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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    ABSTRACT: Cell invasion through extracellular matrix (ECM) is a hallmark of the metastatic cascade. Cancer cells require adhesion to surrounding tissues for efficient migration to occur, which is mediated through the integrin family of receptors. Alterations in expression levels of β1 and β3 integrins have previously been reported in a number of human cancers. However, whether there are specific roles for these ubiquitous receptors in mediating cell invasion remains unclear. Here we demonstrate that loss of β1 but not β3 integrins leads to increased spread cell area and focal adhesion number in cells on 2D immobilized fibronectin. Increased adhesion numbers in β1 knockdown cells correlated with decreased cell migration on 2D surfaces. Conversely, cells depleted of β1 integrins showed increased migration speed on 3D cell-derived matrix as well as in 3D organotypic cultures and inverted invasion assays. This increased invasive potential was also seen in cells lacking β3 integrin but only in 3D cultures containing fibroblasts. Mechanistically, in situ analysis using FRET biosensors revealed that enhanced invasion in cells lacking β1 integrins was directly coupled with reduced activation of focal adhesion kinase (FAK) and the small GTPase RhoA resulting in formation of enhanced dynamic protrusions and increased invasion. These reductions in FAK-RhoA signal activationwere not detected in β3 knockdown cells under the same conditions. This data demonstrates a specific role for β1 integrins in the modulation of a FAK-RhoA-actomyosin signaling axis to regulate cell invasion through complex ECM environments.
    PLoS ONE 09/2013; 8(9):e74659. DOI:10.1371/journal.pone.0074659 · 3.23 Impact Factor
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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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    ABSTRACT: Tumor-associated immune cells often lack immune effector activities, and instead they present protumoral functions. To understand how tumors promote this immunological switch, invasive and noninvasive breast cancer cell (BRC) lines were cocultured with a promonocytic cell line in a Matrigel-based 3D system. We hypothesized that if communication exists between tumor and immune cells, coculturing would result in augmented expression of genes associated with tumor malignancy. Upregulation of proteases MMP1 and MMP9 and inflammatory COX2 genes was found likely in response to soluble factors. Interestingly, changes were more apparent in promonocytes and correlated with the aggressiveness of the BRC line. Increased gene expression was confirmed by collagen degradation assays and immunocytochemistry of prostaglandin 2, a product of COX2 activity. Untransformed MCF-10A cells were then used as a sensor of soluble factors with transformation-like capabilities, finding that acini formed in the presence of supernatants of the highly aggressive BRC/promonocyte cocultures often exhibited total loss of the normal architecture. These data support that tumor cells can modify immune cell gene expression and tumor aggressiveness may importantly reside in this capacity. Modeling interactions in the tumor stroma will allow the identification of genes useful as cancer prognostic markers and therapy targets.
    05/2013; 2013(1):279505. DOI:10.1155/2013/279505
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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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    ABSTRACT: We adapted a microfluidic system used previously to generate durotactic gradients of stiffness in a 3D collagen gel, to produce haptotactic gradients of adhesive ligands through the collagen gel. Oligopeptide sequences that included bioactive peptide sequences from laminin, YIGSR, or IKVAV, were grafted separately onto type I collagen using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Solutions of peptide-grafted collagen and untreated collagen were then used as source and sink input solutions, respectively, in an H-shaped microfluidic network fabricated using traditional soft lithography. One-dimensional gradients of the peptide-grafted collagen solution were generated in the channel that connected the source and sink channels, and these gradients became immobilized upon self-assembly of the collagen into a 3D fibrillar gel. The slope and average concentration of the gradients were adjusted by changing the concentration of the source solutions and by changing the length of the cross-channel. A separate, underlying channel in the microfluidic construct allowed the introduction of a chick embryo dorsal root ganglion into the network. Neurites from these explants grew significantly longer up steep gradients of YIGSR, but shallow gradients of IKVAV in comparison to untreated collagen controls. When these two gradients were presented in combination, the bias in growth acceleration was the largest and most consistent. No differences were observed in the number of neurites choosing to grow up or down the gradients in any condition. These results suggest that the incorporation of distinct gradients of multiple bioactive ligands can improve directional acceleration of regenerating axons.
    Journal of neurotrauma 04/2011; 28(11):2377-87. DOI:10.1089/neu.2010.1606 · 3.71 Impact Factor
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