Mapping tumor heterogeneity and hypoxia within a living intact organism is essential for understanding the processes involved in cancer progression and assessing long-term responses to therapies. Efficient investigations into tumor hypoxia mechanisms have been hindered by the lack of intravital imaging tools capable of multi-parametric probing of entire solid tumors with high spatial and temporal resolution. Here we exploit volumetric multi-spectral optoacoustic tomography (vMSOT) for accurate, label-free delineation of tumor heterogeneity and dynamic oxygenation behavior. Mice bearing orthotopic MDA-MB-231 breast cancer xenografts were imaged non-invasively during rest and oxygen stress challenge, attaining time-lapse 3D oxygenation maps across entire tumors with 100µm spatial resolution. Volumetric quantification of the hypoxic fraction rendered values of 3.9-21.2% whereas the oxygen saturation (sO2) rate declined at 1.7-2.3% per mm in all tumors when approaching their core. Three distinct functional areas (the rim, hypoxic, and normoxic cores) were clearly discernible based on spatial sO2 profiles and responses to oxygen challenge. Notably, while sO2 readings were responsive to the challenge, deoxyhemoglobin (HbR) trends exhibited little to no variations in all mice. Dynamic analysis further revealed the presence of cyclic hypoxia patterns with a 21% average discrepancy between cyclic fractions analyzed by sO2 (42.2±17.3%) and HbR fluctuations (63±14.1%) observed in the hypoxic core. These findings corroborate the strong potential of multi-spectral optoacoustic tomography for advancing pre-clinical imaging of cancer and informing clinical decisions on therapeutic interventions.

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