Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches

Abstract

Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor microenvironment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

Keywords: Confocal microscopy; Fluorescent imaging; High-content imaging; Proteolysis; Screening assays.

Fig. 1

Pathomimetic avatars: schematic diagrams and representative images of 8-day cultures. Left: MDA-MB-231 human breast carcinoma cells in reconstituted basement membrane (rBM) + 2% rBM overlay. Middle: 231 cells in a top layer of reconstituted basement membrane (rBM) overlaid with 2% rBM and WS12Ti human breast carcinoma-associated fibroblasts embedded in a lower layer of collagen I. Right: tumor cells and fibroblasts mixed and plated together in rBM and overlaid with 2% rBM. Quenched fluorescent protein substrates (DQ-collagens IV and I) are mixed with rBM and collagen I, respectively. Note the more extensive degradation and increased size of 231 structures in the presence of fibroblasts. Red, magenta, and green represent 231 tumor cells, fibroblasts, and fluorescent cleavage products of the substrates, respectively

Fig. 2

Schematic cross-sectional view of custom chambers for 3D growth and live-cell imaging of pathomimetic avatars. In this chamber design, each two wells are linked by an open channel to allow cell migration and sharing of factors secreted into culture media by cells in each well

Fig. 3

Degradation of DQ-collagen IV by pathomimetic avatars of MDA-MB-231 human breast carcinoma cells. Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Left panel is DMSO control and middle panel is a mixture of cathepsin B cysteine protease inhibitors (5 μM each of CA074 + CA074Me). Bar graph is quantification of degraded DQ-collagen IV per cell in MDA-MB-231 structures exposed to DMSO (negative control) and CA074/CA074Me (5 μM each). Data shown are from three independent experiments (48 fields); * ≤0.05; mean ± SD. Adapted from Ramalho et al. [10]

Fig. 4

Quantification of degradation of DQ-collagen IV by pathomimetic avatars of HCT116 human colon carcinoma cells. (a) Single optical section of 3D pathomimetic avatar at equatorial plane showing fluorescence of cells (magenta) used for cytoplasmic binarized mask, nuclei (blue) used for counting cells, and degraded DQ-collagen IV (green). (b) Total degraded DQ-collagen IV in this optical section. (c) Pericellular degraded DQ-collagen IV in this section. (d) Intracellular degraded DQ-collagen IV in this section. Quantification of degraded DQ-collagen IV is done in each optical section of 3D volume, totaled and normalized to the number of cells. Image arithmetic is used to separate total proteolysis (magenta bar) into intracellular (yellow bar) and pericellular (cyan bar) components