Abstract 2073: Technological benchmarking of integrated multiome analysis using multiplex immunofluorescence and spatial transcriptomics across cancer types

Abstract Spatially resolved technologies are transforming cancer research by enabling the simultaneous visualization of molecular and cellular processes within tissue contexts. We performed a comprehensive benchmarking of various multiplex immunofluorescence (IF) platforms, including COMET™ and MILAN, and spatial transcriptomics technologies, including RNAscope™ and Xenium, and their combinations to evaluate their performance, compatibility, and potential for deep phenotypic profiling. Tissues from multiple cancer types were subjected to multiplex IF using the COMET and MILAN platforms. Complementary spatial transcriptomics analyses were performed using RNAscope and Xenium on the same sections. Performance metrics and cross-platform reproducibility were systematically assessed and the added value of evaluating protein and RNA markers simultaneously was also evaluated. To facilitate data processing and integration, we developed a novel bioinformatics software, DISSCOVERY, specifically designed for the integrated analysis of high-dimensional multiplex IF and spatial transcriptomics data. Each multiplex IF and spatial transcriptomics platform demonstrated unique strengths and limitations, both individually and in combination. Initial evaluations focused on the performance of each platform independently, followed by assessments of their combined application on the same tissue section. Notably, the combination of the Xenium and MILAN platforms provided unparalleled flexibility and scalability, enabling high-throughput analysis of both large marker panels and extensive sample sets. The COMET platform, which integrates RNAscope with multiplex IF, also proved highly valuable for its speed and automation, particularly in validating newly discovered mechanisms. Integration of these data through the DISSCOVERY software enabled seamless cross-platform analysis, uncovering spatial co-localization patterns and gene-protein associations. This approach allowed us to identify novel cellular niches and functional interactions across various cancer types, highlighting the powerful synergy of multiplex IF, IHC and spatial transcriptomics for advancing our understanding of cancer biology. Overall, our benchmarking underscores the complementary capabilities of multiplex IF and spatial transcriptomics technologies in dissecting the tumor microenvironment. The DISSCOVERY pipeline streamlines data integration, facilitating multi-omic insights into cancer biology. These findings provide a roadmap for selecting and combining spatial technologies to advance cancer research. Citation Format: Jonathan Chui, Asier Antoranz, Jon Pey, Nikolina Dubroja, Madhavi Dipak Andhari, Chiara Caprioli, Katy Vandereyken, Carmen Bravo González-Blas, Julie Morscio, Alexandre Arnould, Pouya Nazari, Gautam Shankar, Bart de Moor, Thierry Voet, Francesca M. Bosisio, Frederik De Smet. Technological benchmarking of integrated multiome analysis using multiplex immunofluorescence and spatial transcriptomics across cancer types [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 2073.