Abstract 3797: Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis

Abstract Spatial biology techniques have revolutionized our approach towards the study of the tumor microenvironment (TME) and its complex cellular interplay. On the one hand, multiplex immunofluorescence (mIF) methods have enabled a precise profiling of immune cells and other key cellular players of the TME while uncovering their spatial distribution and interactions. On the other hand, in situ hybridization (ISH) technologies have shown to provide complementary information to protein profiling such as the mapping of cytokine- and chemokine-expressing cells, essential for comprehending signaling networks and immune activation statuses. Here, we made use of a novel multiomics approach that combines these two biological inputs by integrating RNAscope™ and sequential immunofluorescence (seqIF™) protocols to achieve same-slide co-detection of RNA and protein targets. The combined workflow is automated on COMET™, an advanced tissue staining and imaging platform. Through precise control over temperature and reagent distribution, the instrument ensures maximum efficiency and reproducibility of the assays. The integrated multiomics protocol allows for up to three RNAscope™ detection cycles combined with twelve seqIF™ cycles, for a final 12-plex RNA + 24-plex protein panel. We first showed the capacity of COMET™ platform to fully automate the RNAscope™ protocol and demonstrated its sensitivity and specificity with the analysis of positive and negative control genes on HeLa cell pellets, highlighting the flexibility of sample selection. To illustrate the potential multiomics in unravelling the complexity of the TME, we designed a panel of 12-RNA-targeting probes for key biomarkers of tumor-infiltrating lymphocytes and their activation status, including multiple secreted molecules (e. g. , cytokines and proteases). This was combined with a 24-antibody panel for the detection of protein biomarkers, selected to enable the single-cell profiling of different players within the TME. We applied this panel to characterize different human FFPE tumor tissues and showed that the co-detection of RNA and protein biomarkers on the same slide allows a better characterization of key cellular components involved in tumor progression and immune response. Our results highlight the potential of spatial multiomics technologies in enhancing the efficiency of investigations of immune cell behavior and generally the understanding of cellular interplay in the TME. Their full automation on a platform like COMET™ will accelerate analysis and increase robustness by minimizing user intervention, and ultimately helping in the development of prognostic and predictive biomarkers, in the refinement of cancer diagnoses, and in the selection of new personalized therapies. Citation Format: Paula Juricic, Arec Manoukian, Alice Comberlato, Pino Bordignon, Alix Failletaz, Anushka Dikshit, Emerald Doolittle, Rose Delvillar, Steve Zhou, Ching-wei Chang, Li-chong Wang, Saska Brajkovic, Maithreyan Srinivasan, Diego Dupouy. Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3797.