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Imaging and quantifying the dynamics of tumor-associated proteolysis

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Abstract

The roles of proteases in cancer are dynamic. Furthermore, the roles or functions of any one protease may differ from one stage of cancer to another. Proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as from tumor cells make important contributions to ‘tumor proteolysis’. Many tumors exhibit increases in expression of proteases at the level of transcripts and protein; however, whether those proteases play causal roles in malignant progression is known for only a handful of proteases. What the critical substrate or substrates that are cleaved in vivo by any given protease is also known for only a few proteases. Therefore, the recent development of techniques and reagents for live cell imaging of protease activity, in conjunction with informed knowledge of critical natural substrates, should help to define protease functions. Here we describe live cell assays for imaging proteolysis, protocols for quantifying proteolysis and the use of such assays to follow the dynamics of proteolysis by tumor cells alone and tumor cells interacting with other cells found in the tumor microenvironment. In addition, we describe an in vitro model that recapitulates the architecture of the mammary gland, a model designed to determine the effects of dynamic interactions with the surrounding microenvironment on ‘tumor proteolysis’ and the respective contributions of various cell types to ‘tumor proteolysis’. The assays and models described here could serve as screening platforms for the identification of proteolytic pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use.

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Abbreviations

10A:

MCF-10A human breast epithelial cell line

3D:

Three dimensions

4D:

Three dimensions + time

AT1:

MCF10.AT1 atypical hyperplastic cell line

BSA:

Bovine serum albumin

CA1d:

MCF10.CA1d breast carcinoma cell line

DQ:

Dye-quenched

DCIS1:

MCF10.DCIS breast ductal carcinoma in situ cell line

ECM:

Extracellular matrix

FITC:

Fluorescein isothiocyanate

HUVEC:

Human umbilical vein endothelial cells

MAME:

Mammary architecture and microenvironment engineering

MMP:

Matrix metalloproteinase

neoT:

MCF-10AneoT ras-transfected breast epithelial cell line

rBM:

Reconstituted basement membrane

uPA:

Urokinase plasminogen activator

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Acknowledgments

This work was supported by U.S. Public Health Service Grant CA 56586 and the following awards from the Department of Defense: a Breast Cancer Center of Excellence (DAMD17-02-1-0693) and BC051230 predoctoral fellowship (CJ). The Microscopy and Imaging Resources Laboratory is supported by National Institutes of Health Center Grants P30ES06639 and P30CA22453 and a Roadmap Grant U54RR020843.

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Author notes

  1. Stefanie R. Mullins

    Present address: Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA, 19111, USA

Authors and Affiliations

  1. Department of Pharmacology, School of Medicine, Wayne State University, 540 E. Canfield, Detroit, MI, 48201, USA

    Mansoureh Sameni, Dora Cavallo-Medved, Julie Dosescu, Christopher Jedeszko, Kamiar Moin, Stefanie R. Mullins, Mary B. Olive, Deborah Rudy & Bonnie F. Sloane

  2. Barbara Ann Karmanos Cancer Institute, Wayne State University, 540 E. Canfield, Detroit, MI, 48201, USA

    Dora Cavallo-Medved, Kamiar Moin, Stefanie R. Mullins & Bonnie F. Sloane

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  1. Mansoureh Sameni
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  2. Dora Cavallo-Medved
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Correspondence to Bonnie F. Sloane.

Electronic supplementary material

Supplemental Fig. 4 movie

Time-course for degradation of DQ-collagen IV as HUVECs form cord-like structures in vitro. HUVEC were grown for 18 h on glass coverslips coated with rBM containing 25 μg/ml DQ-collagen IV. The movie depicts confocal image in real-time of proteolysis by live HUVECs over a time period from 2 – 18 h after plating. DQ-collagen IV degradation products (green) are seen surrounding the cord-like structures (MOV 5448 kb)

Time-course for degradation of DQ-collagen I as human breast fibroblasts migrate on a matrix of collagen I containing DQ-collagen I. Movie depicts confocal image in real-time of proteolysis by live fibroblasts over a 90 min period starting 2 hr after plating. Discrete dots of fluorescent degradation products (green) appear as the cells detach from the matrix (MP4 205 kb)

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Sameni, M., Cavallo-Medved, D., Dosescu, J. et al. Imaging and quantifying the dynamics of tumor-associated proteolysis. Clin Exp Metastasis 26, 299–309 (2009). https://doi.org/10.1007/s10585-008-9218-7

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