Protocol for tumor detection in mice using β-glucuronidase targeted breath analysis

Early tumor detection is critical for improving cure rates. Here, we present a protocol for tumor detection in mice using a targeted breath-analysis strategy in which phenyl-β-D-glucuronide specifically targets β-glucuronidase, which is highly expressed in the tumor microenvironment. We describe steps for drug screening, enzymatic reaction, drug stability and toxicity evaluation, and construction of the mouse exhaled-breath extraction device. We then detail procedures for animal-model breath analysis, enabling highly sensitive early tumor detection. For complete details on the use and execution of this protocol, please refer to Xue et al.¹.