M2-type tumor-associated macrophages promote invasion of canine breast cancer through ADAM9 upregulation.

Introduction: Tumor-associated macrophages (TAMs) represent the most abundant immune cell population within the tumor microenvironment and play a critical role in cancer progression. However, the molecular mediators underlying TAM-driven tumor invasion remain incompletely defined. This study investigated whether ADAM9 functions as a key effector of pro-invasive TAM polarization using a canine mammary tumor model integrated with human transcriptomic datasets. Methods: Transcriptomic analyses were performed using canine and publicly available human datasets. Single-cell RNA sequencing was used to determine cellular localization of ADAM9. IL-4-induced M2 macrophages were evaluated for ADAM9 expression, tumor migration and invasion capacity, extracellular matrix (ECM) degradation, cytoskeletal remodeling, and spheroid destabilization. ADAM9 knockdown and cancer stem cell (CSC)-conditioned medium experiments were conducted to assess mechanistic involvement. Results: ADAM9 was consistently enriched in M2-polarized TAMs across species and was confirmed to be expressed in TAM populations by single-cell RNA sequencing. IL-4-induced M2 macrophages upregulated ADAM9 and significantly enhanced tumor migration and invasion. ADAM9 knockdown attenuated ECM degradation, reduced MMP9 expression, and disrupted F-actin remodeling. CSC-conditioned medium further induced ADAM9 expression, suggesting its role as a convergent mediator of CSC-macrophage crosstalk. In spheroid models, ADAM9 depletion prevented TAM-mediated spheroid destabilization and suppressed collective invasion. Conclusion: These findings identify ADAM9 as a key effector of TAM-driven tumor invasion through ECM remodeling and cytoskeletal regulation, and highlight ADAM9 as a promising therapeutic target within the tumor immune microenvironment.