Direct visualization of protease activity on cells migrating in three-dimensions

Abstract

Determining the specific role(s) of proteases in cell migration and invasion will require high-resolution imaging of sites of protease activity during live-cell migration through extracellular matrices. We have designed a novel fluorescent biosensor to detect localized extracellular sites of protease activity and to test requirements for matrix metalloprotease (MMP) function as cells migrate and invade three-dimensional collagen matrices. This probe fluoresces after cleavage of a peptide site present in interstitial collagen by a variety of proteases including MMP-2, -9, and -14 (MT1-MMP) without requiring transfection or modification of the cells being characterized. Using matrices derivatized with this biosensor, we show that protease activity is localized at the polarized leading edge of migrating tumor cells rather than further back on the cell body. This protease activity is essential for cell migration in native cross-linked but not pepsin-treated collagen matrices. The new type of high-resolution probe described in this study provides site-specific reporting of protease activity and insights into mechanisms by which cells migrate through extracellular matrices; it also helps to clarify discrepancies between previous studies regarding the contributions of proteases to metastasis.

Fig. 1

Fluorogenic probe for detection of protease activity in solution, on a 2-dimensional substratum, or in a 3-dimensional extracellular matrix. (a) The GPLGIAG cleavage site from interstitial collagen is incorporated into a peptidyl backbone (yellow) with constrained conformation (Packard and Komoriya, 2008). The 18 amino acid peptide is then homodoubly labeled with rhodamine 6G fluorophores (red) that self-quench by formation of an intramolecular H-type excitonic dimer (Packard et al., 1996). After cleavage of the GPLGIAG site (between blue and purple in the diagram), two fragments are generated, which separate (one fragment remains immobilized and the other diffuses away), thereby disrupting the intramolecular quenching to generate fluorescence (bright red). The probe can be used to detect protease activities in solution or localized proteolytic activity if immobilized with a biotin-streptavidin linker (green) on a surface such as collagen (gray triple helix). (b) Migrating A2 cells [a clone of breast carcinoma MDA-MD-231 cells expressing MT1-MMP with an enhanced GFP marker] (green) produce fluorescence patterns (red) resulting from cleavage of the probe immobilized on a gelatin substratum. (c) Solution kinetics of cleavage by MT1-MMP show stereospecificity of the probe containing all l-amino acids (red) compared to the control with all d-amino acids (blue). (d) Stereospecificity of l- versus d-peptide is retained after immobilization. Cleavage and fluorescence of the probe (red) by A2 cells (green) can be seen in the left column, whereas the right column shows the refractoriness to cleavage of the control (d-amino acids) peptide. Degradation of the gelatin substratum by migrating cells detected by loss of fluorescence (dark areas in blue background) is equivalent. (e) Cleavage of probe (red) by A2 cells (green) on a gelatin substratum is inhibited strongly by 100 nM TIMP-2 compared with untreated control and 100 nM TIMP-1, but migratory morphology is unaffected. Scale bars, 20 μm.

Fig. 2

Imaging of protease activity on the surface of tumor cells migrating in a 3-dimensional collagen matrix. (a) Although eGFP-MT1-MMP (green) is expressed not only at the polarized leading edge of an A2 cell invading through the collagen matrix, most protease activity (red) localizes to the leading edge. (b and c) A2 cells migrating through 3-dimensional collagen matrices labeled with the protease probe show strong fluorescence (red) and accumulation of intracellular vesicles at leading edges. (d) BT549 breast carcinoma cells migrating through a 3-dimensional collagen matrix show a similar localization to the leading edge. In the top row both proteolysis and accumulation of intracellular vesicles are on the left side of the cell whereas 105 minutes later (bottom row) both have relocated to the right. (e) PC-3 prostate carcinoma cells invading a collagen gel also show localization to the leading edge of the migrating cell. (f) Effects of collagen density/concentration on tumor cell motility. HT-1080 fibrosarcoma cells were embedded in type I crosslinked collagen at different collagen concentrations (1.6 to 5.0 mg/ml). After 48 hours, migration of cells was measured at 10 minute intervals for an additional 48 hour period. Note the inverse correlation between average cell velocity and collagen concentration. However, there was no significant difference between cell velocities in 1.6 and 2.5 mg/ml collagen (P > 0.05 by Student-Newman-Keuls Multiple Comparisons Test), but larger differences in concentration affected migration velocity (P<0.001 for 1.6 versus 3.8 mg/ml; P<0.01 for 2.7 versus 5.0 mg/ml). (g) HT-1080 cells in 3-dimensional collagen matrices labeled with the l-peptide probe show extensive non-polarized extracellular proteolysis consistent with their high, relatively random protrusiveness. (h) HT-1080 cells in collagen matrices labeled with the d-isomer control show no detectable fluorescence. Scale bars, 10 μm.

Fig. 3

Fluorescence from GPLGIAG probe cleavage versus DQ- and Alexa 647-labeled collagen. (a) PC-3 cells display a slight asymmetry during migration with intracellular vesicles (see DIC) and fluorescence from cleavage of the GPLGIAG probe (red) at the polarized leading edge. In contrast, the DQ-collagen cleavage signal (green) is present at both the leading edge and in substantial amounts elsewhere in the matrix. The fourth panel represents merged images of GPLGIAG probe (red) and DQ-collagen (green); the fifth panel shows merged DIC (gray), GPLGIAG probe (red), and DQ-collagen (green). (b) Fluorescence from cleavage of the GPLGIAG probe (red) at the site of matrix invasion by a BT549 cell while DQ-collagen (green) and collagen directly labeled with Alexa 647 (blue) co-localize throughout the matrix. Fifth panel shows merged DQ-collagen (green) and Alexa 647 (blue). Scale bars, 10 μm.

Fig. 4

Invasion of tumor cells in 3-dimensional collagen gels. (a) Cleavage and fluorescence of the GPLGIAG probe documents proteolysis of the collagen matrix where a PC-3 cell migrated in zig-zag fashion over a 4 day period. (b) Over a four day time course, similar protease activity patterns are observed for an HT-1080 cell. (c) Video time-lapse tracking of migration paths of HT-1080 cells during 48 hours in a 3-dimensional collagen sandwich gel. Scale bars, 20 μm.

Fig. 5

Effect of MMP inhibitors on proteolysis and velocity of HT-1080 cells. (a) Control showing protease activity (red). (b) TIMP-2 (200 nM) inhibited cleavage of the GPLGIAG probe (red) accompanied by rounder cell morphology. (c) TIMP-1 (200 nM) was a substantially less effective inhibitor as determined by fluorescence intensity, except at some leading lamellae. (d) Comparison of effects of TIMP-1 and TIMP-2 on tumor cell migration/invasion through acid-extracted, native crosslinked type I collagen gels. (e) Comparisons of velocity (top row) and migration paths (bottom) of individual cells though crosslinked collagen. (f) Comparison of effects of TIMP-1 and TIMP-2 on tumor cell migration/invasion through non-crosslinked, type I pepsinized collagen gels. (g) Comparisons of velocity (top row) and migration pattern (bottom) of individual cells through this telopeptide-free non-crosslinked collagen. Scale bars, 10 μm.