Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions

Jedeszko, Christopher; Sameni, Mansoureh; Olive, Mary B.; Moin, Kamiar; Sloane, Bonnie F.

doi:10.1002/0471143030.cb0420s39

Cited by 56 publications

(80 citation statements)

References 14 publications

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“…We have established methods for quantifying the fluorescent cleavage products that result from DQ-collagen degradation 2,3 . We normalize degradation products throughout the entire 3D volume of the MAME cocultures on a per cell basis by labeling and counting cell nuclei.…”

Section: Representative Resultsmentioning

confidence: 99%

“…We normalize degradation products throughout the entire 3D volume of the MAME cocultures on a per cell basis by labeling and counting cell nuclei. Furthermore, we can localize the degradation to extracellular and intracellular compartments and quantify the total, intracellular and extracellular degradation products as we have described previously 2 . The green fluorescence in Figures 2 and 3 represents cleavage products of the two DQ-collagen substrates.…”

Section: Representative Resultsmentioning

confidence: 99%

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MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Sameni

Anbalagan

Olive

et al. 2012

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We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers.Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches. Video LinkThe video component of this article can be found at http://www.jove.com/video/3661/ Protocol Prepare DQ-substratesIt may be necessary to agitate DQ-substrate in an ultrasonic water bath for~5 min and heat to 50°C to facilitate dispersion. Allow lyophilized DQ-substrates to warm to room temperature before opening vials, prepare stock solution of 1 mg/ml of DQ-substrate in deionized water, ...

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Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Sameni

Anbalagan

Olive

et al. 2012

JoVE

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“…This collagen allows for the microscopic analysis of proteolysis because it has an excessive amount of fluorescent dyes conjugated to it which causes a quenching effect. After it has been enzymatically degraded, the quenching is reduced and the local area becomes fluorescent (Jedeszko et al, 2008). Therefore, areas of DQ collagen fluorescence indicate collagenase proteolytic activity, which would be mediated by many of the invadopodia-associated MMP enzymes, including MMP-2.…”

Section: Mechanical Stimulation Increases the Expression And Activitymentioning

confidence: 99%

Transient mechanical strain promotes the maturation of invadopodia and enhances cancer cell invasion in vitro

Gasparski

Ozarkar

Beningo

2017

Journal of Cell Science

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Cancer cell invasion is influenced by various biomechanical forces found within the microenvironment. We have previously found that invasion is enhanced in fibrosarcoma cells when transient mechanical stimulation is applied within an in vitro mechano-invasion assay. This enhancement of invasion is dependent on cofilin (CFL1), a known regulator of invadopodia maturation. Invadopodia are actin-rich structures present in invasive cancer cells that are enzymatically active and degrade the surrounding extracellular matrix to facilitate invasion. In this study, we examine changes in gene expression in response to tugging on matrix fibers. Interestingly, we find that integrin β3 expression is downregulated and leads to an increase in cofilin activity, as evidenced by a reduction in its Ser3 phosphorylation levels. As a result, invadopodia lengthen and have increased enzymatic activity, indicating that transient mechanical stimulation promotes the maturation of invadopodia leading to increased levels of cell invasion. Our results are unique in defining an invasive mechanism specific to the invasive process of cancer cells that is triggered by tugging forces in the microenvironment, as opposed to rigidity, compression or stretch forces.

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“…Using a live cell imaging assay for proteolysis developed in our laboratory [19][20][21][22], we have shown that co-culturing macrophages with breast carcinoma cells increases degradation of the basement membrane protein type IV collagen and that this degradation is reduced by inhibitors of MMPs and CTSB [21]. Therefore, our aim in the present study was to determine how breast cancer cells modulate expression and activity of the proteases CTSB, MMP-2 and MMP-9 in human monocytes.…”

Section: Introductionmentioning

confidence: 96%

Interleukin-6 Increases Expression and Secretion of Cathepsin B by Breast Tumor-Associated Monocytes

Mohamed¹,

Rudy²,

Anbalagan³

et al. 2010

Cell Physiol Biochem

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In the tumor microenvironment, monocytes respond to paracrine stimuli from breast cancer cells by secreting molecules that participate in breast cancer growth, invasion, intravasation and metastasis. Here we examined the effects of media conditioned by MDA-MB-231 human breast carcinoma cells (231-CM) on expression and secretion of proteases and secretion of cytokines by U937 human monocytes. We found that 231-CM increased U937: 1) proliferation; 2) expression, activity and secretion of the cysteine protease cathepsin B (CTSB); 3) secretion of matrix metalloproteinases (MMP)-2 and -9; and 4) secretion of interleukin-6 (IL-6) and insulinlike growth factor binding protein-1 (IGFBP-1). We further demonstrated by western blotting and enzymatic activity assays that the increases in CTSB secretion and activity induced by 231-CM could be reduced by neutralizing antibodies against IL-6. Our data suggest a role for IL-6 in

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Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions

Cited by 56 publications

References 14 publications

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Transient mechanical strain promotes the maturation of invadopodia and enhances cancer cell invasion in vitro

Interleukin-6 Increases Expression and Secretion of Cathepsin B by Breast Tumor-Associated Monocytes

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