Investigating the Immune Effects of Radiation Dose Enhancement Using Internalized Gold Nanoparticles

Bhoomika Muruvekere Lakshmisha, BE (Advisor: Sunil Krishnan, MD)

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 radiation dose enhancement, achieved here using gold nanospheres (GNS), is boosted by a greater degree of free radical formation, resulting in greater DNA damage with GNS + radiation compared to radiation alone. Recent evidence shows that unrepaired DNA damage can lead to chromosome mis-segregation 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 the radiation dose amplification by GNS may also lead to enhanced immune responses. To test this hypothesis, we used GNS that are internalized by colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR) via receptor-mediated internalization. Our prototype construct, cetuximab-conjugated gold nanospheres (cGNS), conjugates cetuximab (a monoclonal antibody targeting EGFR) to 30 nm gold nanospheres via thiolated polyethylene glycol (PEG) to improve biocompatibility. Two murine colorectal cancer cell lines engineered to overexpress human EGFR, CT26-hEGFR and MC38-hEGFR, were used to test cGNS, a pegylated control (pGNS), and an IgG-conjugated control (iGNS). Cellular uptake of the GNSs was measured by Inductively Coupled Plasma Mass Spectrometry and visualized by dark-field microscopy. Clonogenic survival assays assessed radiosensitization, while immune activation was examined using RT-qPCR, immunoblotting, ELISA, cytokine array, and macrophage polarization assays. Following radiation dose enhancement, we observed upregulation of type-1 interferon gene expression and pro-inflammatory cytokines. Conditioned media applied to RAW 264.7 macrophages increased the M1/M2 macrophage ratio, indicating enhanced innate immune activation. In vivo studies assessing GNS biodistribution, efficacy, and systemic immune activation 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.

Advisory Committee:

• Sunil Krishnan, MD, Chair
• Kendra Carmon, PhD
• Sang Hyun Cho, PhD
• Xian Chang Li, MD, PhD
• Deepa Sampath, PhD