Integrative spatial omics to identify determinants of response to neoadjuvant chemo-immunotherapy in triple-negative breast cancer.

e12650 Background: Triple-negative breast cancer (TNBC) demonstrates heterogeneous response to neoadjuvant chemotherapy. Pembrolizumab increases pathologic complete response (pCR) rates, yet the biological features underlying sensitivity or resistance remain unclear. Spatial transcriptomics and single-cell RNA sequencing enable high-resolution profiling of immune and stromal ecosystems after therapy. We analyzed spatially resolved immune, stromal, and transcriptional programs associated with pCR versus non-pCR in TNBC treated with neoadjuvant chemo-immunotherapy. Methods: Pre-treatment biopsies (n = 6) and paired post-treatment specimens (n = 3) from patients receiving chemotherapy plus pembrolizumab underwent spatial transcriptomic analysis and immune deconvolution (ESTIMATE, CIBERSORT). Cell composition, immune activation, stromal states, and differentially expressed genes were compared between pCR and non-pCR tumors. Results: Immune deconvolution of pre-treatment samples (4 pCR, 2 non-pCR) showed that non-pCR tumors had lower ESTIMATE immune and stromal scores. Among post-treatment spatial transcriptomic samples (1 pCR, 2 non-pCR), the pCR tumor demonstrated complete loss of malignant epithelial cells and was dominated by immune and stromal populations with strong type I/II interferon responses, cytotoxic lymphocyte activation, and enhanced antigen-presentation signatures. Enriched populations included CD8⁺ T cells, NK cells, dendritic cells, and plasma cells expressing cytotoxic mediators and immune-checkpoint molecules. In contrast, non-pCR tumors showed reduced immune infiltration and a dense collagen-rich stroma, with upregulation of extracellular matrix genes ( COL1A1 , COL3A1 ). Myeloid antigen-presentation pathways were suppressed, including downregulation of CD74 and ferritin-light-chain–associated metabolic programs, indicating an immunosuppressive, metabolically reprogrammed microenvironment. Fibroblast expansion and matrix-remodeling states were prominent in non-pCR tissue. Conclusions: Effective neoadjuvant chemo-immunotherapy in TNBC is marked by dominant interferon-driven immune activation, high cytotoxic lymphocyte infiltration, and preserved antigen presentation, whereas non-pCR tumors display stromal fibrosis and immune suppression. Spatial and single-cell profiling identifies complementary immune and extracellular matrix signatures that may serve as biomarkers to predict response and guide treatment escalation for patients at risk of non-pCR. Immune deconvulation among 6 pre-treatment samples (4pCR and 2 non-pCR). Immune deconvoluation non-pCR non-pCR pCR pCR pCR pCR Stromal score -1396.66 -454.59 -200.80 1859.89 1510.23 2421.45 Immune score -1620.83 -1054.47 -126.42 2235.97 1901.97 2909.68 ESTIMATE score -3017.49 -1509.06 -327.22 4095.86 3412.21 5331.13