Objective: HER2-positive breast cancer (HER2+ BC) remains an aggressive subtype with limited treatment efficacy due to therapeutic resistance and systemic toxicities. CD73, an ectoenzyme producing extracellular adenosine, promotes tumor progression by enhancing angiogenesis, immune evasion, and metastasis. Exosomes derived from menstrual blood mesenchymal stem cells (Exo-Mens) exhibit anti-tumor and anti-angiogenic effects through bioactive cargo delivery. This study investigated the synergistic therapeutic potential of CD73 inhibition (using APCP) combined with Exo-Mens in HER2+ BC, with particular focus on their effects on angiogenesis-related pathways and on the regulation of miR-20a and miR-422a. Materials and Methods: SKBR3 HER2+ BC cells were treated with Exo-Mens, APCP, and their combination. Exosomes were isolated and characterized by TEM, DLS, and Western blotting. Cytotoxicity assays determined IC 5 0 values and synergy indices. Functional assays (colony formation, scratch migration assay), flow cytometry for apoptosis, and qRT-PCR analysis of pro-angiogenic (HIF-1α, KDR), pro-invasive (NF-κB, MMP9), and microRNAs (miR-20a, miR-422a) were performed. Results: APCP showed higher cytotoxic potency (IC 5 0 = 12.41 μg/mL) than Exo-Mens (IC 5 0 = 61.84 μg/mL). Combination therapy demonstrated strong synergy (combination index < 1), significantly reducing colony formation (10.41% vs. 100% in controls), and cell migration (26.13% wound closure vs. 100% control). Both treatments downregulated angiogenesis factors (HIF-1α, KDR), while invasion markers (NF-κB, MMP9) showed treatment-specific responses. Notably, miR-422a was significantly upregulated across treatments, with the highest fold-change in the combination (3.05 ± 0.05), whereas miR-20a showed only modest changes. Combination therapy also enhanced apoptosis (viable cells reduced to 46.2% vs. 89.1% control). Conclusion: The combination of APCP and Exo-Mens demonstrated synergistic anti-tumor effects in HER2+ BC cells by targeting pathways related to angiogenesis, proliferation, and apoptosis. In this context, the upregulation of miR-422a suggests a potential tumor-suppressive role, warranting further investigation. Plain Language Summary: CD73 inhibition combined with menstrual blood stem cell-derived exosomes synergistically suppressed proliferation, migration, and angiogenesis in HER2-positive breast cancer cells. The combinational treatment downregulated HIF-1α and KDR expression while enhancing apoptosis, indicating multi-pathway anti-tumor effects. miR-422a was significantly upregulated by all treatments, especially the combined approach, suggesting its potential role as a tumor-suppressive regulator in HER2-positive breast cancer. Keywords: HER2-positive breast cancer, CD73 inhibitor, MenSC-exosomes, angiogenesis, miR-422a, synergistic therapy
Iravani et al. (Wed,) studied this question.