Therapeutically Engineering Exosomes to Target CD206⁺ M2-Macrophage to Prevent the Development of Primary and Distal Metastasis in Breast Cancers

Background/objective: Approximately 90% of breast cancer–related deaths result from recurrence and metastasis. Emerging evidence indicates that tumor recurrence, invasion, and metastatic spread are strongly influenced by both the tumor microenvironment (TME) and the metastatic niche. M2 macrophages promote immune suppression, inhibit inflammation, and facilitate epithelial-to-mesenchymal transition, invasion, angiogenesis, and tumor progression—effects that are particularly pronounced in triple-negative breast cancer (TNBC). The objectives of this study were to develop engineered exosomes to selectively deplete M2 macrophages to delay the growth of primary tumor and distal metastasis and enhance overall survival. Methods: Engineered exosomes were developed using our invented platform to selectively target and deplete alternatively activated CD206⁺ M2 macrophages in primary and metastatic TMEs via antibody-dependent cell-mediated cytotoxicity (ADCC). Engineered exosomes were characterized for size, zeta potential, and successful incorporation of targeting peptides and proteins. Whole-body and tumor-specific biodistribution were assessed. In vitro and in vivo experiments were conducted to evaluate targeting specificity. Toxicity and immunogenicity were examined in immune-competent animal models. Two treatment paradigms were employed. Results: Engineered exosomes containing M2-macrophage targeting peptides and Fc-mIgG2b were successfully made and there were no significant size differences between engineered and control exosomes. Both in vitro and in vivo studies confirmed the specificity of the engineered exosomes. Biodistribution studies showed no significant uptake to the resident macrophages in the lung and liver. No significant immune activation, based on cytokine profiling, or organ-specific toxicity was observed in immune-competent models. Flowcytometry studies using splenocytes showed significant depletion of M2-macrophages following treatments with engineered exosomes, however, there was no effect on the distribution of T-cells. M2-targeting therapeutic exosomes significantly delayed the growth of primary tumors and metastatic lesions. Conclusion: These findings support the potential of precision exosome-based strategies for enhancing therapeutic outcomes in breast cancer.