Receptor-targeted gold nanospheres strengthen radiation efficacy and reprogram tumor immunity

Radiation therapy is an essential component of colorectal cancer management. However, it is limited by anatomical constraints, toxicity, and modest immune activation. When tumors are laden with high atomic number (Z) elements and exposed to ionizing radiation, a higher radiation dose is deposited within the tumor. This radiosensitization, achieved here using gold nanospheres (GNS), is boosted by a greater degree of free radical formation resulting in greater DNA damage with GNS + radiation. Recent evidence shows that unrepaired DNA damage can lead to chromosome missegregation and the formation of immature nuclei called micronuclei. The poorly formed nuclear envelope around these micronuclei exposes DNA to cytoplasmic sensors, triggering a type I interferon response and innate immune activation. Based on this, we hypothesized that GNS amplifies X-ray therapy and elicits unique immune responses. To improve receptor-mediated internalization into colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR), we employed our prototype, cetuximab-conjugated gold nanospheres (cGNS). Cetuximab (monoclonal antibody targeting EGFR) was attached to 30 nm gold nanospheres using thiol polyethylene glycol (PEG) for improved biocompatibility. Two murine colorectal cancer cell lines engineered to overexpress human EGFR, CT26-EGFR and MC38-EGFR, were used to test cGNS and a pegylated control (pGNS). Cellular uptake of the GNSs was shown by Inductively Coupled Plasma Mass Spectrometry and dark-field microscopy. Clonogenic survival assays assessed radiosensitization, while immune activation was examined using flow cytometry, immunoblotting, cytokine arrays, and macrophage polarization assays. Following radiosensitization, we observed upregulation of the cGAS-STING pathway and pro-inflammatory cytokines. Conditioned media applied to RAW 264.7 macrophages increased the M1/M2 macrophage ratio, indicating enhanced innate immune activation. We next used the CT26 syngeneic mouse model to assess GNS biodistribution, the extent of radiosensitization, immune cell infiltration, and potential abscopal effects. Efficacy studies demonstrated that cGNS outperformed pGNS in combination with radiation. Immune studies incorporating immune checkpoint inhibitors are currently underway. By integrating nanotechnology with radiation oncology and immunotherapy, this project lays the groundwork for advancing nanomaterial-based radio-immunotherapy toward clinical translation.