Abstract 167: Novel spatial tagging of single nuclei spatializes single-cell ATAC-seq and V(D)J sequencing with gene expression for tumor microenvironment analysis

Abstract Advances in single-cell multiomic technologies such as RNA-seq, ATAC-seq, or V(D)J sequencing have enriched our understanding of tumor gene expression, chromatin accessibility, and immune receptor diversity, but they fall short in capturing the critical spatial context of cells within the tumor microenvironment (TME). This limitation hinders our understanding of how tumor cells organize and interact with surrounding cells during tumor progression, metastasis, and treatment response. Existing spatial methods, whether microscopy- or sequencing-based, primarily focus on gene or protein expression only. Additionally, they often lack true single-cell resolution, requiring computationally involved cell segmentation or deconvolution, and offer limited molecular complexity. To address this gap, Curio Trekker single-cell spatial mapping kit, based on Slide-tags technology, provides a paradigm-shifting solution by transforming single-cell sequencing assays into spatially resolved single-cell data. Curio Trekker spatially labels individual cells within a tissue section by releasing the DNA barcodes from a spatially indexed bead array surface into nearby nuclei. The spatially tagged nuclei are then dissociated from the array and processed on conventional single-cell sequencing assays and platforms. The technique expands spatial measurement beyond gene expression alone, enabling spatial multiomic analyses such as chromatin and V(D)J profiling. Moreover, given the spatial measurements are at true single-cell resolution, the resulting datasets are easier to analyze and interpret. In this study, we applied Curio Trekker to existing single-nuclei (sn) ATACseq and sn-V(D)J assays combined with gene expression to spatially analyze human melanoma fresh frozen tissues. These previously unattainable combinations enable the simultaneous spatial exploration of gene expression and epigenetic regulation within the spatial context of tumors and the mapping of immune receptor clones within TME. Joint unsupervised clustering on sn-RNAseq and sn-ATACseq data identified spatially defined tumor, stromal, immune, and vasculature cell types driven by both transcriptomic and epigenetic signatures. These cell types were further classified spatially into tumor zones and tumor-free areas, determined by their spatial locations and CNV profiles. We linked tumor-zone-specific transcriptional factors with accessible chromatin domains and binding motifs. Tumor-stromal interactions were inferred within their native spatial context using spatially aware ligand-receptor analysis. V(D)J and immune composition analysis revealed spatial distribution of different T cell clones. Our findings demonstrate the potential of Curio Trekker to advance oncology and immunotherapy by providing a comprehensive spatial view of tumor ecosystems. Citation Format: Wanxin Wang, Julie Wilhelmy, Christina Chang, Christina Fan. Novel spatial tagging of single nuclei spatializes single-cell ATAC-seq and V(D)J sequencing with gene expression for tumor microenvironment analysis [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 167.