Optical Imaging of Matrix Metalloproteinase-7 Activity in Vivo Using a Proteolytic Nanobeacon

Abstract

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor) and AF750 as an internal reference to monitor relative substrate concentration (reference). In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APC Min) mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APC Min mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APC Min adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

Imaging matrix metalloproteinases in cancer

“…Using a simple gamma camera SPECT is capable of measuring the biodistribution of small concentrations of biomolecules in vivo and quantifying the molecular kinetic processes. SPECT also offers 3-D reconstruction similar to other tomographic techniques, but its unique ability to [21] for details image relatively slow kinetic processes (due to the long half-life of the commonly used isotopes) sets this modality apart from PET.…”

“…The PB demonstrated differential response between MMP7 positive vs. MMP7 negative xenograft tumors. Recently, we have developed a similar construct using NIR FRET pairs to reduce absorption and scattering of the fluorescent signal improving response of the proteolytic beacon [21]. The selectivity for MMP7 and enhanced sensitivity of the NIR-PB was assessed by ex-vivo imaging of benign tumors in the intestines of adenomatous polyposis coli multiple intestinal neoplasia (APC Min ) mice injected systemically with the PB; the images revealed a significant increase in Sensor/Reference ratios in the adenomas of APC Min mice compared to control intestinal tissue or adenomas from MMP7-null Min mice (see Fig.…”

Intravital microscopy: new insights into metastasis of tumors

“…The injectable probes that have been used to measure protease activity in tumors include autoquenched protease substrates that fluoresce upon cleavage (Weissleder et al, 1999), proteolytic beacons that lose FRET upon cleavage (e.g. McIntyre et al, 2010;Scherer et al, 2008) and fluorescently labeled activatable cell-penetrating peptides (ACCPs) that translocate into cells upon cleavage (Olson et al, 2009;Olson et al, 2010). The disadvantage of these probes is that chemical knowledge is required to synthesize them, although recent commercialization of some of these probes has overcome this problem.…”

Oral Protein Therapy for the Future – Transport of Glycolipid-Modified Proteins: Vision or Fiction

“…The overproduction of enzymes, such as the matrix metalloproteinases, is also common for most proliferating malignant cells, since they are essential for angiogenesis, metastasis, and other extracellular signalling events involved in tumour propagation [59] . Overexpression of metalloproteinases has been explored as a way of targeting and 'turning on' of imaging agents for the allocation of tumours or other lesions [62][63][64] .…”