Abstract 4939: Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo
Authors: Peterson Jeffrey D. , Ho Guojie , Morin Jeffrey , Delaney Jeannine , Yared Wael , Rajopadhye Milind , Kossodo Sylvie C. 
Published: May 2015

Abstract
Prostate-Specific Antigen (PSA) is a 237 amino acid, 33 kDa, extracellular serine protease expressed in prostate epithelial tissue. In the normal prostate, high concentrations of PSA are stored in the prostatic ductal network. However, cancer leads to the disruption of normal tissue architecture and subsequent leakage of PSA into the tissue interstitium and circulation. Enzymatically active PSA is only present in prostate tissue and at other sites of prostate cancer growth. In circulation, active PSA immediately forms complexes with the serum protease inhibitor alpha-1-antichymotrypsin (ACT), while the inactive forms remains “free”. Traditional assays relied on testing for the presence of total PSA (mostly inactive and complexed) and do not provide information regarding the amount of enzymatically active PSA, a more biologically relevant tumor biomarker. To fill this gap, we developed a novel near infrared (NIR) fluorescent agent, designed to detect active PSA with no detection of inactive or complexed PSA. This agent, PSA750 FAST, contains a PSA-cleavable peptide sequence labeled with NIR fluorophores (ex/em 750/770 nm) and coupled to a pharmacokinetic modifier designed to improve its plasma availability. In its native state, the agent is nearly completely optically quenched (&gt; 95%); and only becomes fluorescent upon enzymatic cleavage with active PSA, yielding a 300-1800 fold increase in signal as compared to the signal obtained using inactive or complexed PSA. Proteolytic cleavage was also selective for PSA over a large panel of enzymes, including Kallikrein 1, Cathepsin B, MMP-9, MMP-12, MMP-13, uPA, chymotrypsin and thrombin. We hypothesized that the enzymatic activity of PSA could be non-invasively and quantitatively monitored in 3D using Fluorescence Molecular Tomography (FMT), a powerful near-infrared imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals. LNCaP (PSA positive) and PC3 (PSA negative) cells were implanted in the chest area of male nude mice. Real time imaging was performed after systemic administration of PSA750 FAST when tumors reached the desired size. Our results demonstrated a significantly higher fluorescent signal in LNCaP tumors as compared the adjoining muscle (34.1 +/- 3.8 nM versus 7.2 +/- 0.6 nM, p=0.0043). Likewise, fluorescence concentration in tumors was significantly higher in LNCaP as compared to PC3 tumors (34.1 +/- 3.8 nM vs 17.14 +/- 3.48 nM, p=0.0191; 12.99 +/- 3.7 vs 2.8 +/- 0.8 pmoles, p=0.0233). This is the first report that demonstrates the feasibility of non-invasive, real-time, molecular imaging of active PSA in vivo in a relevant tumor model. These findings demonstrate the effectiveness of this agent in conjunction with optical imaging as a functional platform for molecular imaging in the field of prostate cancer.
Citation Format: Jeffrey D. Peterson, Guojie Ho, Jeffrey Morin, Jeannine Delaney, Wael Yared, Milind Rajopadhye, Sylvie C. Kossodo. Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2014-4939