Abstract 2779: Whole transcriptome spatial exploration of cultured tumor cells at subcellular resolution

Abstract Cancer research has long sought to understand the complex tumor microenvironment and the molecular underpinnings of tumor progression. Traditional methods often fail to capture the spatial organization and cell-cell interactions within tumors. The CosMx® Spatial Molecular Imager (SMI) with its CosMx Whole Transcriptome (WTX) panel offers a transformative solution, providing spatially resolved, single-cell transcriptomics with nanometer-scale precision. This platform enables the mapping of RNA molecules within their native tissue context, which is essential for uncovering critical biological insights related to cancer cell behavior, tumor microenvironment interactions, and therapeutic responses. We developed and optimized a tailored protocol for in vitro cell-based assays using the CosMx WTX platform. Key steps, including permeabilization and enzymatic digestion, were fine-tuned to preserve RNA integrity and cellular morphology, ensuring high-quality transcript detection. Cancer cell lines, U2OS (osteosarcoma) and SH-SY5Y (neuroblastoma), were cultured and analyzed using CosMx WTX to map the spatial distribution of RNA transcripts. The data were compared to bulk RNA-seq results to validate reproducibility and accuracy. The assay’s spatial resolution allows for precise mapping of individual RNA molecules within their spatial context, facilitating phenotype-associated spatial transcriptomics. In our analysis, the CosMx WTX platform successfully quantified approximately 7,300 transcripts per cell in U2OS cells and 1,900 transcripts per cell in SH-SY5Y cells, showing high reproducibility and strong agreement with bulk RNA-seq data. This high-throughput capability allows for robust transcriptomic profiling of diverse cancer cell types. By correlating gene expression data with morphological and functional phenotypes, we were able to visualize the spatial organization of transcripts within cells. The platform’s nanometer-scale resolution also supports mapping of cancer organoids, offering insights into intratumoral heterogeneity, treatment resistance, and metastatic processes. The CosMx WTX cell-based assay represents a significant advancement in spatially resolved transcriptomics. By enabling high-resolution analyses, this platform provides valuable insights into cancer biology and offers a powerful tool for drug screening and companion diagnostic development. The integration of spatial transcriptomics with protein-level data holds the potential to transform our approach to cancer diagnostics and therapy, advancing precision medicine and improving patient outcomes. Citation Format: Yi Cui, Margaret Hoang, Shanshan He, Rachel Liu, Tushar Rane, Rustem Khafizov, Haiyan Zhai, Christine Kang, Michael Rhodes, Joseph Beechem. Whole transcriptome spatial exploration of cultured tumor cells at subcellular resolution [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 2779.