Spatial PK/PD profiling in human ex vivo tumor slices to identify exposure-limited niches driving heterogeneous response to a TROP2-targeting ADC in NSCLC.

e15040 Background: TROP2-targeting antibody–drug conjugates (ADCs) have demonstrated promising clinical activity in non-small cell lung cancer (NSCLC); however, substantial inter- and intra-tumoral response heterogeneity remains poorly explained by bulk TROP2 expression alone. We hypothesized that spatial heterogeneity of ADC exposure and payload distribution within intact tumor microenvironments represents a key determinant of pharmacodynamic response. Methods: Fresh human NSCLC tumor specimens (n = 12) were processed into precision-cut ex vivo tissue slices (300–400 µm) and maintained under short-term culture conditions. Tissue slices were treated with a TROP2-targeting ADC or vehicle control for up to 48 hours. Following treatment, slices were fixed, embedded, and re-sectioned into 4–5 µm sections for spatial analysis. ADC localization and target expression were assessed using multiplex immunofluorescence and imaging mass cytometry. Pharmacodynamic responses were evaluated through spatial profiling of DNA damage (γH2AX), apoptosis (cleaved caspase-3), and proliferation (Ki67), together with markers of tumor microenvironmental architecture including vasculature (CD31), stroma (αSMA), and hypoxia (CAIX). Spatial co-localization analyses were performed to map relationships between ADC exposure, tissue compartments, and pharmacodynamic effects. Results: Spatial analysis revealed marked heterogeneity in ADC distribution across tumor regions, with reduced exposure in stromal-rich and hypoxic compartments despite relatively homogeneous TROP2 expression. High-exposure regions exhibited increased γH2AX and cleaved caspase-3 signaling, consistent with treatment-induced DNA damage and apoptosis, whereas low-exposure regions maintained elevated Ki67 expression, indicating persistent proliferative activity. Importantly, bulk TROP2 expression did not correlate with spatial pharmacodynamic response, while compartment-level ADC exposure metrics were strongly associated with treatment-induced biological effects. Conclusions: These findings demonstrate that spatial PK/PD profiling in human ex vivo NSCLC tumor slices provides mechanistic insight into TROP2-ADC efficacy beyond conventional biomarker approaches. Identification of exposure-limited tumor niches may explain heterogeneous responses to ADC therapy and supports the development of spatial biomarkers to guide patient stratification and rational ADC combination strategies.