{
  "abstract": "Background NK cells are key players in anti-tumor response by their production of pro-inflammatory cytokines as well as their cytotoxic activity. In non-small cell lung cancer (NSCLC), several studies have shown that NK cells are scarce and dysfunctional. 1 2 Indeed, the tumor microenvironment (TME) reshapes NK-cell phenotype, reducing their cytotoxicity potential and promoting pro-tumoral behavior through the secretion of angiogenic factors and the expression of inhibitory immune checkpoints.3–6 Two main subsets of NK cells have been described in NSCLC: tissue-resident NK cells, characterized by the expression of residency markers such as CD69, CD49a and CD103, and circulating NK cells (CD56dim and CD56bright subsets) which lack these markers.5–9 However, little is known about their spatial distribution in the TME and how this may influence their functions and interactions with other components of TME.Methods To address these questions, this study integrates spatial transcriptomic [CosMx (Nanostring), Xenium Prime (10X Genomics)], high-plex immunofluorescence (IF) (PhenoCycler, Akoya), and in vitro assays modeling NK-TME interactions.Results Using publicly available spatial transcriptomic datasets from eight NSCLC samples (960-gene panel, CosMx) 10 we identified the presence of SELL+CCR7+GZMK+ NK cells in the vicinity of tertiary lymphoid structures (TLS) – lymphoid organ-like structures enriched in B cells, T cells and dendritic cells. The presence of NKp46+ NK Cells in proximity of TLS was also confirmed by IF (figure 1). The transcriptomic profile of NK cells associated with TLS niche resembled that of previously described circulating CD56bright NK cell subset, also found in secondary lymphoid organs where they are attracted and retained via CCR7 and SELL receptor expression.Furthermore, we observed a lower expression of tissue-resident transcriptomic signature in NK cells in the proximity of TLS compared to NK cells in the tumor margin or in the stroma. Moreover, ligands attracting and retaining tissue-resident NK cells were more abundant in the stromal and tumor margin niches. This supports the hypothesis that tissue-resident NK cells are likely excluded from TLS and instead attracted to the stroma and to the tumor interface.Spatial inference of communication networks between NK cells and the TME cells revealed both immuno-suppressive and immune-stimulating signaling, as well as signatures involved in cellular attraction and retention.Conclusions These results, along with ongoing in-depth phenotypic characterization of NK cell subsets by Xenium Prime spatial transcriptomics and high-plex immunofluorescence (Phenocycler), aim to elucidate how spatial context dictates NK cell function in the NSCLC tumor microenvironment.References Platonova S, Cherfils-Vicini J, Damotte D, Crozet L, Vieillard V, Validire P, André P, Dieu-Nosjean MC, Alifano M, Régnard JF, Fridman WH, Sautès-Fridman C, Cremer I. Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma. Cancer Res. 2011;71:5412-5422.Carrega P, Morandi B, Costa R, Frumento G, Forte G, Altavilla G, Ratto GB, Mingari MC, Moretta L, Ferlazzo G. Natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in CD56 bright CD16(-) cells and display an impaired capability to kill tumor cells. Cancer. 2008;112:863-875.Russick J, Joubert PE, Gillard-Bocquet M, Torset C, Meylan M, Petitprez F, Dragon-Durey MA, Marmier S, Varthaman A, Josseaume N, Germain C, Goc J, Dieu-Nosjean MC, Validire P, Fournel L, Zitvogel L, Bindea G, Lupo A, Damotte D, Alifano M, Cremer I. Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions. J Immunother Cancer 2020;8:e001054.Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neoplasia. 2013;15:133-142.Brownlie D, von Kries A, Valenzano G, Wild N, Yilmaz E, Säfholm J, Al-Ameri M, Alici E, Ljunggren HG, Schliemann I, Aricak O, Haglund de Flon F, Michaëlsson J, Marquardt N. Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors. Oncoimmunology 2023;12:2233402.Chen X, Chen Y, Xin Z, Lin M, Hao Z, Chen D, He T, Zhao L, Wu D, Wu P, Chai Y. Tissue-resident CD69+CXCR6+ Natural Killer cells with exhausted phenotype accumulate in human non-small cell lung cancer. Eur J Immunol. 2022;52:1993-2005.Brownlie D, Scharenberg M, Mold JE, Hård J, Kekäläinen E, Buggert M, Nguyen S, Wilson JN, Al-Ameri M, Ljunggren HG, Marquardt N, Michaëlsson J. Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood. Proc Natl Acad Sci U S A. 2021;118:e2016580118.Marquardt N, Kekäläinen E, Chen P, Kvedaraite E, Wilson JN, Ivarsson MA, Mjösberg J, Berglin L, Säfholm J, Manson ML, Adner M, Al-Ameri M, Bergman P, Orre AC, Svensson M, Dahlén B, Dahlén SE, Ljunggren HG, Michaëlsson J. Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69−CD56dim cells. J Allergy Clin Immunol. 2017;139:1321-1330.e4.Marquardt N, Kekäläinen E, Chen P, Lourda M, Wilson JN, Scharenberg M, Bergman P, Al-Ameri M, Hård J, Mold JE, Ljunggren HG, Michaëlsson J. Unique transcriptional and protein-expression signature in human lung tissue-resident NK cells. Nat Commun. 2019;10:3841.He S, Bhatt R, Brown C, Brown EA, Buhr DL, Chantranuvatana K, Danaher P, Dunaway D, Garrison RG, Geiss G, Gregory MT, Hoang ML, Khafizov R, Killingbeck EE, Kim D, Kim TK, Kim Y, Klock A, Korukonda M, Kutchma A, Lewis ZR, Liang Y, Nelson JS, Ong GT, Perillo EP, Phan JC, Phan-Everson T, Piazza E, Rane T, Reitz Z, Rhodes M, Rosenbloom A, Ross D, Sato H, Wardhani AW, Williams-Wietzikoski CA, Wu L, Beechem JM. High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging. Nat Biotechnol. Nature Publishing Group; 2022 Dec;40(12):1794–1806.Ethics Approval All the patients gave an informed consent prior to inclusion. The study was conducted with the agreement of the French ethic committee (number 2012 06 12 IRB00001072) in application with the article L. 1121-1 of French law, according to the recommendations in the Declaration of Helsinki.Abstract 806 Figure 1Demonstration of NK cell presence in the vicinity of Tertiary Lymphoid Structures (TLS) by immunofluorescence in non-small cell lung cancer tissue section (representative section). Formalin-fixed paraffin-embedded non-small cell lung cancer tumor sections were stained using Opal™-TSA technique. TLS are defined as aggregate of CD20+ B cells (yellow), NK cells are defined as NKp46+ cell (orange) and are highlighted by red arrows",
  "authors": [
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France",
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France"
      ],
      "name": "Rayan El-Kholdi"
    },
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France"
      ],
      "name": "Jing Liu"
    },
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France"
      ],
      "name": "Clément Levin"
    },
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France"
      ],
      "name": "Eric Parmantier"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France"
      ],
      "name": "Pierre-Alexis Da Costa"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France",
        "Departments of Pathology and Thoracic Surgery, Hospital Cochin Assistance Publique Hôpitaux de Paris, Paris, France"
      ],
      "name": "Audrey Mansuet-Lupo"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France",
        "Departments of Pathology and Thoracic Surgery, Hospital Cochin Assistance Publique Hôpitaux de Paris, Paris, France"
      ],
      "name": "Mathilde Prieto"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France",
        "Departments of Pathology and Thoracic Surgery, Hospital Cochin Assistance Publique Hôpitaux de Paris, Paris, France"
      ],
      "name": "Marco Alifano"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France",
        "Departments of Pathology and Thoracic Surgery, Hospital Cochin Assistance Publique Hôpitaux de Paris, Paris, France"
      ],
      "name": "Diane Damotte"
    },
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France"
      ],
      "name": "Maud Leonetti"
    },
    {
      "affiliations": [
        "Sanofi Research and Development, Sanofi, Vitry-sur-Seine, France"
      ],
      "name": "Marielle Chiron"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France"
      ],
      "name": "Isabelle Cremer"
    },
    {
      "affiliations": [
        "Centre de recherche des Cordeliers, INSERM U1138, Sorbonne Université, Université Paris Cité, Paris, France"
      ],
      "name": "Sophie Sibéril"
    }
  ],
  "title": "806 Unraveling the spatial distribution and interactions of NK cell subsets in the tumor microenvironment of non-small cell lung cancer using spatial transcriptomics",
  "uid": "a8fe31bc-b194-5bf0-a93b-6405f45afb35"
}