Abstract
Abstract Objective: Molecular degradation in cancer specimens may lead to misinterpretation of patients’ biological information. We have recently shown that utilization of the quantitative ratio between Tissue Degradation Indicators’ (TDIs’) intact form and their breakdown form(s) accurately reveals the stage of degradation in cancer specimens. We here propose to develop an enzymatic device built on one of the TDIs - alpha II spectrin (SPTAN1) - to monitor the degradation of cancer specimens, starting at the time of surgical resection. The device will allow specimen-users to assess the degradation stage of individual specimens prior to laboratory analyses. Approach: Proteolytic enzyme screening was conducted to identify the enzyme responsible for degradation-induced SPTAN1 breakdown. An in vitro enzymatic reaction was set up using the identified enzyme to catalyze overexpressed SPTAN1. The cleavage speed was monitored under various ambient temperatures at assigned time points. Correlation was studied between in vitro SPTAN1 breakdown and respective ambient conditions. A Fluorescence Resonance Energy Transfer (FRET)-based SPTAN1 peptide containing the proteolytic site was synthesized to replace the overexpressed SPTAN1 in above reaction. The enzymatic device was attached to experimental specimens and underwent assigned ambient exposures. SPTAN1 cleavage was measured by spectrophotometry. Correlation was studied among the SPTAN1 breakdown-induced fluorescence, the ambient impact, and the intrinsic TDI status. Further, FRET- SPTAN1 was injected into specimens in situ and the breakdown-induced fluorescent changes (catalyzed by local enzymes) were examined by fluorescence microscopy. Results: Calpain I was identified to be responsible for degradation-induced spectrin proteolysis. The in vitro enzymatic reaction can accurately reveal (following certain mathematic algorithm) the ambient temperature alteration and the exposure length via displaying a dynamic ratio between the breakdown and the intact SPTAN1. The introduction of FRET technology greatly facilitates the detection of spectrin breakdown reflected by breakdown-induced fluorescent signals. The FRET-device can properly reveal the ambient impact and the corresponding degradation inside the attached specimens. Local proteolytic enzymes in specimens are able to catalyze injected FRET- SPTAN1 and thereby the in situ approach imitates the in vitro enzymatic reaction and detects the ambient impact in specimens’ microenvironment. Conclusions: The prototype device yields a potentially transformative platform dedicated to specimen quality control. This foundational device may be attractive for commercialization and widely used throughout the basic and clinical cancer research communities. This effort also addresses critical, yet unmet needs for developing a universal standard for specimen degradation measurement. Citation Format: Jie Li, Catherine Kil, Jia Sun, Michael Stankewich, Alexander O. Vortmeyer. A prototype device for quality control of cancer specimens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 730. doi:10.1158/1538-7445.AM2013-730
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