Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors.

Molecular fluorescence imaging is effective for tumor diagnosis but limited by low-depth profiling, which can be addressed by photoacoustic (PA) imaging. However, PA imaging has low sensitivity due to microenvironment-induced effects on exogenous contrast agents. Accordingly, the temporal and biophysical determinants of PA contrast in Cetuximab-IRDye800 conjugates should be performed to complement fluorescence-based diagnostics. In this study, we compare the temporal dynamics of PA and fluorescence signals from a Cetuximab-IRDye800 conjugate in a tumor xenograft model. We demonstrate that while fluorescence signal increases steadily over time after administration of Cetuximab-IRDye800, PA signal peaks early (~75% higher at 3 hours), followed by a decrease (~24% higher at 24 hours). Mechanistic analysis revealed formation of H-aggregates with Cetuximab-IRDye800 conjugation, which results in enhanced PA contrast, while receptor-mediated endocytosis disrupts these aggregates, reducing PA signal intensity over time. These findings underscore the complementary nature of PA and fluorescence imaging and emphasize timing as a critical factor for capturing peak PA contrast for tumor diagnostics.