이수민 (메테오바이오텍)

이충원 (메테오바이오텍)

이수민 (메테오바이오텍)

이지혜 (메테오바이오텍)

홍정우 (메테오바이오텍)

신동윤 (CHA University School of Medicine)

박준호 (CHA University School of Medicine)

이충원 (메테오바이오텍)

There is a growing need to understand spatial molecular heterogeneity within formalin-fixed paraffin-embedded (FFPE) tissues, which remain a cornerstone of clinical pathology. However, extracting high-quality transcriptomic and proteomic information from the same spatially defined region of FFPE samples remains technically challenging. While spatial transcriptomics and low-input proteomics have each made significant advances, few methods allow their combined application in a truly co-localized, spatially resolved manner. This gap limits our ability to capture coordinated regulatory dynamics across RNA and protein layers within the native tissue context. To meet this need, we developed a spatial co-profiling workflow that enables parallel transcriptomic and proteomic analysis from spatially matched regions in FFPE samples, leveraging Spatially-resolved Laser Activated Cell Sorting (SLACS) for precise region isolation. HER2-positive breast cancer FFPE tissues were IHC-stained to identify adjacent HER2-high and HER2-low regions. Pairs of ~ 200 um x 200 um regions were microdissected using SLACS and divided for RNA and protein extractionFull-length transcriptomic profiling was performed using Smart-seq2, enabling robust gene detection from limited RNA input. Proteomic analysis was conducted using an ultra-low input LC-MS/MS workflow optimized for FFPE tissue, achieving deep proteome coverage from the same spatial regions. Results: From each ~1 mm² region of interest (ROI), over 6,000 proteins were identified by LC-MS/MS with high depth and reproducibility, while transcriptomic profiling yielded approximately 3,000 genes per ROI using Smart-seq2. ERBB2 expression was consistently elevated in HER2-high regions at both RNA and protein levels, confirming spatial specificity. Co-profiling revealed spatially concordant upregulation of oncogenic pathways, including ERBB2 signaling and cell cycle–related networks, in HER2-high areas. In contrast, discordant regulation was observed in immune-related and stromal remodeling pathways—where RNA and protein levels exhibited differing spatial patterns—highlighting complex post-transcriptional and microenvironmental influences. This study demonstrates a robust spatial co-profiling approach for FFPE tissue, enabling parallel transcriptome and proteome analysis from matched microdissected regions. Leveraging SLACS for spatial precision, this workflow provides new insights into tumor heterogeneity and localized biology in archived clinical specimens.​