{
  "abstract": "Background CMS4 colorectal cancer features an immunosuppressive tumour microenvironment rich in cancer-associated fibroblasts (CAFs) and TGF-β, contributing to immunotherapy resistance.NK cells serve as a promising candidate for cell-based immunotherapy due to natural cytotoxicity, antigen independence and no requirement for co-stimulation. However, there are still several barriers to NK cell-based therapies, including tumour-derived extracellular vesicles (T-EVs). T-EVs have been shown to alter NK cell phenotype, inhibit NK cell activation and cause NK cell exhaustion.1 2 The impact of T-EVs on NK cell immunity in stroma rich tumours is still widely unexplored.This study investigates the immunomodulatory ligand profile of CMS4 T-EVs and their impact on NK cells to determine whether targeting T-EV release is a viable strategy at restoring NK anti-tumour immunity.Methods EVs were isolated from CMS4 cancer cells (HCT116 and SW480) ±TGF-β1 and analysed for expression of cancer associated immunomodulatory ligands by spectral flow cytometry and super high-resolution microscopy.Healthy donor PBMC-derived NK cells were incubated with ±TGF-β1 SW480 or HCT116 T-EVs. NK phenotypic changes were tracked at early (0–4 h) and late (24–72 h) time points to capture temporal variation in receptor modulation. Cell phenotype was examined via flow cytometry.To investigate NK cytotoxicity, T-EV treated NK cells were incubated with SW480 cells or primary human colorectal tumour CAFs. Cell killing was monitored via live cell imaging.Results The addition of TGF-β1 induced higher particle production in CMS4 T-EVs consistently expressed Siglec-7/9/10 ligands, PD-L1, and CD24.T-EVs induced significant and varying phenotypic changes to NK cells depending on exposure time. After just 30 minutes, activation marker NKG2D was downregulated especially with TGF-β1 treatment, while inhibitory markers KIR2DL, Siglec 9 and 10 were upregulated. These changes were not static. Instead, receptor expression fluctuated over time, suggesting a complex and temporally dynamic NK cell response to T-EV exposure.After 72h of exposure, T-EVs especially from TGF-β1-treated cancer cells maintained a suppressive NK phenotype, significantly reducing NKG2D and DNAM-1 expression while upregulating KIR2DL, CD96 and Siglec 10, suggesting that CMS4 T-EVs, particularly those generated in the presence of TGF-β1, induce an evolving immunosuppressive phenotype in NK cells.Interestingly, T-EV treatment impacted NK cytotoxicity against CAFs but not SW480s.Conclusions My findings demonstrate that EVs are key mediators of the immunomodulatory effects of NK cells in the CMS4 tumour microenvironment. This underscores their role as critical drivers of cancer-mediated immune evasion and resistance to immunotherapy, positioning them as promising targets for future therapeutic intervention.References Azambuja JH, Ludwig N, Yerneni S, Rao A, Braganhol E, Whiteside TL. Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes. Neuro-Oncology Advances 2020;2(1). doi:10.1093/noajnl/vdaa056Zhang PF, Gao C, Huang XY, et al. Cancer cell-derived exosomal circUHRF1 induces natural killer cell exhaustion and may cause resistance to anti-PD1 therapy in hepatocellular carcinoma. Mol Cancer 2020 Jun 27;19(1):110. doi: 10.1186/s12943-020-01222-5Ethics Approval Ethical approval is in place from the Research Ethics Committee at the University of Galway. Informed consent has been obtained from all blood and tissue donors as part of this study.",
  "authors": [
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Anastasija Walsh"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Lei Lei"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Norashikin Zakaria"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Nial O’Reilly"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Sean Hynes"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Aisling Hogan"
    },
    {
      "affiliations": [
        "University Hospital Galway, Galway, Ireland"
      ],
      "name": "Margaret Sheehan"
    },
    {
      "affiliations": [
        "University Hospital Galway, Galway, Ireland"
      ],
      "name": "Aoife Canney"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Roisin M Dwyer"
    },
    {
      "affiliations": [
        "University of Galway, Galway, Ireland"
      ],
      "name": "Aideen E Ryan"
    }
  ],
  "title": "813 Exploring the immunomodulatory role of extracellular vesicles on NK cells in stroma dense colorectal tumours",
  "uid": "e8363dff-5222-5c82-b39d-976c57834423"
}