Radiation and Gold Nanoparticle Increase the Expression of Immunogenic Cell Death Markers in MDA MB 231 Breast Cancer Model

Abstract

Tumor immunogenic cell death (ICD) plays a major role in stimulating host anti-cancer immune response that can determine the success of cancer radiotherapy (RT). Additionally, ICD can also be induced by radiation itself. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Two breast cancer (BCa) DAMPs involved in phagocytosis and dendritic cell antigen presentation are Calreticulin (CLR) and HMGB1; loss of HMGB1 expression is linked to poor clinical prognosis. Therefore, these molecules are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BCa therapies, the effect of RT and radiosensitizers, such as gold nanoparticles (AuNPs), on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CLR and HMGB-1 expression in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BCa in vitro and in vivo models. We hypothesized that AuNP modulates radiation induced ICD by altering CLR and HMGB1 expression. In vitro: MDA MB 231 cells were incubated for 3h with 14nm AuNPs and irradiated. After 48h and 72h the percentages of CLR and HMGB1 positive cells were determined by flow cytometry. In vivo: Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CLR antibodies and analyzed by light microscopy. In vitro, radiation induced increase in the percentages of CLR and HMGB1 positive cells was potentiated by AuNP at 48h and 72h time points. In control cells, AuNP alone increased the percent of HMGB1 positive cells at both time points. The percent of CLR positive cells was increased at 72h only. In vivo, in animals receiving RT or AuNP only, CLR expression was increased relative to control animals and CLR was detected extracellularly after RT, and extra- and intracellularly after AuNP alone. However, after combined RT and AuNP, CLR expression was higher than after single treatments and was detected both, extra- and intracellularly. In BCa patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single irradiation dose in BCa cells and mouse model. This effect was measured by an increase in the expression of CLR and HMGB1, DAMPs critical to the response of BCa. In addition, AuNP alone exhibited an immunogenic effect similar to RT alone. These findings support that BCa depends on ICD and provide a platform for designing multimodal BCa RT formulations with novel radiosensitizers or immunotherapy.