. The positive signal was CD163 (green) + YAP (red), CD8 (green) + YAP (red). The blue was nuclear staining signal. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05 vs the paratumor tissues group. t‐test.

FIGURE 2

Inhibition of YAP reversed M2 macrophage polarization induced by the supernatant of BC cells. (A) RT‐qPCR and WB were chosen to detect YAP expression in cell lines. (B) M1 and M2 macrophages were identified with flow cytometry with markers of CD16/32⁺F4/80⁺ and CD206⁺F4/80⁺, respectively. (C) The expression of PD‐L1 protein in cell lines was detected through WB. (D) The iNOS, IL‐12, IL‐10, TGF‐β, Arg‐1, and CCL‐22 expressions in cell lines were tested with ELISA. (E) The expression of YAP in cell lines was examined with IF staining. The green signal indicated positive staining for YAP, while the blue signal indicated nuclear staining. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05 vs the control group, ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 vs the TAMs + si‐NC group. one‐way ANOVA (A–E).

FIGURE 3

YAP/STAT3 regulated M2 macrophage polarization induced by BC cell supernatant. (A) YAP, STAT3, and the p‐STAT3 expression level in cell lines were detected by WB. (B and C) M1 and M2 macrophages were identified using flow cytometry. (D) PD‐L1 expression level in cell lines was detected by WB. (E) ELISA was adopted to test iNOS, IL‐12, IL‐10, TGF‐β, Arg‐1, and CCL‐22 expression in cell lines. (F) RT‐qPCR was used to detect the expression of VEGF and VEGFR‐2 in cell lines. (G) CoIP verified that YAP directly binds to STAT3 protein in cell lines. ***p < 0.001, **p < 0.01, *p < 0.05 vs the oe‐NC group, ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 vs the oe‐YAP + si‐NC group. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. one‐way ANOVA.

FIGURE 4

YAP/STAT3 promoted M2 polarization of TAMs and inhabited CD8⁺ T cell viability. (A) WB and RT‐PCR were chosen to test the expression of YAP in cell lines. (B) CCK8 was selected to examine the proliferation of T cells in cell lines. (C) M1‐ and M2‐type macrophages in cell lines were identified using flow cytometry. (D) T‐cell‐mediated cytotoxicity in cell lines was investigated by the LDH release assay. ***p < 0.001, **p < 0.01, *p < 0.05 vs the si‐NC (1:3) group, ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 vs the si‐NC (1:5) group, ^&&& p < 0.001, ^&& p < 0.01, ^& p < 0.05 vs the si‐NC (1:10) group. One‐way ANOVA. (E) CCK8 was selected to examine the proliferation of T cells in cell lines. (F) M1‐ and M2‐type macrophages in cell lines were identified using flow cytometry. (G) T cell‐mediated cytotoxicity in cell lines was investigated by the LDH release assay. ***p < 0.001, **p < 0.01, *p < 0.05 vs the si‐YAP + NC‐oe‐STAT3. t‐test. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate.

FIGURE 5

Inhibition of YAP could affect TME and tumor proliferation through the STAT3/VEGF/VEGFR‐2 axis. (A) The detection of volume and weight of tumors of mice. (B) The tumor morphological changes of mice were observed by HE staining. (C) YAP expression of tumor tissues was tested by RT‐qPCR. (D) CD3⁺CD4⁺, CD3⁺CD8⁺, and treg cells in tumor tissues were examined by flow cytometry. (E) The ratio of M1 and M2 macrophages in tumor tissues was tested by flow cytometry. (F) The iNOS, IL‐12, IL‐10, TGF‐β, Arg‐1, and CCL‐22 expressions were tested by ELISA. (G) IF staining was selected to examine the YAP, STAT3, and p‐STAT3 expression levels in tumor tissues. The red signal represented positive staining for YAP, p‐STAT3, and STAT3, while the blue signal indicated nuclear staining. (H) The levels of YAP, STAT3, p‐STAT3, VEGF, VEGFR‐2, and PD‐L1 in tumor tissues were examined by WB. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05 vs the model group. One‐way ANOVA (A, left and B–H). Two‐way ANOVA (A, right).