Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array
Authors: Stewart Morgan , Quentel Arnaud , Mira Jose Montoya , Manalo-Hall Elise , Ranganathan Srivathsan , Branchaud Bruce , Civitci Fehmi , Tu Eugene , Fischer Jared , Chiu Yu-Jui , Yildirim Adem 
Published: March 2024

Abstract
Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers.
Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1021.