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.
$These authors contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Edwards D, Høyer-Hansen G, Blasi F, Sloane BF (2008) The cancer degradome: proteases and cancer biology. Springer-Verlag, New York
Sloane BF, List K, Fingleton B, Matrisian L (2013) Proteases in cancer—significance for invasion and metastasis. In: Brix K, Stoecker W (eds) Proteases—structure and function. Springer-Verlag Wien, Vienna, pp 491–550
Lopez-Otin C, Matrisian LM (2007) Emerging roles of proteases in tumour suppression. Nat Rev Cancer 7(10):800–808
Lopez-Otin C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283(45):30433–30437
Schwartz DR, Moin K, Yao B, Matrisian LM, Coussens LM, Bugge TH, Fingleton B, Acuff HB, Sinnamon M, Nassar H, Platts AE, Krawetz SA, Linebaugh BE, Sloane BF (2007) Hu/Mu ProtIn oligonucleotide microarray: dual-species array for profiling protease and protease inhibitor gene expression in tumors and their microenvironment. Mol Cancer Res 5(5):443–454
Kappelhoff R, Auf dem Keller U, Overall CM (2010) Analysis of the degradome with the CLIP-CHIP microarray. Methods Mol Biol 622:175–193
Acuff HB, Sinnamon M, Fingleton B, Boone B, Levy SE, Chen X, Pozzi A, Carbone DP, Schwartz DR, Moin K, Sloane BF, Matrisian LM (2006) Analysis of host- and tumor-derived proteinases using a custom dual species microarray reveals a protective role for stromal matrix metalloproteinase-12 in non-small cell lung cancer. Cancer Res 66(16):7968–7975
Jedeszko C, Sameni M, Olive MB, Moin K, Sloane BF. 2008 Visualizing protease activity in living cells: from two dimensions to four dimensions. Curr Protoc Cell Biol Chapter 4:Unit 4.20.
Sameni M, Tovar EA, Essenburg CJ, Chalasani A, Linklater ES, Borgman A, Cherba DM, Anbalagan A, Winn ME, Graveel CR, Sloane BF (2016) Cabozantinib (XL184) inhibits growth and invasion of preclinical TNBC models. Clin Cancer Res 22(4):923–934
Ramalho SD, Sharma R, White JK, Aggarwal N, Chalasani A, Sameni M, Moin K, Vieira PC, Turro C, Kodanko JJ, Sloane BF (2015) Imaging sites of inhibition of proteolysis in pathomimetic human breast cancer cultures by light-activated ruthenium compound. PLoS One 10(11):e0142527
Acknowledgment
We would like to thank members of the Sloane laboratory for their discussions and contributions to the development of pathomimetic avatars and Dr. Yong Xu’s lab for fabrication of TAME chambers. This work was supported in part by R01 CA131990 and R21 CA175931 from the National Institutes of Health to Dr. Sloane and an award from the President’s Research Enhancement Program of Wayne State University to Drs. Sloane and Xu. Imaging was performed in the Microscopy, Imaging and Cytometry Resources Core, which is supported, in part, by NIH Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University, and the Perinatology Research Branch of the National Institutes of Child Health and Development at Wayne State University.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Chalasani$, A. et al. (2017). Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches. In: Schilling, O. (eds) Protein Terminal Profiling. Methods in Molecular Biology, vol 1574. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6850-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6850-3_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6849-7
Online ISBN: 978-1-4939-6850-3
eBook Packages: Springer Protocols