spheroids overnight at 37uC. After spheroid formation a solution of 1.5 mg/ml of rat tail collagen type-I (BD Biosciences, UK) was prepared in the appropriate amount of EGM-2 medium and pH neutralized by drop wise addition of 1 M NaOH. An initial layer was deposited in the centre of the wells of a 12 well plate as a droplet and allowed to set at 37uC. The spheroids were resuspended in an equivalent solution of collagen type-I and deposited over the first layer, and incubated at 37uC for 1.5 h-2 h to set. After allowing the collagen gels to set, 1.5 ml of EGM-2 medium including angiogenesis inhibitors or stimulants were added to the wells and the spheroids were allowed to form sprouts for 2 days before fixation with 4% PFA (w/v) in HBSS with Ca2+ and Mg2+ (Invitrogen). Function blocking antibodies were added within the collagen matrix. For longer term experiments spheroids were incubated for 7 days with medium changes every 2 days before fixation with 4% PFA (w/v) in HBSS with Ca2+ and Mg2+. Immunocytochemistry of spheroid co-cultures After fixing the co-cultures, the PFA was quenched with 100 mM Glycine, pH 7.4 after which the spheroids were blocked for 1 h with 1% BSA (w/v) in PBS at room temperature (RT). Primary antibodies were added at the appropriate dilutions in 1% BSA in PBS and incubated at RT overnight. The spheroids were subsequently washed for at least 8 h in 1% BSA (w/v) in PBS with 0.1% Tween (v/v), with hourly changes of the washing solution, followed by incubation with the appropriate donkey secondary antibody (Jackson ImmunoResearch, Stratech, UK) overnight at RT. Finally the co-cultures were washed again with 1% BSA (w/v) in PBS with 1% Tween (v/v) and stored in PBS at 4uC for before imaging. Confocal microscopy and image analysis Spheroid sprouting was imaged with a Nikon C1Si confocal inverted microscope (Nikon UK limited, UK), using 106 magnification. 10 spheroids from 2 different wells were imaged per condition. Green channel confocal images corresponding to the pre-dyed endothelial cell sprout formation were subsequently quantified using the Metamorph (Molecular Devices, Berkshire, UK) image analysis software. In short, a proprietary Metamorph plug-in for Neurite Outgrowth analysis was used, which masks the spheroid outgrowth area, separating spheroid body from sprouts. This method provided automated measurements of Sprout Length and Number of Sprouts for each spheroid. The quantification method was optimized and validated, showing reproducibility between experiments (Figure S1). For Multiphoton microscopy, spheroids were imaged on a Leica confocal TCS SP5 microscope using a Titanium Sapphire laser (100 fs pulses at 80 MHz, Chameleon model from Coherent). Samples were excited with 880 nm pulses. An oil objective lens was used for excitation and detection at the appropriate wavelengths (206 magnification = 0.7). Z-stack images were obtained from individual spher- oids, and maximum projections were created with the LAS AF Leica imaging software (Leica Microsystems GmbH, Germany). Further 3D image treatment and analysis was performed using VolocityH software (Improvision, Perkin-Elmer, USA). Electron Microscopy Minitumour spheroids cultured for 40 h or 7 days were washed in 0.9% saline solution and fixed in 2.5% (w/v) glutaraldehyde with one volume H2O2 at 4uC for 8 h, followed by 6 washes in 0.9% saline solution. They were treated with 1% (w/v)osmium ferricyanide for 1 h, rinsed 4 times in DIW and bulk stained in 2% (w/v) uranyle acetate in 0.05 M maleate buffer at pH 5.5 for 1 h. A 3D Spheroid Model of Tumour Angiogenesis They were rinsed 4 times in DIW and dehydrated in an ascending series of ethanol solutions from 70% to 100% (v/v). They were rinsed twice in dry acetonitrile and incubated in 50:50 acetonitrile and araldite epoxy resin overnight. This mixture was replaced with 25:75 acetonitrile and araldite for 6 h followed by 4 changes in pure araldide over 48 h. The resin castings were cured at 65uC for 48 h. One micrometre sections were cut with a histodiamond knife (Diatome, Switzerland) on a Leica Ultracut UCT ultramicrotome. They were stained with methylene blue and identified areas were cut at 60 nm with a 45u wedge angle diamond knife (Diatome, Switzerland), mounted on 200 mesh copper grids and stained with uranyle acetate and lead citrate. They were viewed on a FEI Tecnai G2 TEM operated at 120 kV. Images were captured with an AMT XR60B digital camera running Deben software. Lentiviral transduction of shRNA Lentivirus expressing shRNAs from the U6 promoter in the pLKO.1-puro vector containing a puromycin resistance marker were purchased from the Sigma MissionH TRC shRNA library and used to infect subconfluent cells in 6 well plates. Antibiotic free medium was added to cells containing lentivirus particles at an MOI of 2 for 4 h and replaced with fresh medium overnight. shRNA expressing cells were subsequently selected using the appropriate puromycin concentrations (HUVEC 0.3 mg/ml, NHDF 1.2 mg/ml, MDA-MB-231 0.6 mg/ml). Puromycin was removed from the cells 48 h before each experiment. QRT-PCR analysis confirmed that knock-down is not only specific but also did not elicit an interferon dependent non-targeted effect (data not shown), as no significant differences were detected in expression values of other MT-MMPs or interferon-response genes OAS1 and Mx1. Luciferase-based measurement of tumour cell proliferation Minitumour spheroids were prepared using the previously described protocol, using MDA-MB-231-luc2 cells, a MDA-MB- 231 cell line expressing the firefly luciferase (luc2) from the ubiquitin C promoter (Caliper Life Sciences, USA). At the end of spheroid incubation, medium was replaced with 500 mL of fresh medium containing 1% of a 30 mg/ml luciferin stock solution prepared from D-Luciferin potassium salt (Caliper Life Sciences). Spheroid luminescence was imaged using the IVISH 200 imaging system (Caliper Life Sciences). A 96 well plate containing serial concentration dilutions of MDA-MB-231-luc2 cells in triplicates was also imaged as a control for signal stability and linearity with cell number. The luminescence signal was analysed as photons/ second using the Living ImageH 3.2 software from Caliper Life Sciences. Western blotting Protein extracts were obtained from cell monolayers using an SDS lysis buffer containing 50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS (w/v), and CompleteTM EDTA-free proteinase inhibitor cocktail (Roche Diagnostics GmbH, Switzerland). Extracts were homogenized by sonication and cleared by centrifugation for 15 min at 14800 RPM. Total protein concen- tration was determined using a Bicinchoninic Acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein were incubated at 100uC for 5 min in a loading buffer containing 100 mM Dithiothreitol (DTT) (Melford Laboratories, UK) and separated by 10% SDS-PAGE using standard protocols before transferring to a nitrocellulose membrane using a transblot semi- PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30753