title: Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.
abstract: Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.
authors.forename: Albert G
authors.initials: AG
authors.lastname: Remacle
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Shiryaev
authors.forename: Vladislav S
authors.initials: VS
authors.lastname: Golubkov
authors.forename: John N
authors.initials: JN
authors.lastname: Freskos
authors.forename: Michael A
authors.initials: MA
authors.lastname: Brown
authors.forename: Amolkumar S
authors.initials: AS
authors.lastname: Karwa
authors.forename: Arati D
authors.initials: AD
authors.lastname: Naik
authors.forename: Carol P
authors.initials: CP
authors.lastname: Howard
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Sympson
authors.forename: Alex Y
authors.initials: AY
authors.lastname: Strongin
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D061986:MCF-7 Cells; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D009154:Mutation; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D061848:Optical Imaging / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D011485:Protein Binding; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism
chemical_list: C417664:Alexa594; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D009930:Organic Chemicals; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C024098:6-carboxyfluorescein; D053511:Matrix Metalloproteinase 14
keywords: Breast Cancer; Cancer Biology; Drug Delivery; Enzyme Inhibitors; Hydroxamate Inhibitors; Imaging; Liposomes; MT1-MMP; Matrix Metalloproteinase (MMP); Protease Inhibitor