Abstract
Tumor growth and progression is the result of a complex process controlled not only by malignant cancer cells but also by the surrounding tumor microenvironment (TME). Cancer associated fibroblasts (CAFs), the most abundant cellular component of TME, play an active role in tumor invasion and metastasis by promoting cancer cell invasion through cell-cell interactions and secretion of pro-invasive factors such as extracellular matrix (ECM)-degrading proteases. Due to their tumor-promoting activities, there is an emerging interest in investigating CAFs biology and its potential as drug targets for cancer therapies. Here we describe an easy and highly reproducible quantitative method to analyze CAF invasive activity by forming multicellular spheroids embedded into a three-dimensional (3D) matrix that mimics in vivo ECM. Subsequently, invasion is monitored over time using a time-lapse microscope. We also provide an automated image analysis system that enables the rapid quantification of the spheroid area increase (invasive area) over time. The use of a 96-well plate format with one CAF spheroid per well and the automated analysis provides a method suitable for drug screening test, such as protease inhibitors.
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References
Kalluri R (2016) The biology and function of fibroblasts in cancer. Nat Rev Cancer 16(9):582–598
Gaggioli C, Hooper S, Hidalgo-Carcedo C et al (2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9(12):1392–1400
Labernadie A, Kato T, Brugués A et al (2017) A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat Cell Biol 19(3):224–237
Xing F, Saidou J, Watabe K (2011) Cancer associated fibroblasts in tumour microenvironment. Front Biosci 15:166–179
Vinci M, Box C, Eccles SA (2015) Three-dimensional (3D) tumour spheroid invasion assay. J Vis Exp 99:e52686
Vinci M, Gowan S, Boxall F et al (2012) Advances in establishment and analysis of three-dimensional tumour spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:29
Kramer N, Walzl A, Unger C et al (2013) In vitro cell migration and invasion assays. Mutat Res 752(1):10–24
Timmins NE, Nielsen LK (2007) Generation of multicellular tumour spheroid. Methods Mol Med 140:141–151
Katt ME, Placone AL, Wong AD et al (2016) In Vitro tumour models: advantages, disadvantages, variables, and selecting the right platform. Front Bioeng Biotechnol 4:12
Ivanov DP, Parker TL, Walker DA et al (2014) Multiplexing spheroid volume, Resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres. PLoS One 9(8):e103817
Bonnans C, Chou J, Werb Z (2014) Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15(12):786–801
Miles FL, Sikes RA (2014) Insidious changes in stromal matrix fuel cancer progression. Mol Cancer Res 12(3):297–312
Artym VV, Matsumoto K (2010) Imaging cells in three-dimensional collagen matrix. Curr Protoc Cell Biol Chapter 10:Unit 10.18.1–20
Fontanil T, Álvarez-Teijeiro S, Villaronga MA et al (2017) Cleavage of Fibulin-2 by the aggrecanases ADAMTS-4 and ADAMTS-5 contributes to the tumorigenic potential of breast cancer cells. Oncotarget 8(8):13716–13729
Acknowledgments
This study was supported by the Instituto de Salud Carlos III-FEDER (Funding Program from the European Union) (PI13/00259 and PI16/00280 to J.M.G.P. and CD13/00157 to M.A.V.), Consorcio CIBERONC (CB16/12/00390 to J.M.G.P.), and the Cancer Research UK (C.R.U.K.) (C33043/A12065 to V.S.M.).
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Villaronga, M.Á., Teijeiro, S.Á., Hermida-Prado, F., Garzón-Arango, M., Sanz-Moreno, V., García-Pedrero, J.M. (2018). Analysis of Invasive Activity of CAF Spheroids into Three Dimensional (3D) Collagen Matrices. In: Cal, S., Obaya, A. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 1731. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7595-2_14
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DOI: https://doi.org/10.1007/978-1-4939-7595-2_14
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Online ISBN: 978-1-4939-7595-2
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