Visualizing protease activity in living cells: from two dimensions to four dimensions

Abstract

Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal microscopy and advanced imaging software, the fluorescence is detected and accurate measurements of proteolytic degradation in three and four dimensions can be assessed.

Figure 4.20.1

Culture with live cells is required to cause release of green fluorescence from DQ-substrates. Confocal images of green fluorescent channel at a z-level below the ECM-medium border. Cultures containing either collagen I with DQ-collagen I (A,B) or rBM containing DQ-collagen IV (C,D) were imaged after 3 days of culture without cells (A,C), with human breast cancer cells (B) or with human breast epithelial cells (D). Bar, 50 µm.

Figure 4.20.2

Examples of cellular morphologies observed on different ECM. Phase contrast or DIC images of: (A) human breast cancer cells forming a simple monolayer on gelatin; (B) human prostate cancer cells forming clusters and sheets on collagen type I; (C) human colon carcinoma cells forming a 3-D spheroid of polarized cells surrounding a hollow lumen in rBM; and (D) human umbilical vein endothelial cells forming a tubular network of cellular cords on rBM.

Figure 4.20.3

Frame captures of 3-D reconstructions of human breast cancer cells grown in rBM containing DQ-collagen IV. Image stacks containing the DQ-substrate channel (green), the nuclei channel (blue) and the CellTracker channel (red) were used to create graphical 3-D reconstructions/projections using the QVTR feature of the Volocity imaging software suite. The 3-D image can be rotated 360° in order to (A) view proteolysis of DQ-substrates (green) in relation to the cells (red) or (B) view only the nuclei for the purpose of counting cells.

Figure 4.20.4

Quantification of proteolysis and discrimination of intracellular and extracellular localization of degradation fragments in human breast epithelial cells grown in rBM containing DQ-collagen IV. (A) Single optical section at equatorial plane showing fluorescence of the CellTracker Orange channel. (B) Same optical section but a binarized image of the CellTracker Orange and DNA dye channels. (C) Total (extracellular and intracellular) degradation fragments of DQ-collagen IV in same optical section. (D) Extracellular degradation fragments of DQ-collagen IV, obtained by masking image of panel C with the image from panel B to eliminate all signal from intracellular areas. (E) Intracellular degradation fragments of DQ-collagen IV. (F) Total degradation fragments of DQ-collagen IV in all optical sections were quantified as normalized integrated intensity per cell. Total fluorescence separated into intracellular and pericellular components using image arithmetic in MetaMorph software.