Oxygen‐sensitive MRI has been extensively used to investigate tumor oxygenation based on the response (R₂* and/or R₁) to a gas breathing challenge. Most studies have reported response to hyperoxic gas indicating potential biomarkers of hypoxia. Few studies have examined hypoxic gas breathing and we have now evaluated acute dynamic changes in rat breast tumors.

Rats bearing syngeneic subcutaneous (n = 15) or orthotopic (n = 7) 13762NF breast tumors were exposed to a 16% O₂ gas breathing challenge and monitored using blood oxygen level dependent (BOLD) R₂* and tissue oxygen level dependent (TOLD) T₁‐weighted measurements at 4.7 T. As a control, we used a traditional hyperoxic gas breathing challenge with 100% O₂ on a subset of the subcutaneous tumor bearing rats (n = 6). Tumor subregions identified as responsive on the basis of R₂* dynamics coincided with the viable tumor area as judged by subsequent H&E staining. As expected, R₂* decreased and T₁‐weighted signal increased in response to 100% O₂ breathing challenge. Meanwhile, 16% O₂ breathing elicited an increase in R₂*, but divergent response (increase or decrease) in T₁‐weighted signal. The T₁‐weighted signal increase may signify a dominating BOLD effect triggered by 16% O₂ in the relatively more hypoxic tumors, whereby the influence of increased paramagnetic deoxyhemoglobin outweighs decreased pO₂. The results emphasize the importance of combined BOLD and TOLD measurements for the correct interpretation of tumor oxygenation properties.