Quantitative FRET Imaging to Visualize the Invasiveness of Live Breast Cancer Cells

Shaoying Lu,Yijia Pan,He Huang,Eric J Chaney,Yi Wang,Stephen A Boppart,Howard Ozer,Yingxiao Wang,Alex Y Strongin,Fan Yuan

doi:10.1371/journal.pone.0058569

Abstract

Matrix metalloproteinases (MMPs) remodel tumor microenvironment and promote cancer metastasis. Among the MMP family proteases, the proteolytic activity of the pro-tumorigenic and pro-metastatic membrane-type 1 (MT1)-MMP constitutes a promising and targetable biomarker of aggressive cancer tumors. In this study, we systematically developed and characterized several highly sensitive and specific biosensors based on fluorescence resonant energy transfer (FRET), for visualizing MT1-MMP activity in live cells. The sensitivity of the AHLR-MT1-MMP biosensor was the highest and five times that of a reported version. Hence, the AHLR biosensor was employed to quantitatively profile the MT1-MMP activity in multiple breast cancer cell lines, and to visualize the spatiotemporal MT1-MMP activity simultaneously with the underlying collagen matrix at the single cell level. We detected a significantly higher level of MT1-MMP activity in invasive cancer cells than those in benign or non-invasive cells. Our results further show that the high MT1-MMP activity was stimulated by the adhesion of invasive cancer cells onto the extracellular matrix, which is precisely correlated with the cell’s ability to degrade the collagen matrix. Thus, we systematically optimized a FRET-based biosensor, which provides a powerful tool to detect the pro-invasive MT1-MMP activity at single cell levels. This readout can be applied to profile the invasiveness of single cells from clinical samples, and to serve as an indicator for screening anti-cancer inhibitors.

Highlights

Matrix metalloproteinases (MMPs) are a family of zincdependent proteases, which play important roles in tumor metastasis, inflammation, and development [1,2]
When the purified biosensors were co-incubated with the catalytic domain of membrane-type 1 (MT1)-MMP (MT1-CAT) to allow cleavage, the enhanced cyan fluorescence protein (ECFP)/fluorescence resonant energy transfer (FRET) ratio of the biosensor with the AHLR sequence flanked by the GGSGGT linker increased 7-fold, with a response 38% and 109% higher than the biosensor with the GGS linker[30] and that without the linker (AHLR sequence alone), respectively (Fig. 1A)
The RHLR biosensor with the GGSGGT linker was superior when compared to the RHLR biosensors with the GGS linker or without the linker (RHLR sequence alone) (Fig. 1B). These results indicate that the flexible linkers GGSGGT can improve the sensitivity of membrane type-1 MMP (MT1-MMP) biosensors, possibly by allowing the active MT1-CAT more space to access and cleave the substrate sequence

Summary

Introduction

Matrix metalloproteinases (MMPs) are a family of zincdependent proteases, which play important roles in tumor metastasis, inflammation, and development [1,2]. It is essential to investigate specific roles played by individual MMPs at the cellular level, as well as to evaluate the effectiveness of specific MMP inhibitors, for the purpose of developing anti-MMP cancer therapy. Among the MMP family proteases, membrane type-1 MMP (MT1-MMP; called MMP-14) is essential for the peri-cellular proteolysis of collagen matrix in the basement membrane, as well as tumor cell invasion and metastasis [7,8,9,10,11]. Furin-like convertases and MMPdependent proteolysis can cause the removal of the prodomain and the subsequent activation of MT1-MMP [15,16,17]. The activated MT1-MMP can localize at the cellular surface, internalized via the clathrin-dependent pathway [18], and recycled back to the plasma membrane via exocytosis [19]

Methods

Results

Conclusion