Integrating hematoxylin & eosin histology with multiplexed imaging mass cytometry for spatial proteomic profiling of antibody-drug conjugate targeted biomarkers

Hematoxylin and Eosin (H&E) staining remains the cornerstone of histopathology, providing essential morphological context for tissue architecture and disease characterization. However, traditional H&E lacks the ability to deliver comprehensive molecular insights required for precision medicine application. Imaging Mass Cytometry (IMC) addresses this gap by enabling simultaneous detection of 40+ biomarkers at subcellular resolution using metal-tagged antibodies. Unlike fluorescence-based multiplexing, IMC is a quantitative, high-throughput spatial proteomics methodology with a huge (5 orders of magnitude) linear dynamic range, making it a perfect match for ADC work. Applying IMC directly to H&E-processed slides and aligning the resulting images combines the familiarity of histology with the power of spatial proteomics, allowing researchers and pathologists to overlay molecular data on conventional morphology without compromising tissue integrity. We implemented a workflow integrating H&E-stained sections with IMC antibody panels targeting biomarkers relevant to antibody-drug conjugate (ADCs) development. Regions of interest were selected based on histological features, followed by IMC acquisition and computational analysis for cell phenotyping and spatial mapping. Alignment of images and integration of H&E morphology with IMC data enabled spatially resolved correlation of histopathological features with multiplexed protein expression on the same tissue section. Using a computational pipeline, H&E-derived regions of interest were aligned with IMC segmentation maps, achieving high registration accuracy across diverse tissue architectures. IMC uses metal-labeled primary antibodies without amplification and maintains the sensitivity to quantitate the intensity of biomarkers. Quantitative analysis demonstrated the ability to identify and enumerate cells expressing ADC targets (HER2, TROP2, EGFR, etc.) within specific histological contexts, providing insights into spatial patterns of biomarker distribution. Overall, this integrated workflow improved cell-type annotation and spatial interpretation, uncovering clinically relevant microenvironmental heterogeneity not apparent from morphology alone. Integrating IMC with H&E histology provides a powerful platform for ADC development, offering unparalleled capability to link tissue architecture, biomarker expression, and immune contexture and enables the use of archival material for spatial proteomic studies. Clinically, this approach could inform patient stratification, guide ADC selection, and predict therapeutic response by correlating immune infiltration with target expression, ultimately supporting personalized oncology strategies. For Research Use Only. Not for use in diagnostic procedures.