A "turn-off" photoacoustic contrast for urokinase-type plasminogen activator activity

The ability to distinguish cancerous lesions based on aggressiveness using noninvasive molecular imaging techniques enables more precise and accurate diagnosis. In colorectal cancer screening, current approaches such as blood or fecal tests and endoscopic examination are widely used. However, reliably differentiating malignant adenomatous lesions from benign lesions, particularly those ≤5 mm in size, remains a significant clinical challenge. We synthesized, optimized, and validated a small-molecule near-infrared (NIR) activated photoacoustic dye conjugated to a tripeptide substrate specific for urokinase plasminogen activator (uPA), a protease that is upregulated in colorectal cancer tissues. The probe was designed to produce an "ON-OFF" photoacoustic signal upon activation by uPA. Specificity of the probe towards aggressiveness was evaluated using two colorectal cancer cell lines with differential uPA and cathepsin B expression. The uPA-responsive photoacoustic probe demonstrated high sensitivity and a clear "ON-OFF" activation signal in response to uPA activity. It showed strong specificity between colorectal cancer cell lines with different levels of uPA expression, confirming its selective activation. Noninvasive monitoring of extracellular uPA activity using photoacoustic imaging shows promise as a predictive screening approach for distinguishing malignant and premalignant colorectal lesions, particularly those that are small and difficult to classify using current screening methods.