<p>Trastuzumab-based HER2-targeted therapy remains the cornerstone treatment for HER2-positive breast cancer. However, its clinical efficacy is significantly modulated by the tumor microenvironment (TME). Our single-cell sequencing analysis of clinical samples revealed that patients with poor radiologic response after trastuzumab-based neoadjuvant therapy presented significant enrichment of TIGIT⁺ NK cells with high immune checkpoint expression, exhausted CD8⁺ T cells, and immunosuppressive regulatory T cells (Tregs). Further analyses leveraging cell-cell communication, spatial transcriptomics, and multiplex immunofluorescence showed that SPP1⁺ tumor-associated macrophages (SPP1⁺ TAMs) enrichment was associated with dysfunctional NK- and T-cell states in tumors from patients with poor radiologic response. Functional validation studies revealed that SPP1⁺ TAMs actively induced exhaustion phenotypes in both NK cells and CD8⁺ T cells, thereby impairing trastuzumab-dependent antibody-dependent cellular cytotoxicity (ADCC) and adaptive immune responses. In vivo experiments using humanized NCG murine models further confirmed the SPP1⁺ TAMs-mediated suppression of NK cell function. Significantly, HER2-positive breast cancer patients with elevated SPP1⁺ TAMs levels experienced both reduced efficacy of trastuzumab neoadjuvant therapy and diminished long-term survival prospects. In summary, our findings provide the first systematic characterization of TME remodeling following trastuzumab therapy, identifying SPP1⁺ TAMs as a potential driver of trastuzumab resistance. This work advances our understanding of microenvironmental mechanisms underlying trastuzumab resistance and suggests new therapeutic strategies targeting TAM-mediated immunosuppression.</p>