Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

Abstract

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

Figure 1

A) Solid-phase synthesis of LS276-THP (“O” represents 4-hydroxy-l-proline). B) Chemical structure of LS276.

Figure 2

Fluorescence spectra of LS276 (solid) and LS276-THP (dashed), where λ_excitation = 780 nm and λ_emission = 760-850 nm.

Figure 3

A) Absorbance of LS276-THP with varying temperature. B) Thermal transition curve for LS276-THP.

Figure 4

Proteolysis of increasing concentrations of LS276-THP by MMP-2. Lineweaver-Burk analysis is provided in the inset. Error bars indicating standard deviation are too narrow to be visualized on this plot.

Figure 5

Representative in vivo whole-body images of mice bearing HT1080 tumor xenografts 24 h after injection of (A) LS276-THP; n=4, (B) LS276-THP and inhibitor; n=3, or (C) MMPSense™ 680; n=3. Tumors (arrows) and kidney (K) regions are marked. (D) The ratio of tumor and contralateral thigh ROI fluorescence with respect to time show the time dependent activation of the molecular probes. (E) Ex vivo fluorescence biodistribution confirmed the high fluorescence in the non-inhibited tumors and the high retention of LS276-THP in the mouse kidneys relative to the larger MMPSense™ 680. Error bars represent standard deviation; au = arbitrary units.

Figure 6

Immunohistochemistry for MMP-2 (A), MMP-9 (B), and secondary antibody control (C) with hematoxylin counterstain in HT1080 xenograft cryosections. (D) Fluorescence imaging of HT1080 tumor xenograft 24 h after injection of the LS276-THP probe.