{
  "abstract": "Background Spatial Multiomics allows for context-based study of the tumor microenvironment (TME). TME consists of diverse cells that collectively influence tumor progression, therapeutic response, and immune activation. CD8 T-cells, essential for antitumor immunity, often become exhausted in the TME. Recent studies show that TCF1+ stem-like CD8 T-cells retain differentiation potential and can be rejuvenated through cytokine signaling and immune checkpoint modulation. This detailed spatial profiling of RNA and protein expression in the TME is crucial to understanding immune cell function, activation, and differentiation.Methods Here we introduce a protease-free workflow for RNAscope™ Multiplex Fluorescent v2 assay combining RNA in-situ hybridization (ISH) and protein immunofluorescence to study markers of T cell activation and exhaustion. We used cell marker proteins (CD8, PD1,CD3) with RNA targets (TNFA, TCF7, IFNG, CTLA4, LAG3,TIM3) in a tumor microarray (TMA).The manual multiplex protease-free workflow enables simultaneous RNA and protein visualization without enzymatic disruption, preserving antigen integrity and tissue morphology.Results Using this assay we studied CD8 T-cell differentiation and activation. Co-detection of TNFA, TCF7, and IFNG transcripts with CD8 protein provides insights into its activation status while PD1 protein and LAG3,TIM3 and CTLA4 transcripts provide insights into its exhaustion status. Data revealed robust co-expression patterns of these targets, particularly in the tumor-adjacent stromal and immune cells in breast, cervical and stomach cancers, reflecting activation and exhaustion across distinct TME niches.Conclusions This methodology lays a foundation for refining immunotherapeutic strategies targeting T-cells, enhancing effector functions, and advancing precision oncology interventions.",
  "authors": [
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne Brand, Newark, CA, USA"
      ],
      "name": "Anushka Dikshit"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, Minneapolis, MN, USA"
      ],
      "name": "Steve Zhou"
    },
    {
      "affiliations": [],
      "name": "Yifan Wang"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne Brand, Newark, CA, USA"
      ],
      "name": "Debia Wakhloo"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne Brand, Newark, CA, USA"
      ],
      "name": "Li-Chong Wang"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne Brand, Newark, CA, USA"
      ],
      "name": "Maithreyan Srinivasan"
    }
  ],
  "title": "73 Spatial detection reveals T-cell activation and exhaustion states in the tumor microenvironment",
  "uid": "39af6b41-4eac-59ed-8499-449dacf68ffe"
}