{
  "abstract": "Background The tumor microenvironment (TME) in non-small cell lung cancer (NSCLC) is shaped by both immune infiltration and metabolic reprogramming, which together modulate therapeutic response. We applied Akoya’s PhenoCode™ Discovery IO60 Panel, combined with a spike-in PhenoCode Metabolic Module, to spatially characterize immunometabolic interactions in FFPE NSCLC samples, including both adenocarcinoma and squamous cell carcinoma (SqCLC) subtypes. AI-powered image analysis and spatially resolved cellular phenotyping were performed using Visiopharm®, enabling high-dimensional, quantitative assessment of cell types and their spatial relationships within the immunometabolic contexture.Methods Experiments were performed on the PhenoCycler®-Fusion platform, which supports the simultaneous detection of 100+ markers per tissue section. This capability enabled us to spike in key metabolic markers—GLUT1, LDHA, HK1, G6PD, ASCT2, CPT1A, ATP5A, SDHA, and IDH2—without sacrificing panel resolution. These markers span major metabolic pathways, including glycolysis, oxidative phosphorylation, fatty acid oxidation, and glutamine metabolism.Image analysis was conducted using Visiopharm’s Phenoplex™ spatial tissue analysis software, enabling high-throughput single-cell segmentation, immune cell phenotyping, spatial proximity mapping, and co-expression analysis across millions of cells. This approach allowed us to resolve distinct immunometabolic niches in situ. Glycolytic tumor regions (GLUT1+, LDHA+) were associated with exclusion of CD8+ T cells and enrichment of FoxP3+ Tregs and CD163+ macrophages, while oxidative and FAO-rich zones (ATP5A+, CPT1A+) supported suppressive myeloid populations with reduced effector T cell presence. CD8+ T cells expressing metabolic fitness markers (HK1+, ATP5A+) and effector molecules (Granzyme B+, Perforin+) were confined to metabolically permissive regions.Results Comparative analysis revealed subtype-specific trends: adenocarcinomas displayed increased checkpoint expression (PD-1, PD-L1) in glycolytic zones, whereas SqCLC tumors exhibited broader FAO signatures and deeper immune exclusion. These findings underscore how spatial metabolic heterogeneity can shape immune cell function and distribution.Conclusions Together, this work demonstrates the power of combining ultrahigh-plex spatial proteomics with advanced spatial analysis via Visiopharm’s AI-powered Phenoplex platform to uncover metabolic drivers of immune suppression. Spike-in strategies on the PhenoCycler-Fusion platform offer a flexible approach for integrated biomarker discovery in complex tissues.Ethics Approval Human tissue samples used in this study were obtained from a commercial vendor specializing in ethically sourced biospecimens. All samples were collected under Institutional Review Board (IRB)-approved protocols with informed donor consent, and were fully de-identified prior to receipt. The procurement and use of these samples complied with all applicable ethical guidelines and regulatory standards, including the principles outlined in the Belmont Report and the Common Rule (45 CFR 46).",
  "authors": [
    {
      "affiliations": [
        "Akoya Biosciences, Marlborough, MA, USA"
      ],
      "name": "Alyssa Whitley"
    },
    {
      "affiliations": [
        "Visiopharm Corp, Broomfield, CO, USA"
      ],
      "name": "Daniel Winkowski"
    },
    {
      "affiliations": [
        "Akoya Biosciences, Marlborough, MA, USA"
      ],
      "name": "Yue Hou"
    },
    {
      "affiliations": [
        "Akoya Biosciences, Marlborough, MA, USA"
      ],
      "name": "Matthew Moore"
    },
    {
      "affiliations": [
        "Akoya Biosciences, Marlborough, MA, USA"
      ],
      "name": "Ritu Mihani"
    },
    {
      "affiliations": [
        "Visiopharm Corp, Broomfield, CO, USA"
      ],
      "name": "Regan Baird"
    },
    {
      "affiliations": [
        "Visiopharm Corp, Broomfield, CO, USA"
      ],
      "name": "Katherine J Hales"
    }
  ],
  "title": "81 Spatial immunometabolic profiling of non-small cell lung cancer reveals distinct immune exclusion patterns and functional niches",
  "uid": "871d2394-9802-574c-983a-9cd4d934ce21"
}