Site-Specific Immune and Stromal Architecture Drive Resistance to Trastuzumab Deruxtecan in HER2+ Metastatic Breast Cancer

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) that has demonstrated remarkable efficacy in HER2+ and HER2-low metastatic breast cancer (mBC). Yet, 40-50% of patients fail to respond, and mechanisms underlying resistance, particularly those involving the immune microenvironment, remain poorly understood. To address this gap, we conducted spatial proteomic profiling of metastatic lesions from 25 HER2+ mBC patients treated with T-DXd, using the Bruker GeoMX Digital Spatial Profiler (DSP) platform. Regions of interest (ROIs) were segmented by CD45 expression to distinguish immune-rich versus immune-poor compartments, and spatial marker expression was compared between responders (R) and non-responders (NR). Spearman correlation analysis was used to assess spatial coordination among HER2, fibronectin, and immune markers. Non-responders exhibited greater HER2 heterogeneity, lower CD45+ immune infiltration, and consistent upregulation of fibronectin and granzyme B. In both bone and brain metastases, NR tissues showed strong correlations between fibronectin and T cell markers, indicating spatial immune exclusion. In contrast, NR lymph node metastases displayed negative correlations between fibronectin and immune markers and uncoordinated B-T cell clustering, suggesting an alternative resistance mechanism rooted in immune disorganization rather than exclusion. Collectively, these results implicate HER2 heterogeneity, fibrotic immune exclusion, and immune suppression as key drivers of T-DXd resistance across metastatic sites. By integrating spatial proteomics with clinical outcomes, this study provides novel insights into the spatial immune landscape of ADC resistance and identifies actionable biomarkers for patient stratification and combination therapy design, laying a critical foundation for developing biomarker-guided therapies and personalized treatment strategies to overcome ADC resistance in metastatic breast cancer. Translational SignificanceT-DXd represents a major advance in antibody-drug conjugate (ADC) therapies to address HER2 heterogeneity in tumors through bystander killing. However, treatment options following resistance to T-DXd remain limited, leaving a critical unmet need. As new ADCs continue to emerge, resistance will likely remain a persistent challenge across tumor types. This work extends beyond T-DXd resistance--it provides a framework to address resistance mechanisms common to other ADCs. By investigating immune-mediated mechanisms of ADC resistance, this research identifies therapeutic targets that can be leveraged to develop new treatment strategies. By integrating spatial biology with clinical data, this study offers unprecedented insights into immune-tumor interactions within the resistant tumor microenvironment. This approach deepens mechanistic understanding and enables personalized treatment strategies guided by spatially resolved biomarkers. This novel combination of spatial mechanistic research with clinical data represents advancement toward overcoming ADC resistance and improving outcomes for patients with few therapeutic options.