Spatial Multiomics Reveals T-cell Activation and Exhaustion States in the Tumor Microenvironment

Spatial multiomics has emerged as a transformative approach for deciphering the tumor microenvironment (TME), enabling the study of cellular heterogeneity and functional states within their native spatial context. The TME comprises diverse cell populations, including immune cells like CD8 T-cells, which play a pivotal role in antitumor immunity but often exhibit exhaustion in chronic antigen exposure. Recent advances highlight the therapeutic potential of PD1+ TCF1+ 
stem-like CD8 T-cells, which retain regenerative capacity and can be reinvigorated through cytokine signaling and checkpoint modulation. Understanding these dynamics requires precise spatial profiling of RNA and protein expression to map immune cell activation, exhaustion, and 
differentiation states.

We developed a protease-free workflow for the RNAscope™ Multiplex Fluorescent v2 assay to address this need, integrating RNA in situ hybridization (ISH) with protein immunofluorescence. This method enables the simultaneous detection of key RNA targets (TNFA, TCF7, IFNG) and protein markers (CD8, PD1) in tumor microarrays (TMAs) without enzymatic disruption, preserving tissue morphology and antigen integrity. By co-mapping these biomarkers, we reveal distinct CD8 T-cell phenotypes and their spatial distribution across breast, cervical, and gastric cancers, offering insights into immune activation and exhaustion within specific TME niches. This approach provides a robust platform to refine immunotherapeutic strategies targeting PD1+ TCF7+ CD8 T-cells, ultimately 
advancing precision oncology.

RNAscope Multiplex Fluorescent v2 protease-free Assay enabled simultaneous spatial profiling of RNA and protein markers in FFPE tissues, preserving antigen integrity and epitope recognition.

This poster was presented at the AACR 2025 meeting.