Abstract 6604: Characterizing the tumor microenvironment using spatially barcoded archival FFPE tissue: converting single-nucleus RNA-seq into spatial transcriptomics
Authors: Cedric R. Uytingco, Julie Wilhelmy, Bryant Ngo, Jonathan Sakkos, Wanxin Wang, Christina Chang, Christina Fan
Journal: Cancer Research (2025)

Abstract Identifying the contribution and the role of each individual cell in the spatial context of the tumor microenvironment (TME) is crucial in determining tumor progression, examining the extent of immune infiltration, and identifying potential therapeutic targets. While single-cell sequencing-based techniques help reveal cellular heterogeneity in a cell population, they do not provide spatial context. In turn, recently emerged spatial transcriptomics techniques are challenged by the difficulty in pinpointing the spatial measurement to single cells, requiring either complex cell segmentation or deconvolution algorithms in the data processing. To address these limitations, we introduce Curio Trekker single-cell spatial mapping kit, a unique platform that turns a single-cell experiment into a spatial experiment, essentially providing a spatial measurement that has true single-cell resolution. It is based on the Slide-tags methodology (Russel et al, 2023) that spatially tags each nucleus within its native tissue environment. This approach utilizes existing single-nuclei sequencing infrastructure to generate high-resolution spatial transcriptomics data. Using archived FFPE samples, we demonstrate the effectiveness of Curio Trekker by aligning 25-30 µm tissue sections to spatially barcoded tiles. Spatial barcodes are cleaved and hybridized to cells and nuclei in the intact tissue, followed by tissue dissociation and nuclei isolation. The tagged nuclei are processed with established snRNA-seq workflows (e.g., 10x Chromium FLEX), producing gene expression and spatial libraries for the individual nuclei in the tissue section. This spatial tagging allows each nucleus to be bioinformatically mapped back to its original tissue location. We applied this solution to breast cancer biospecimens from different patients using whole transcriptome probes (10x Genomics). The resulting data allowed for detailed characterization of the TME, confidently identifying epithelial-malignant, stromal, and immune cells from each biospecimen. This enabled marker-guided regional selection and differential gene expression analysis based on both cell type and spatial location. Overall, the Curio Trekker platform provides a streamlined, high-resolution solution for profiling cellular heterogeneity and interactions within tumors, seamlessly integrating with existing workflows to generate rapid insights into disease mechanisms and therapeutic development. Citation Format: Cedric R. Uytingco, Julie Wilhelmy, Bryant Ngo, Jonathan Sakkos, Wanxin Wang, Christina Chang, Christina Fan. Characterizing the tumor microenvironment using spatially barcoded archival FFPE tissue: converting single-nucleus RNA-seq into spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6604.