{
  "abstract": "Background Unrepaired DNA damage may lead to mis-segregation of chromosomes during cell division and formation of smaller immature nuclei called micronuclei. 1 The poorly formed nuclear envelope around these micronuclei exposes DNA to cytoplasmic sensors.1 2 DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), produces the second messenger cyclic GMP-AMP (cGAMP).1 cGAMP binds and activates the adaptor protein STING (stimulator of interferon genes), triggering a type-I interferon response, leading to innate immune cell-mediated clearance of the damaged cell.2 We hypothesize that radiation-induced genomic DNA damage, amplified by gold nanoparticles (GNP), leads to cGAS activation and stimulation of an inflammatory response.Methods We used our prototype GNP, cetuximab-conjugated gold nanospheres (GNSs), that enhances receptor-mediated internalization into colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR). We evaluated GNSs conjugated with polyethylene glycol (pGNS), control antibody IgG (iGNS), or cetuximab (cGNS) in two murine colorectal cancer cell lines overexpressing human EGFR, CT26-EGFR and MC38-EGFR. We confirmed the cellular uptake of the GNSs using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and dark-field microscopy. Clonogenic survival assay and immunofluorescence techniques assessed the radiosensitization ability. The effects on immune activation were evaluated by flow cytometry, immunoblotting, qRT-PCR, cytokine array, ELISA, and macrophage polarization assays.Results Firstly, the highest cellular uptake was observed with cGNS compared to controls, which decreased upon pre-treatment (receptor blockade) with cetuximab. Secondly, immunofluorescence staining for γH2Ax foci noted that irradiation of cGNS-treated cells resulted in persisting unrepaired DNA damage at early and late time points. Parallelly, we observed that the number of micronuclei was higher in cGNS-treated cells than controls. Further, cGAS-STING activation was more pronounced with cGNS and irradiation. Preliminary results from a cytokine array experiment suggest increased production of CXCL10 upon cGNS+radiation combination treatment, indicative of an increased inflammatory response. Lastly, the downstream consequence of type 1 interferon response induction was studied by treating RAW 264.7 cells with the conditioned media from cGNS+radiation treatments. This showed significantly higher M1/M2 macrophage phenotype ratio compared to the controls, consistent with induction of a pro-inflammatory anti-tumor phenotype.Conclusions These preliminary experiments suggest that increased radiosensitization with cGNSs is associated with increased DNA damage and increased induction of the cGAS-STING pathway to stimulate a robust innate immune response in vitro. C57Bl/6 mice bearing bilateral ovalbumin-expressing MC38 thigh tumors will be utilized to investigate the potential of immune activation and abscopal effect. This investigation advances our understanding of nanomaterial-biological interactions, revealing novel opportunities for therapeutic development.Acknowledgements The project is supported by the National Institutes of Health (NIH) R01 to SK (5R01CA257241-05).References Harding SM, Benci JL, Irianto J, Discher DE, Minn AJ, Greenberg RA. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature. 2017;548(7668):466–470.Mackenzie KJ, Carroll P, Martin CA, Murina O, Fluteau A, Simpson DJ, Olova N, Sutcliffe H, Rainger JK, Leitch A, Osborn RT, Wheeler AP, Nowotny M, Gilbert N, Chandra T, Reijns MAM, Jackson AP. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature. 2017;548(7668):461–465.",
  "authors": [
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA",
        "The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA"
      ],
      "name": "Bhoomika Muruvekere Lakshmisha"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA",
        "The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA"
      ],
      "name": "Ngoc Tuyet Tra"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA",
        "The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA"
      ],
      "name": "PM Quan Mai"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA"
      ],
      "name": "Ayobami Fidelix"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA"
      ],
      "name": "Prapannajeet Biswal"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA"
      ],
      "name": "Sai Kumar Samala"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA"
      ],
      "name": "Khadijeh Koushki"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA"
      ],
      "name": "Yuri Mackeyev"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA",
        "The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA"
      ],
      "name": "Geraldine Raja"
    },
    {
      "affiliations": [
        "The University of Texas Health Science Center at Houston, Houston, TX, USA",
        "The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA"
      ],
      "name": "Sunil Krishnan"
    }
  ],
  "title": "876 Investigating the immune effects of radiation dose enhancement using internalized gold nanoparticles",
  "uid": "c8cf0a80-222e-5be8-8c51-726a03216968"
}