Fig 2: Validation of ANXA9 expression in BCA. (A) ANXA9 expression in adjacent non‐tumor tissues and BCA tissues was detected by IHC staining. (B) RT‐qPCR assay was to measure ANXA9 expression in adjacent non‐tumor tissues and BCA tissues in 30 patients. (C) Western blot analysis of ANXA9 protein levels in adjacent non‐tumor and tumor tissues of five patients. GAPDH was used as an internal control. (D) RT‐qPCR and (E) western blot analysis of ANXA9 mRNA and ANXA9 protein levels in normal MCF10A cells, and BCA cell lines MCF7, SK‐BR‐3, and T‐47D cells. Significance: **p < 0.01; ***p < 0.001.

Fig 3: ANXA9 is secreted by BCA tissue‐derived exosomes. (A) Exosomes at different magnifications by transmission electron microscope. (B) Western blot was utilized to detect exosomal markers CD63, CD81, and CD9 protein levels in MCF10A, MCF, and T‐47D cells cultured with or without exosomes. (C) ANXA9 mRNA expression level in MCF10A, MCF7, and T‐47D cells cultured with or without exosomes was tested by RT‐qPCR. (D, E) Proteinase and RNase protection assays were utilized to test ANXA9 mRNA and ANXA9 protein expression levels in MCF7 and T‐47D cells cultured with exosomes and treated with/without RNase/Protease/Triton X‐100. Significance: **p < 0.01; ***p < 0.001.

Fig 4: ANXA9 silencing suppresses BCA progression in vitro and in vivo. (A) Colony formation assay was applied for estimating cell proliferation when ANXA9 was silenced. (B) Cell apoptotic capability was determined by flow cytometry analysis when ANXA9 was knocked down. (C, D) Transwell assay was employed for measuring cell migratory and invasive capabilities when ANXA9 was knocked down. (E) RT‐qPCR analysis of ANXA9 expression in tumor tissues of mice injected with MCF7 cells transfected with LV‐NC or LV‐sh‐ANXA9. (F, H) Tumor size, volume, and weight in indicated groups. (I) IHC staining assay was performed to measure Ki‐97 expression in mice injected with LV‐NC or LV‐sh‐ANXA9 transfected MCF7 cells. (J) TUNEL assay was utilized for measuring cell apoptosis in mice injected with LV‐NC or LV‐sh‐ANXA9 transfected MCF7 cells. Significance: **p < 0.01; ***p < 0.001.

Fig 5: Relationship between ANXA9 expression and clinicopathological characteristics of BCA patients. (A–F) ANXA9 mRNA expression levels in different BCA patients based on clinical parameters (race, age, cancer stage, subclasses, metastasis, and menopause status) were obtained from the UALCAN database. Significance: *p < 0.05; **p < 0.01; ***p < 0.001.