Abstract 7466: Advancing tumor characterization and immune phenotyping through multiomic in situ profiling of RNA and proteins

Abstract Cancer continues to be one of the leading causes of death and financial burden on public health worldwide. Despite advances in detection and treatments, there is still a significant level of uncertainty in predicting how an individual's cancer will respond to treatment due to tumor heterogeneity among different patients and multiple subclones from the same individual. A more comprehensive understanding of cancer cell populations, their molecular interactions, and their microenvironment can offer valuable insights into optimal treatment strategies and patient stratification. This can be achieved through the integrated detection of both RNA and protein targets, enabling a deeper exploration of the molecular mechanisms driving cancer. Here, we analyzed formalin-fixed, paraffin-embedded (FFPE) human tissue sections from diverse tissue types using spatial multi-omics technologies from 10x Genomics. A new add-on capability enables visualization and analysis of protein expression for key immuno-oncology markers including markers for immune cell typing, cell structure, proliferation, and differentiation, as well as immune checkpoint markers (e.g., PD-1, PD-L1, LAG-3, and VISTA). Simultaneous spatial profiling of 480 genes and 28 proteins, integrated with H&E imaging, provided detailed insights into tissue architecture, tumor origins, immune cell infiltration, and the immune status of the tumor microenvironment. The combined RNA and protein profiling method is a powerful approach for validating RNA-based findings and providing definitive evidence at the functional biological level. Protein data also offers critical insights into the expression and function of different isoforms that are challenging to discern at the RNA level. For instance, isoforms such as CD45RA and CD45RO, which arise from alternative splicing of the PTPRC gene, are often indistinguishable using RNA profiling alone due to the shared exonic sequences in their transcripts. However, protein-level analysis can differentiate these isoforms and enables a more precise understanding of their distinct roles in the immune response, including the delineation of naïve and memory T-cell subsets. Furthermore, RNA expression does not always directly correlate with protein levels, underscoring the importance of integrating both data types. For instance, discrepancies between RNA and protein expression were observed for β-catenin, encoded by the CTNNB1 gene, across tissues. β-catenin plays a pivotal role in tumor progression through its dual functions in cell adhesion and signaling. By integrating RNA and protein data, researchers can uncover the complexities of post-transcriptional regulation, subcellular localization, and their combined influence on cancer progression, providing deeper insights into disease mechanisms and potential therapeutic targets. Citation Format: Thanutra Zhang, Smritee Dadhwal, Joshua Thao, Anatalia Robles, Cheyenne Christopherson, Suyog Pathare, Kristen Pham, Minkyung Park, Hsin Wen Chang, Janine Hensel, Cedric Espenel, Hayato Ikoma, Gabriele Girelli, Paul Ryvkin, Veronica Gonzalez Muñoz. Advancing tumor characterization and immune phenotyping through multiomic in situ profiling of RNA and proteins [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 7466.