Live-cell imaging of tumor proteolysis: impact of cellular and non-cellular microenvironment

Abstract

Our laboratory has had a longstanding interest in how the interactions between tumors and their microenvironment affect malignant progression. Recently, we have focused on defining the proteolytic pathways that function in the transition of breast cancer from the pre-invasive lesions of ductal carcinoma in situ (DCIS) to invasive ductal carcinomas (IDCs). We use live-cell imaging to visualize, localize and quantify proteolysis as it occurs in real-time and thereby have established roles for lysosomal cysteine proteases both pericellularly and intracellularly in tumor proteolysis. To facilitate these studies, we have developed and optimized 3D organotypic co-culture models that recapitulate the in vivo interactions of mammary epithelial cells or tumor cells with stromal and inflammatory cells. Here we will discuss the background that led to our present studies as well as the techniques and models that we employ. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Figure 1. Caveolae (A) and invadopodia (B) cluster proteases in networks at the cell surface

There are structural similarities between caveolae and invadopodia e.g. both require caveolin-1 and lipid rafts for the formation. (A) Caveolae are a subset of lipid rafts in the plasma membrane, which form flask-shaped invaginations as a result of the interactions of dimerized caveolin-1. Many proteases associated with extracellular matrix degradation are localized to caveolae, e.g., plasmin[ogen], MMP-2, -9 and 14 and cathepsin B (CTSB) via binding to receptors, e.g., uPA to uPAR, plasmin[ogen] and CTSB to the annexin II heterotetramer. MMP-14 is a transmembrane MMP, which is also known as MT1-MMP, whereas uPAR is inserted into the membrane by a GPI anchor and is also part of a trimeric complex with β1-integrin and uPARAP, an endocytic receptor associated with collagen uptake. (B) Invadopodia are membrane protrusions associated with filamentous actin and extracellular matrix degradation. MMP-14 and fibroblast activation protein (FAP) or seprase accumulate in the membrane of invadopodia. In addition, there is increased secretion of MMP-2 and lysosomal cysteine cathepsins (e.g., CTSK, CTSL, CTSX) from invadopodia. Depicted here is a lysosome releasing several cysteine cathepsins following trafficking to the membrane on a microtubule (grey). The hexagon shapes represent MMPs with the associated number in the frame; pro-MMPs have an additional swoosh representing their pro-peptide.

Figure 2. An acidic microenvironment is generated by tumors and enhances invasion and degradation of the extracellular matrix

The scissors indicates cleavage of the basement membrane protein collagen IV. In response to the pericellular acidic pH, lysosomes redistribute to the cell periphery via a microtubule-dependent process [111], possibly leading to the observed secretion of both active and pro forms of cathepsin B (Rothberg, Sloane, unpublished data) as well as other lysosomal enzymes from breast cancer cells. This also includes lysosomal glyosidases such as hyaluronase-2 (Hyal-2) [176], which cleaves hyaluronan and release of bioactive molecules bound to this matrix component (stars).

Figure 3. At pH 6.8, degradation of collagen IV is significantly increased

After 2 days in culture at either pH 7.4 and 6.8, degradation products (green) accumulate pericellularly around structures formed by MDA-MB-231-RFP human breast carcinoma cells. Fluorescent degradation products are increased at the acidic pH. Red and blue represent expression of RFP in the cytoplasm and nuclear staining, respectively. 3D reconstructions of confocal image stacks were generated with Volocity software (Perkin Elmer). The increase in proteolysis at acidic pH was significant (p ≤ 0.01; x ± SD; n=3) as illustrated by the graph; proteolysis was quantified following our published protocols [127].

Figure 4. Schematic diagram illustrating representative components of MAME (mammary architecture and microenvironment engineering) tripartite (A) and mixed (B) co-cultures

(A) Plastic coverslips are coated with collagen I, containing DQ-collagen I and fibroblasts (red). A 2^nd layer of reconstituted basement membrane (rBM) containing DQ-collagen IV is added and tumor cells (red) are plated on top. The cultures are then overlaid with a 3^rd layer of 2% rBM, which also is included in subsequent changes of media. (B) Coverslips are coated with rBM containing DQ-collagen IV and a mixture of fibroblasts (red) and tumor cells (red) are plated on top. MAME co-cultures are imaged live using an immersion lens in order to follow changes in morphogenesis and collagen degradation, which is depicted here as green.

Figure 5. Live-cell imaging of tumor proteolysis in 4D

Extensive proteolysis (green fluorescence) is observed as MAME tripartite co-cultures of MCF10.CA1d human breast carcinoma cells and WS-12T_i human breast carcinoma-associated fibroblasts grow and invade through the surrounding matrix over a 23-day period. Over this time period, fibroblasts become incorporated into the structures. Top (left panels), side (middle panels) and bottom (right panels) views of representative co-cultures reconstructed in 3D are illustrated. Co-cultures were stained with CellTracker Orange to label the carcinoma cells and fibroblasts (red) and then imaged by confocal microscopy at 3 (top row), 16 (middle row) and 23 (bottom row) days. Magnification: top panel, 40X; and middle and bottom panels, 10X.