Investigation of hypoxia/reoxygenation in spheroids following radiation and SN30000 treatment using a novel click chemistry based hypoxia probe

Abstract

Hypoxia is a significant issue in cancer therapy due to the resistance of hypoxic cells to radiation, therefore supporting the use of combination treatment to achieve greater tumour control by targeting these radioresistant cell populations. SN30000, is one of many hypoxia activated prodrugs developed to eliminate hypoxic cells in tumours, leading to spatial complementarity with radiation. Multicellular tumour spheroids are increasingly used in cancer pre-clinical pharmacology because they mimic tumour environments, including hypoxic gradients, and appear ideal for studying spatial complementarity. However, interpretation of results is often difficult. To address this, we have developed an agent based computational model of spheroid growth in response to drug and radiation therapy. Our model and others predict rapid reoxygenation of spheroids following radiation or combined radiation/SN30000 treatment. SN33267, a novel 2-nitroimidazole hypoxia marker based on click chemistry, developed at the ACSRC, does not require antibody conjugated fluorophores, thus enabling analysis of whole spheroids by confocal microscopy or widefield fluorescence imaging. However, comparison to histological section has highlighted the major obstacle of light penetration in thicker specimens in confocal microscopy. Methods were developed to optimize histological processing of spheroids to produce a reference set of images of hypoxia using EF5 and SN33267. Determination of the refractive index of spheroids with optical coherence tomography enabled selection of ScaleViewA2 as an immersion medium better matched to spheroids, thus achieving greater depths of imaging. Confocal and widefield microscopy were then used to investigate the effect of radiation and SN30000 in spheroids by imaging hypoxia using SN33267. Both modalities produced similar trends, demonstrating a clear delay in growth, with only a small decrease in hypoxia relative to spheroid size. This disagreed with the ABMs that assume cells apoptose rapidly or undergo reduced metabolic capacity after receiving lethal doses of radiation. This represents a failure of the model to adequately simulate radiation induced cell cycle growth delay. This study confirmed the utility of SN33267 to investigate hypoxia in whole spheroids. Further investigation of hypoxia/reoxygenation in spheroids is necessary to improve the ABM, and has relevance for assumptions about hypoxia/reoxygenation in dose scheduling experiments investigating fractionated radiation therapy in spheroids.