Abstract
Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell–cell and cell–matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease–matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.
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References
López-Otín C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433–30437
Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3:362–374
Madzharova E, Kastl P, Sabino F et al (2019) Post-translational modification-dependent activity of matrix metalloproteinases. Int J Mol Sci 20:3077
Turk B (2006) Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5:785–799
Vizovisek M, Ristanovic D, Menghini S et al (2021) The tumor proteolytic landscape: a challenging frontier in cancer diagnosis and therapy. Int J Mol Sci 22:2514
Carter EP, Roozitalab R, Gibson SV et al (2021) Tumour microenvironment 3D-modelling: simplicity to complexity and back again. Trends Cancer 7:1033–1046
Gibson SV, Madzharova E, Tan AC, Allen MD, auf dem Keller U, Jones JL, Carter EP, Grose RP (2023). ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation. Matrix Biology 121:74–89
Gibson SV, Tomas Bort E, Rodríguez-Fernández L, Allen MD, Gomm J, Goulding I, auf dem Keller U, Agnoletto A, Brisken C, Peck B, Cameron AJ, Marshall JF, Jones JL, Carter EP, Grose RP (2023). TGFβ-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression. NPJ Breast Cancer 9(1):9
Bartek J, Bartkova J, Kyprianou N et al (1991) Efficient immortalization of luminal epithelial cells from human mammary gland by introduction of simian virus 40 large tumor antigen with a recombinant retrovirus. Proc Natl Acad Sci U S A 88:3520–3524
Allen MD, Thomas GJ, Clark S et al (2014) Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of alphavbeta6 integrin in DCIS identifies high-risk patients and predicts recurrence. Clin Cancer Res 20:344–357
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to imageJ: 25 years of image analysis. Nat Methods 9:671–675
Adriance MC, Inman JL, Petersen OW et al (2005) Myoepithelial cells: good fences make good neighbors. Breast Cancer Res 7:190–197
Polyak K, Hu M (2005) Do myoepithelial cells hold the key for breast tumor progression? J Mammary Gland Biol Neoplasia 10:231–247
Hayward M-K, Allen MD, Gomm JJ et al (2022) Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion. NPJ Breast Cancer 8:109
Acknowledgments
This work was supported by Cancer Research UK (CRUK) (C10847/A27781), Breast Cancer Now (2017NovPR988), Barts Charity (MRC0173), Rosetrees Trust (M501-F1), and CRUK Microscopy Core Service Grant (C16420/A18066) and by a CRUK UK Centre Grant to Barts Cancer Institute (C355/A25137). We would like to dedicate this manuscript in memory of Professor Ulrich auf dem Keller, a wonderful collaborator and inspiring scientist.
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Gibson, S.V., Carter, E.P., Grose, R.P. (2024). Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models. In: Santamaria, S. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 2747. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3589-6_15
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DOI: https://doi.org/10.1007/978-1-0716-3589-6_15
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