Functional live-cell imaging demonstrates that β1-integrin promotes type IV collagen degradation by breast and prostate cancer cells

Department of Pharmacology and Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.
Molecular Imaging (Impact Factor: 1.96). 11/2007; 7(5):199-213.
Source: PubMed


The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as beta1-integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating beta1-integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of beta1-integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of beta1-integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells.

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    • "In addition to crosstalk with growth factors, integrins have previously been reported to regulate positioning and activation of the matrix metalloproteinase (MMP) family of ECM proteases [12]. MMP’s play a key role in the control of specific local ECM degradation and biochemical complexes between both β1 and β3 integrin families have previously been reported in a range of cell types [13], [14], [15], [16], [17]. Integrins are therefore a potential key nodal point at which growth factor, ECM and protease signaling can converge. "
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    • "The 3D culture system used was a modification of the Debnath and Sameni overlay method [31] [35] [36]. To analyze mRNA and protein expression, cells were cultured as follows: 10 í µí¼‡L containing 2.5 × 10 5 cells/well (MCF-7 or MDA-MB-231) in single cell suspensions were seeded in 4-well plates (Lab-Tek Chamber Slide System; Nalge Nunc International, Rochester, NY, USA) on 55 í µí¼‡L of a solidified layer of Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA); after 15–20 min, a suspension of 1.25 × 10 5 U937 cells in 40 í µí¼‡L assay medium (supplemented RPMI 1640 with 60% Matrigel) was added. "
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    • "If receptors are highly expressed and/or ligand concentration is too high, adhesion strength is too great, and the cell-substrate interactions are essentially too adhesive to allow migration. Additionally, the biochemical signaling induced by ligand-adhesion receptor binding contributes by regulating expression of factors important in migration such as cytoskeletal proteins, cell adhesion receptors, extracellular matrix, and matrix metalloproteinases, and dictates the profile of the bimodal response curve with respect to ligand and receptor concentrations (Borrirukwanit et al., 2007; Lamar et al., 2008; Sameni et al., 2008; Vicente-Manzanares et al., 2009). In the context of axon regeneration, adhesion strength is important because the formation of stable adhesive contacts from the growth cone to the substrate is essential for growth cone steering and subsequent stimulation of neurite growth (Schmidt and Leach, 2003). "
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