Enhancing cancer radiotherapy efficacy using NanOx, a novel oxygenating nanoemulsion that reverses tumour hypoxia.

Abstract

Radiotherapy is used to treat over 50% of cancer patients. It is often used in combination with surgery, chemotherapy, and immunotherapy, for cancers of the breast, lung, oesophagus, and rectum. Ionising radiation predominantly exerts its anti-cancer effect through both direct DNA damage and indirectly via water radiolysis and the production of reactive oxygen species. This DNA damage is made permanent in the presence of molecular oxygen; however, it is reversible under hypoxia. Therefore, hypoxia confers significant radiotherapy resistance and given that it is a common feature of most solid tumours it offers a unique tumour vulnerability to exploit to improve radiotherapy efficacy. Many efforts to increase radiotherapy efficacy by oxygen delivery have failed due to limited efficacy and toxicity. To address this, we have developed a biocompatible, oxygenating perfluorocarbon nanoemulsion (nPFC) with imaging capacity via microCT for locoregional delivery. We have demonstrated that this nPFC is biocompatible using an in vitro 3D liver hepatotoxicity model and in vivo using a developmental zebrafish embryo model, as well as in our efficacy model system, an isogenic model of acquired radioresistance in oesophageal adenocarcinoma (OAC). We have also shown that our nPFC can load and deliver a significant amount of molecular oxygen and reverse hypoxia regardless of prior oxygen loading. We have shown that this oxygen-loaded nPFC enhances cellular radiosensitivity under hypoxia using an established in vitro isogenic model of acquired radioresistance in OAC, in accordance with the oxygen enhancement effect. This effect was characterised by reduced clonogenicity, as well as increased 53BP1 foci persistence, cell cycle accumulation at G2/M, and apoptosis. Furthermore, this oxygen-loaded nPFC alters other features of radioresistance, including reduced oxidative phosphorylation (OXPHOS), alterations in the inflammatory secretome, and changes in DNA repair protein expression. We also tested the effects of our novel nPFC on ex vivo GI tumour explants under hypoxia. Tumour explants from upper and lower GI were included in this study, and the oxidative stress levels of these tumour explants were significantly correlated with the level of several inflammatory mediators. Oxygen-loaded nPFC treatment significantly reduced OXPHOS and significantly altered the secretome in the tumour-conditioned media of GI tumour explants differentially depending on combination with radiotherapy. Some of the key mediators altered include IFN-�, IL-12p70, IL-10, IL-13, IL-4, IL-2, TARC, and IP-10. The level of lactate dehydrogenase (LDH), an indirect measure of cell viability, was significantly increased following oxygen-loaded nPFC treatment alone, and interestingly this was increased to a greater extent than that of the combination treatment (oxygen-loaded nPFC and radiotherapy). In conclusion, we have developed a novel hypoxia-modifying agent that has significant oxygen loading and assimilation capacity and have demonstrated its radiosensitising effect in vitro using OAC cells with acquired radioresistance under hypoxia. Furthermore, we have assessed the impact of oxygen-loaded nPFC treatment on key features of radioresistance in ex vivo GI tumour explants. This drug may offer an alternative treatment approach to improve radiotherapy treatment efficacy in oesophageal adenocarcinoma patients who do not response to current treatment strategies.