FRET Sensor-Modified Synthetic Hydrogels for Real-Time Monitoring of Cell-Derived Matrix Metalloproteinase Activity using Fluorescence Lifetime Imaging

Matrix remodeling plays central roles in a range of physiological and pathological processes and is driven predominantly by the activity of matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) proteins. How MMPs regulate cell and tissue dynamics is not well understood as in vivo approaches are lacking and many in vitro strategies cannot provide high-resolution, quantitative measures of enzyme activity in situ within tissue-like 3D microenvironments. Here, a Forster resonance energy transfer (FRET) sensor of MMP activity is incorporated into fully synthetic hydrogels that mimic many properties of the native ECM. Fluorescence lifetime imaging is then used to provide a real-time, fluorophore concentration-independent quantification of MMP activity, establishing a highly accurate, readily adaptable platform for studying MMP dynamics in situ. MCF7 human breast cancer cells encapsulated within hydrogels are then used to detect MMP activity both locally, at the sub-micron level, and within the bulk hydrogel. This versatile platform may find use in a range of biological studies to explore questions in the dynamics of cancer metastasis, development, and tissue repair by providing high-resolution, quantitative, and in situ readouts of local MMP activity within native tissue-like environments. MMPs play central roles in development and disease, but how MMPs regulate tissue dynamics is poorly understood. Here, a FRET sensor of MMP activity is incorporated into synthetic hydrogels, which mimic native extracellular matrices. Fluorescence lifetime imaging is then used to provallows for real-time, high-resolution, quantitative, and in situ monitoring of MMP activity around encapsulated cells without the need for genetic modification. image