Spatial analysis of tumor immune microenvironment of TNBC with different neoadjuvant chemotherapy outcomes using multiplex Immunofluorescence

Triple-negative breast cancer (TNBC) is characterized by aggressive biological behavior and poor prognosis. However, TNBC exhibits higher immunogenicity than other breast cancer subtypes, making it more responsive to immunotherapy. Neoadjuvant chemotherapy (NAC) is the standard treatment for early high-risk TNBC; however, reliable biomarkers for predicting NAC response remain elusive. Tumor-infiltrating lymphocytes (TIL) are recognized as predictive markers of NAC response in TNBC, yet discordant cases remain, such as tumors with high TIL levels but poor response. This study aimed to further elucidate the immune environment of TNBC by analyzing TIL, programmed death-ligand 1 (PD-L1) expression, and tumor-stroma ratio using pretreatment biopsy tissue slides from 16 patients with TNBC treated with NAC. Multiplexed immunofluorescence for CD8, FOXP3, CD4, CD20, and CK was employed to investigate immune cell (IC) composition and spatial interactions within the tumor immune microenvironment. Cell to cell distance and comparison of subcellular proportion of IC and were analyzed by treatment response, TIL, and PD-L1 status. Significant differences were found in IC composition and distribution between patients achieving pathologic complete response (pCR) and those with residual disease (non-pCR). The pCR group exhibited significant enrichment of cluster of differentiation CD8 + IC and CD20 + IC in both tumor and stromal regions, suggesting their critical role in mediating an effective NAC response. In contrast, non-pCR cases showed higher proportions of immunosuppressive CD4 + FOXP3 + IC, particularly in the tumor region. High TIL levels were associated with pronounced B-T cell interactions, as evidenced by the significant clustering of CD20 + and CD8 + ICs near tumor cells, highlighting their cooperative role in antitumor immunity. In conclusion, our findings suggest that tumoral CD8 + and CD20 + ICs are pivotal determinants of NAC response in TNBC. The enrichment of CD20 + IC under high-TIL conditions underscores the potential role of B-T cell interactions in shaping immune-mediated chemotherapy responses. These insights provide a foundation for leveraging immune-based biomarkers to stratify patients with TNBC and optimize NAC outcomes.