{
  "abstract": "Background Heterogeneous tumors exhibit impaired immune responses 1–3 and inferior responses to immune checkpoint inhibitors.1 4 5 To understand the mechanisms by which heterogenous tumors evade immune control, our lab previously established a novel model of tumor heterogeneity in which we implant mice with tumors comprised of multiple fluorescently labeled tumor populations.6 Using the fluorescent tags to track the spatial locations of specific tumor cell populations, we have demonstrated that tumor cells profoundly impact the organization of the immune microenvironment in their spatial vicinity. Building on this work, we here investigate the mechanisms tumor cells use to shape their local tumor microenvironment.Methods We implanted mice with heterogeneous tumors comprised of 1:1 mixes of fluorescently tagged tumor cells, using two ‘immune hot’ squamous cell skin carcinoma tumor lines (CIT18 and CIT6, tagged RFP+ and YFP+), or with homogeneous tumors comprised of a single population. Tumors were manually dissected into RFP+ and YFP+ regions. T cells in each region were analyzed by flow cytometry or by single cell RNAseq + TCRseq of sorted T cells. Chemokines and cytokines in each region were assayed by a 64-plex ELISA-based array. To complement these approaches, immune cells in each RFP+ and YFP+ regions were analyzed by immunofluorescence microscopy across multiple timepoints.Results We find that the chemokine microenvironment within a heterogeneous tumor is dictated by the local tumor cells. Specifically, in mixed CIT6:CIT18 tumors, spatial regions occupied by CIT18 tumor cells had a chemokine milieu characterized by high levels of pro-inflammatory chemokines CXCL10, CCL5, CXCL11, CCL17 and CCL22, matching homogeneous CIT18 tumors. By contrast, regions of the same heterogeneous tumors occupied by CIT6 tumors lacked this signature, mirroring homogeneous CIT6 tumors. The inflammatory chemokine-rich tumor regions exhibited elevated numbers of CD4+ T cells, whose abundance was significantly correlated with elevated effector function (IFNγ+) of CD8+ T cells. Both T cell metrics were diminished in regions lacking the inflammatory chemokine signature. Using single-cell TCRseq analysis, we additionally mapped the distribution of expanded T cell clonotypes across multiple spatial regions of a single tumor.Conclusions We demonstrate that within heterogeneous tumors, local tumor cells dictate the cytokine and chemokine milieu within their spatial vicinity. This provides a mechanistic underpinning explaining the ability of tumor cells to shape their immune microenvironment on a localized level. Further, we link regions of diminished CD4+ infiltration and effector CD8+ T cell function to spatial regions where tumor cells enforce decreased levels of inflammatory chemokines.References Wolf Y, Bartok O, Patkar S, Eli GB, Cohen S, Litchfield K, et al. UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma. Cell. 2019;179:219-235.e21.Westcott PMK, Muyas F, Hauck H, Smith OC, Sacks NJ, Ely ZA, et al. Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity. Nat Genet. 2023;55:1686–95.Nguyen KB, Roerden M, Copeland CJ, Backlund CM, Klop-Packel NG, Remba T, et al. Decoupled neoantigen cross-presentation by dendritic cells limits anti-tumor immunity against tumors with heterogeneous neoantigen expression. eLife. 2023;12:e85263.McGranahan N, Furness AJS, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science. 2016;351:1463–9.Liu D, Schilling B, Liu D, Sucker A, Livingstone E, Jerby-Arnon L, et al. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat Med. 2019;25:1916–27.Tanaka M, Lum L, Hu KH, Chaudhary P, Hughes S, Ledezma-Soto C, et al. Tumor cell heterogeneity drives spatial organization of the intratumoral immune response. J Exp Med. 2025;222:e20242282.Ethics Approval All animal experiments were approved by the by the University of Utah Office of Comparative Medicine (IACUC #22-02003).",
  "authors": [
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Robert Letchworth"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Savannah Hughes"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Abigail Keku"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Emilio Cortes-Sanchez"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Piyush Chaudhary"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Joshua KH Tay"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Matthew Lieberman"
    },
    {
      "affiliations": [
        "Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA"
      ],
      "name": "Melissa Q Reeves"
    }
  ],
  "title": "742 Tumor cells drive spatial organization of the local chemokine milieu within heterogeneous tumors",
  "uid": "1180f2e7-6e73-5dd3-a338-2572355c5f50"
}