Bimodal fluorogenic sensing of matrix proteolytic signatures in lung cancer.

Alicia Megia-Fernandez,Nikola Krstajić,Chesney Michels,Kevin Dhaliwal,Sunay V Chankeshwara,Bethany Mills,Chris Haslett,Mark Bradley

doi:10.1039/c8ob01790e

Abstract

Optical biosensing based on the activation of fluorescent reporters offers a powerful methodology for the real-time molecular interrogation of pathology. Here we report a first-in-class, bimodal fluorescent reporter strategy for the simultaneous and highly specific detection of two independent proteases (thrombin and matrix metalloproteases (MMPs)) pivotal in the fibroproliferative process surrounding lung cancer, based on a dual, multiplexing, peptide FRET system. This sophisticated synthetic smartprobe, with a molecular weight of 6 kDa, contains two independent fluorophores and quenchers that generate photonic signatures at two specific wavelengths upon activation by target enzymes within human lung cancer tissue.

Highlights

Lung cancer is the leading cause of cancer death in the world and most patients present with late stage disease due to a protracted diagnostic pathway.[1]
Biosensing of enzymatic activity has been at the focus of significant chemical and clinical advances,[6,7,8] with fluorescent probes reporting protease activity in vivo, including the cathepsins,[9,10,11] caspases,[12] enzymes of the coagulation cascade[13] and those responsible for matrix remodelling.[14]
Optical-imaging probes based on fluorescence offer the possibility of optical multiplexing but previous reports utilizing fluorescent probes for multi-analyte detection have principally focused on the sequential activation of a single fluorophore with enzymes working in series[16] or for the detection of metal ions and small molecules such as H2O2, NO or H2S,17,18 with the few reported examples of multienzymatic detection strategies relying on the turn-on of a single caged fluorophore.[19]

Summary

Introduction

Lung cancer is the leading cause of cancer death in the world and most patients present with late stage disease due to a protracted diagnostic pathway.[1]. Biosensing of enzymatic activity has been at the focus of significant chemical and clinical advances,[6,7,8] with fluorescent probes reporting protease activity in vivo, including the cathepsins,[9,10,11] caspases,[12] enzymes of the coagulation cascade[13] and those responsible for matrix remodelling.[14] Many of these probes are peptide-based, often exploiting the FRET (Förster Resonance Energy Transfer) phenomenon to detect enzymatic activity.[13,15] Optical-imaging probes based on fluorescence offer the possibility of optical multiplexing but previous reports utilizing fluorescent probes for multi-analyte detection have principally focused on the sequential activation of a single fluorophore with enzymes working in series[16] or for the detection of metal ions and small molecules such as H2O2, NO or H2S,17,18 with the few reported examples of multienzymatic detection strategies relying on the turn-on of a single caged fluorophore.[19]. Paper which may lead to dysregulation of the extracellular matrix (ECM) They have key roles in early carcinogenesis, in promoting early tumour–platelet interactions and have an association with pulmonary metastases. They are highly overexpressed in the malignant matrix and in fibrosis,[26,27,28] and as such are rational biomarkers for the molecular imaging of the matrix response surrounding cancer

Ethics statement

Results and discussion

Conclusions