Spatial multi-omic profiling to capture functional signatures in HER2-heterogeneous FFPE breast cancer tissue

Intratumoral heterogeneity in human epidermal growth factor receptor 2 (HER2)-positive breast cancer can lead to variable therapeutic responses, but its spatially resolved molecular characteristics in formalin-fixed, paraffin-embedded (FFPE) tissue are not fully characterized. In this study, we applied spatial multi-omic profiling to investigate how regional differences in HER2 expression within the same HER2-positive breast cancer tissue are reflected at the proteome and transcriptome levels. Using hematoxylin and eosin (H&E) and HER2 immunohistochemistry (IHC)-guided spatially resolved laser-activated cell sorting (SLACS), we isolated 200 μm × 200 μm × 10 μm regions of interest (ROIs) corresponding to HER2-high and HER2-low areas from FFPE tumor sections and analyzed them with an optimized trace-level LC-MS workflow. To extend the analysis to the multi-omics level, we additionally performed RNA sequencing on adjacent ROIs from the same tissue sections. This approach consistently quantified an average of more than 4,400 proteins per ROI (approximately 60-80 cells), yielding a total of 6,116 identified proteins and revealing 1,188 differentially expressed proteins (DEPs) between HER2-high and HER2-low regions. Protein-protein interaction network analysis of DEPs upregulated in HER2-high regions revealed tightly connected functional modules mainly related to protein synthesis, metabolism, intracellular trafficking, and cellular stress responses, whereas DEPs upregulated in HER2-low regions were enriched in modules associated with mitochondrial and energy metabolism, extracellular matrix organization, and cellular differentiation and cytoskeletal regulation. Canonical pathway analysis further showed that hallmark HER2 signaling in breast cancer, together with PI3K/AKT and mTOR-related pathways, were predicted to be activated in HER2-high regions, while several tumor microenvironment-associated pathways and sirtuin signaling pathways were predicted to be inhibited, indicating region-specific rewiring of oncogenic and microenvironmental signaling networks. Although HER2-related pathways were enriched in both transcriptomic and proteomic data, pathway activation was more clearly resolved in the proteome, suggesting post-transcriptional regulation and highlighting the added functional information obtained from proteomics. Collectively, these results indicate that spatial multi-omic profiling of HER2-heterogeneous FFPE breast cancer tissue captures region-specific pathway activities and biological functions associated with varying HER2 expression.