A Heterogeneous In Vitro Three Dimensional Model of Tumour-Stroma Interactions Regulating Sprouting Angiogenesis Pedro Correa de Sampaio1, David Auslaender1, Davia Krubasik1, Antonio Virgilio Failla2, Jeremy N. Skepper3, Gillian Murphy1, William R. English1*¤ 1 University of Cambridge, Department of Oncology, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom, 2 Microscopy Unit, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom, 3 University of Cambridge Multi-Imaging Centre, Department of Physiology, Development and Neuroscience, Downing Site, Cambridge, United Kingdom Abstract Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model – a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co- culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre- capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis. Citation: Correa de Sampaio P, Auslaender D, Krubasik D, Failla AV, Skepper JN, et al. (2012) A Heterogeneous In Vitro Three Dimensional Model of Tumour- Stroma Interactions Regulating Sprouting Angiogenesis. PLoS ONE 7(2): e30753. doi:10.1371/journal.pone.0030753 Editor: Donald Gullberg, University of Bergen, Norway Received July 29, 2011; Accepted December 27, 2011; Published February 20, 2012 Copyright: 2012 Correa de Sampaio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Cancer Research UK and Hutchinson Whampoa Limited. PCdS was funded by the Portuguese Foundation for Science and Technology (Fundac¸a˜o para a Cieˆncia e Tecnologia, grant number SFRH/BD/35884/2007). DK was supported by the DAAD (Deutscher Akademischer Austausch Dienst) and Trinity College Cambridge. A British Heart Foundation Intermediate Research Fellowship (grant number FS/03/055/15910) supported WRE during initiation of the project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Work in the Cambridge Research Institute is supported by the corporation Hutchinson Whampoa Limited. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: w.english@sheffield.ac.uk ¤ Current address: Cancer Research UK Tumour Microcirculation Group, Department of Oncology, The University of Sheffield School of Medicine, Sheffield, United Kingdom Introduction Solid tumours are heterogeneous and complex organ-like structures in which the transformed cancer cell co-exists with several other cell types. This microenvironment supports the growth, proliferation, invasion and metastasis of cancer cells through a complex network of signals propagated by interactions that include the extracellular matrix (ECM), other cells, growth factors, chemokines, cytokines and the proteinase system [1,2]. Genetically aberrant cancer cells have been extensively shown to need this permissive framework in order to proliferate and achieve their metastatic potential [3,4]. The observation that tumour growth is often accompanied by neovascularisation has been established since the 70 s, notably through Judah Folkman’s pioneering work [5]. Since then it has been well documented that tumours cannot progress without oxygen and nutrient supply through newly formed vasculature, which is also essential for the metastatic process [6,7,8]. Without this process of neovascularisation tumours remain in their dormant, non-angiogenic form of around 1–2 mm, where proliferation is balanced with apoptosis, maintaining these microtumours quiescent [6]. Strategies for targeting angiogenesis have received significant attention with some degree of clinical success [9,10]. Tumour angiogenesis is thought to occur mostly via sprouting angiogenesis. This is a process through which a single endothelial cell, called the tip cell, is selected from the vasculature, overcoming its quiescent environment, and forming a new vessel. The tip cell migrates towards a chemoattractant angiogenic signal constituted of growth factors that are secreted by the tumour cells and their stroma, which induces endothelial cell mitogenesis and PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30753