Differentiating Aggressive from Nonaggressive Prostate Cancer Using Unconjugated Contrast Agents.

Prostate cancer is one of the most common malignancies among men globally. Early and accurate assessment of tumor aggressiveness is essential for guiding treatment decisions and improving patient outcomes. With growing clinical interest in combining optical, ultrasound, and photoacoustic imaging approaches for cancer detection, we systematically investigated the cellular uptake of three multimodal contrast agents─indocyanine green (ICG), ICG-loaded nanobubbles (ICG-NBs), and ICG-loaded microbubbles (ICG-MBs)─across prostate cancer cell lines with varying aggressiveness (PC-3M > PC-3 > DU-145 > LNCaP). Concentration- and time-dependent assays revealed that after 1 h of incubation, ICG-NBs exhibited 5-fold higher uptake (***p < 0.001) by highly metastatic PC-3M cells compared to the less aggressive and indolent cell lines. In contrast, ICG-MBs showed minimal uptake, detectable only after prolonged incubation (12 h) in PC-3M and PC-3 cells, while free ICG exhibited negligible uptake at 6 h, except in PC-3M cells, but increased over time in all cell lines. Validation across breast, colorectal, and pancreatic cancer cells further confirmed that ICG-NB uptake positively correlates with the metastatic potential of the cancer cell. Mechanistic studies identified macropinocytosis as the primary pathway for ICG-NB internalization, with additional contributions from clathrin-mediated endocytosis. These findings highlight that unconjugated ICG-NBs can selectively differentiate aggressive from indolent cancer phenotypes, offering a promising multimodal contrast agent for cancer theranostics.