{
  "abstract": "Background The tumor microenvironment presents a major challenge to implementing effective immunotherapies because tumors can suppress immune cell functions. Adjuvants that initiate innate immune signaling offer a promising approach to modulate the tumor microenvironment and enhance antitumor immunity. Cyclic GMP-AMP synthase (cGAS) triggers an innate immune signaling cascade resulting in the induction of inflammatory genes upon cytosolic DNA detection. A constitutively active mutant of cGAS encoded as mRNA (cGASΔN) can be encapsulated in lipid nanoparticles (LNPs) and used to induce cytokines, chemokines, and costimulatory molecules which initiate both innate and adaptive immune responses. In vitro 2-D cultures fail to sufficiently model the complex tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) provide a more representative model for evaluating cancer immunotherapeutics. Whether cGASΔN-LNPs can modulate the tumor microenvironment modeled by PDOTS is unknown. This study investigates mRNA-LNP uptake, gene expression changes, and immune activation in endometrial cancer PDOTS using GFP-LNPs and cGASΔN-LNPs.Methods Tumors resected from endometrial cancer patients (n=5) were processed into PDOTS, loaded into microfluidic devices, and treated with GFP-LNPs (10 µg/ml), cGASΔN-LNPs (1 and 10 µg/ml), or αPD-L1 monoclonal antibody (300 µg/ml). GFP-LNP uptake was assessed on days 1 and 2 using fluorescence microscopy. Flow cytometry was used to identify cells internalizing and translating GFP-LNPs. Cytotoxicity was evaluated on day 2 using Hoechst/Propidium Iodide staining. Immune modulation was assessed on day 2 using Nanostring IO-360 gene expression profiling.Results GFP-LNP uptake and translation were observed in both tumor and immune cells, validating the compatibility of the PDOTS culture system and LNPs. Treating PDOTS with cGASΔN-LNP triggered robust interferon-alpha signaling in five PDOTS samples, unlike the GFP-LNP controls. In contrast, αPD-L1 antibody induced immune activation in only two of five samples. cGASΔN-LNP treatment was not cytotoxic within the 2-day treatment period. However, markers of CD8+ T cell and NK cell activation and cytotoxic function were upregulated. cGASΔN-LNPs also enhanced tumor necrosis factor signaling while downregulating tumor proliferation and kinase signaling pathways. Notably, cGASΔN-LNP converted one donor’s ‘cold’ tumor into a ‘hot’ phenotype, as evidenced by increased tumor inflammation signature (TIS) gene expression—an effect not seen with GFP-LNP or αPD-L1 treatment.Conclusions These findings highlight the rapid immunostimulatory effects of cGASΔN-LNP in patient-derived tumors and underscore the utility of the PDOTS platform in elucidating the mechanism of action of immunotherapeutics within the tumor microenvironment. Future studies will explore patient variability and the relationship between immune modulation and cytotoxicity.Ethics Approval This study was approved by the WCG Institutional Review Board (study 1280260). Patients gave informed consent at the clinical site to donate resected tumor tissue.",
  "authors": [
    {
      "affiliations": [
        "Corner Therapeutics, Watertown, MA, USA"
      ],
      "name": "Jonathan Chow"
    },
    {
      "affiliations": [
        "Harvard Medical School, Woburn, MA, USA"
      ],
      "name": "Aaron Goldman"
    },
    {
      "affiliations": [
        "Corner Therapeutics, Watertown, MA, USA"
      ],
      "name": "Emily Gosselin"
    },
    {
      "affiliations": [
        "Corner Therapeutics, Watertown, MA, USA"
      ],
      "name": "Catherine A Gormley"
    },
    {
      "affiliations": [
        "Xsphera Biosciences, Cambridge, MA, USA"
      ],
      "name": "Chunxiao Cui"
    },
    {
      "affiliations": [
        "Xsphera Biosciences, Cambridge, MA, USA"
      ],
      "name": "Anthony Attardo"
    },
    {
      "affiliations": [
        "Xsphera Biosciences, Cambridge, MA, USA"
      ],
      "name": "Alyssa Martin"
    },
    {
      "affiliations": [
        "Xsphera Biosciences, Cambridge, MA, USA"
      ],
      "name": "Michael A Perricone"
    },
    {
      "affiliations": [
        "Corner Therapeutics, Watertown, MA, USA"
      ],
      "name": "Jonathan Kagan"
    }
  ],
  "title": "942 An mRNA-encoded adjuvant delivered via lipid nanoparticles induces interferon signaling and converts immune cold tumors to hot in patient-derived organotypic tumor spheroids",
  "uid": "abf95ae5-e45e-5ac0-8a07-01a94754f94c"
}