Purpose Accurate evaluation of hypoxia is particularly important in patients with nasopharyngeal carcinoma (NPC) undergoing radiotherapy, yet no clinically accepted methodology for imaging hypoxia is available. The aim of this study was to develop a novel imaging strategy for quantitative three-dimensional (3D) evaluation of hypoxia in a small animal model of NPC.

Methods A carbonic anhydrase IX (CAIX)-specific molecular probe (CAIX-800) was developed for imaging of hypoxia. Mouse models of subcutaneous, orthotopic, and spontaneous lymph node metastasis from NPC (5 mice per group) were established to assess the imaging strategy. A multi-modality imaging method that consisted of a hybrid combination of fluorescence molecular tomography-computed tomography (FMT-CT) and multispectral optoacoustic tomography (MSOT) was used for 3D quantitative evaluation of tumour hypoxia. Magnetic resonance imaging, histological examination, and immunohistochemical analysis were used as references for comparison and validation.

Results In the early stage of NPC (2 weeks after implantation), FMT-CT enabled precise 3D localization of the hypoxia biomarker with high contrast. At the advanced stage (6 weeks after implantation), MSOT allowed multispectral analysis of the biomarker and haemoglobin molecules with high resolution. The combination of high sensitivity and high resolution from FMT-CT and MSOT could not only detect hypoxia in small-sized NPCs but also visualise the heterogeneity of hypoxia in 3D.

Conclusions Integration of FMT-CT and MSOT could allow comprehensive and quantifiable evaluation of hypoxia in NPC. These findings may potentially benefit patients with NPC undergoing radiotherapy in the future.