In vivo assessment of protease activity in colorectal cancer by using activatable molecular photoacoustic imaging

Matrix metalloproteinases (MMPs), a family of zinc-dependent proteases, have been recognized as a significant biomarker associated with tumor microenvironment. MMPs has been shown to enhance the expression of pro-angiogenic growth factors, such as VEGF and bFGF in colorectal cancer. A non-invasive imaging method that could enable MMPs assessment in vivo would enhance the understanding of the MMPs activity in tumor micro-environments. In this report, photoacoustic (PA) imaging was used for non-invasive assessment of the MMPs activity in vivo, employing activatable gold nanorods (GNRs) as contrast agent. GNRs and dye IRDye800, which exhibit strong photoacoustic signals at the wavelength of 670 nm and 800 nm, respectively, were conjugated via a MMPs specific cleavable peptide according to standard synthesis protocol. The photoacoustic signal intensity of the GNRs-peptide-IRDye800 compound, GNRs, and IRDye800 were tested individually with human fibrosarcoma HT1080 cells (positively expressing MMPs) and human mammary adenocarcinoma BT20 cells (negatively expressing MMPs) using a PA system (Vevo 2100 LAZR, VisualSonics Inc.) at 670 nm and 800 nm. During in vivo experiments, a subcutaneous COLO 201 xenograft nude mice model was used. The mice (n = 16) were divided into two groups according to tumor size (Group A & B, 8 mice each), small (50-150 mm<;sup>3<;/sup>), and medium (151-250 mm<;sup>3<;/sup>). Each group of mice were intravascularly injected with contrast agent (10 nM, 100 μl) and imaged at different time points (1 h, 3h, 6 h and 24 h) post injection. Then, the PA intensity ratios between 670 nm and 800 nm were calculated. Finally, correlation test was conducted between in vivo PA intensity ratios and ex vivo MMPs immuno-histochemical results. The cell experiments showed that the GNRs-peptide-IRDye800 exhibited strong photoacoustic intensity at the wavelengths of 670 nm and 800 nm before enzyme cleavage by MMPs, owing to the absorption of GNRs and IRDye800, respectively. The statistical analysis showed that the PA intensity ratio was highly correlated with ex vivo immune-histochemical results. After the MMPs expressed in tumor cells, GNRs-peptide-IRDye800 released free IRDye800 which were rapidly washed out from tumor cells as small molecules, leaving GNRs retaining inside the tumor cells for much longer period of time. Thus, the recorded PA signal ratios at wavelengths 800 nm over 670 nm, were remarkably decreased accordingly. The result indicates that photoacoutic imaging with the aid of activatable contrast agent is potentially valuable for non-invasive assessment of protease activity in colorectal cancer in vivo.