Cell lines and culture conditions
For the majority of our studies, four tumor cell lines representative of highly invasive cancers were selected: glioblastoma (U-87 MG, KNS42); oral squamous cell carcinoma (LICR-LON-HN4) and triple negative breast carcinoma (MDA-MB-231). Additional cell lines were included in spheroid formation trials to cover a wide range of tumor types.
Tumor cell lines (Table 1) were grown in tissue culture flasks under standard conditions (37°C, 5% CO2, 95% humidity). All media (Table 1) were as recommended by the supplier of the cells and were supplemented with 10% fetal calf serum (FCS), with the exception of the DLD-1 medium that was additionally supplemented with 1 × non-essential amino acids (NEAA). Media were routinely changed twice weekly. When subconfluent, cell monolayers were passaged using TrypLE Express Phenol Red, (Gibco, Life Technologies Ltd. Paysley, UK).
mES R1 cells were grown on mitomycin-C treated mouse embryonic fibroblast (MEF) feeder layers in ESC medium: Dulbecco's modified Eagle medium (DMEM) (4.5 g/l D-glucose; sodium pyruvate), supplemented with 15% FCS, 1 × penicillin, streptomycin and neomycin antibiotic mix (Gibco, Life Technologies Ltd. Paysley, UK), 100 μM β-mercaptoethanol (Sigma-Aldrich Company Ltd., Dorset, England), 1 × NEAA plus 1,000 U/ml leukemia inhibitor factor (LIF). Medium was changed 24 h after seeding (1.4 × 105 cells/ml, 5 ml/60 mm dish) and cells were passaged 2 to 3 days later. All media and NEAA were purchased from Gibco (Life Technologies Ltd. Paysley, UK), and FCS from Biosera.
Generation and analysis of tumor spheroids
For spheroid generation, 200 μl/well of cell suspensions at optimized densities (0.5 × 104 cells/ml for U-87 MG, KNS42 and LICR-LON-HN4; 1.5 × 104 for MDA-MB-231 P and M variants) were dispensed into ULA 96-well round-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) using a multichannel pipette. Plates were incubated for 4 days at 37°C, 5% CO2, 95% humidity. Where indicated, optimal three-dimensional structures were achieved by addition of 2.5% Matrigel as previously described . Fully automated image analysis of tumor spheroids was carried out on a Celigo cytometer (Cyntellect Inc, San Diego, CA, USA; http://www.cyntellect.com/content/products/celigo/index.html), which is equipped with a 4-megapixel CCD camera with an F-theta scan lens (1 μm/pixel, 0.25 NA, 3.5 ×). Images were acquired and analyzed by using the Colony Counting Embryoid Body application with the option to scan 1/16 field of view/well. The width of 1 field of view (FOV) is 975 pixels/2,057 μm and image file size is 0.41 MB. Further technical details of imaging parameters are shown in Additional file 15.
For the lower throughput method, images were captured using an inverted microscope (Olympus IX 70, (Olympus Microscopy, Southend-on-SeaEssex, UK) equipped with a CCD camera (QImaging, Surrey, BC, Canada and imported into Image-Pro Plus Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA; http://www.mediacy.com/index.aspx?page=IPP) and by either using macros or manually, multiparametric analysis was performed. In both cases, the radius of each tumor spheroid was used to calculate the volume (μm3): V = 4/3 π r3.
For comparison with our system, U-87 MG cells were also used to generate spheroids using conventional methods as follows: (1) agar-coated 96-well flat-bottomed plates (BD Biosciences, Oxford, England) as previously described  (0.5 × 104 cells/ml as in the ULA 96-well round-bottomed plates); (2) poly-Hema-coated 24-well plates (2 × 105 cells/ml, 1 ml/well). A stock solution of 6 mg/ml poly-Hema (Sigma-Aldrich Company Ltd., Dorset, England) in 95% ethanol was prepared and diluted 1:10 in ethanol. A total of 100 μl/well was dispensed and left to dry before cell addition; (3) RCCS (105 cells/ml, 10 ml/disposable vessel).
In all cases, an inverted microscope was used for image analysis as described above.
Tumor spheroid growth kinetics and treatment with test compounds
Spheroids were generated as described above. Growth kinetics and inhibition assays were performed as previously reported  but with significant modifications. Briefly, spheroid size was measured up to 14 days after initiation. A 50% medium replenishment was performed on days 4, 7, 10 and 12 using a multichannel pipette. Image analysis was performed on a Celigo cytometer or by microscopy as described. Where indicated, day 4 tumor spheroids were treated with 17-AAG (Invivogen, San Diego, CA, USA), or PI-103 (Charnwood Molecular Ltd., Loughborough, UK). Control spheroids were treated with appropriate vehicle. Then, 72 h following compound addition, 50% medium replenishment was performed as described above. Responses were evaluated by spheroid volume measurements at regular intervals.
Tumor spheroid-based migration assay on matrix protein
Flat-bottomed, 96-well plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) were coated with 0.1% (v/v) gelatin (Sigma-Aldrich Company Ltd., Dorset, England) in sterile water for 1 h at 37°C. A total of 200 μl/well of culture medium supplemented with 2% (v/v) FCS was then added. For compound evaluation studies, medium contained 1.5 × the final concentration of 17-AAG or PLCγ inhibitor CCT130234, in a dilution series. Controls were treated with vehicle.
A total of 100 μl medium was removed from each well containing 4-day spheroids, and the remaining medium including the spheroids transferred into the prepared 'migration' plate using a multichannel pipette (final volume 300 μl). Spheroids were allowed to adhere and images were obtained at t0, 24, 48 and 72 h, using an inverted microscope as described. Effects of compounds were analyzed by measuring the area covered by migrating cells using Image-Pro Analyzer software to track the migration front. Data were normalized to the initial size of each spheroid at t0. For timelapse experiments, images were recorded every 15 minutes over a period of 70 h and videos edited using AVS Video Editor (Online Media Technologies Ltd., London, Uk).
Tumor spheroid-based Matrigel invasion assay
A total of 100 μl medium was removed from wells containing 4-day spheroids and 100 μl Matrigel was gently added. When Matrigel solidified, 100 μl of culture medium was added on top. For compound evaluation studies, 17-AAG or vehicle was added to both the Matrigel and the overlying medium. U-87 MG spheroids were treated on the day of assay initiation while MDA-MB-231 spheroids were pretreated for 24 h and during the assay. From t0 and at intervals up to 72 h, automated image analysis was carried out for U-87 MG on a Celigo cytometer, using the Cell Counting Confluence application. Due to the central location of the spheroid in each well, only 1/16 of the field of view needed to be imaged. The width for 1 FOV is 1,958 pixels/2,055 μm and image file size is 1.55 MB. A segmentation analysis around the invading cells was produced and invasion measured as the area covered by the invading cells (percentage confluence) in the scanned field of view. Analysis settings must be adjusted for each cell type in order to achieve an optimal image segmentation that also reflects the degree of invasion. Alternatively, images were imported and analyzed on Image-Pro Analyzer software as described above. Finally, to demonstrate that equivalent data can be generated in the absence of a sophisticated cytometer, microscopic imaging and analysis on Image-Pro Analyzer software was performed using MDA-MB-231 M cells. Further technical specifications for both imaging methods are provided in Additional file 15.
EB differentiation and tumor spheroid-EB confrontation culture
EB formation and cell differentiation was performed as described  with some modifications. R1 cells (1 to 5 × 103 cells/ml) in ESC medium containing 50 ng/ml vascular endothelial growth factor (VEGF) (Sigma-Aldrich Company Ltd., Dorset, England) were plated in ULA 96-well round-bottomed plates, 200 μl/well. A 50% medium refresh was performed daily from day 3 to day 14. Automated image analysis was performed using a Celigo cytometer as described and growth curves were generated.
For confrontation culture experiments , day 4 tumor spheroids and day 5 to day 7 EBs were used. Using a multichannel pipette, 100 μl of culture medium was removed from each well of the spheroid plate and replaced with 100 μl of ESC medium. This procedure was repeated three times, and after the last wash, remaining medium containing a single spheroid/well was transferred into the EB ULA plate to give a final volume of 200 μl/well, with one spheroid and one EB in each well. Confrontation cultures were incubated at 37°C in 5% CO2 for 60 h. To exemplify responses to a targeted agent, cocultures were treated with 17-AAG (5 μM) or with vehicle (control). For timelapse studies, videos were acquired on the Olympus microscope as described above. Images were recorded hourly over 65 h. Where indicated, GFP-transduced U-87 MG tumor spheroids were used and brightfield and fluorescence images of the confrontation cultures were obtained over 55 to 60 h on a Celigo cytometer, using the Expression Analysis application with the option to scan 1/16 field of view/well.
Determination of coefficient of variation (CV) and frequency distribution of tumor spheroid size
During assay optimization, the reproducibility of spheroid size was measured for U-87 MG, KNS42 and LICR-LON-HN4 cells by determining their volumes on day 4. The intraplate and interplate CV for each cell line was calculated in at least three separate experiments over three different batches of plates. The frequency of spheroid volumes was plotted to illustrate Gaussian distribution of the sample for U-87 MG spheroids on day 4 (untreated) and on day 14 (controls and compound treated) in a representative growth kinetic study.
Cell viability assay
Cell viability was measured using a CellTiter-Glo Luminescent Cell viability assay (Promega, Madison, WI, USA). In pilot studies to determine optimal incubation times, day 4 U-87 MG spheroids were incubated for 10, 30 and 60 minutes with CellTiter Glo reagents; all gave comparable results, hence a 10 minute incubation time was adopted for later studies. U-87 MG, KNS42, MDA-MB-231 and LICR-LON-HN4 spheroids were established as described. For cell monolayers (two-dimensional), cells were plated into 96-well black-sided flat-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) at the following densities: 600 cells/well (U-87 MG, KNS42); 2,000 cells/well (MDA-MB-231) and 3,000 cells/well (LICR-LON-HN4). The outer wells of the plates were filled with phosphate-buffered saline (PBS) to reduce the effects of evaporation. Then, 4 days later, three-dimensional and two-dimensional cultures were treated with a range of concentrations of 17-AAG, PI-103, CCT130234 or appropriate vehicle. Cultures were incubated for 72 h and the CellTiter-Glo assay kit was used following the manufacturer's instructions. After 10 minutes of incubation with the CellTiter-Glo reagent, three-dimensional cultures were pipette mixed, aspirated with multichannel pipettes and transferred into black-sided, flat-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) for luminescence measurement on a Synergy 2 SL Luminescence microplate reader (BioTek, Potton, UK) to generate GI50 values.
Tumor spheroids (TSs), EBs and TS-EB cocultures were processed for immunohistochemistry as follows. Three-dimensional structures were collected in V-bottomed 15 ml Falcon tubes and allowed to sediment. Supernatant was removed by gentle aspiration and pellets washed once with PBS. After repeated sedimentation, supernatant was removed and 4% paraformaldehyde (PFA) was added and left overnight. The next day, PFA was removed and, using a warmed p1000 pipette tip, 1 ml of liquid agarose (4% w/v in sterile water) was gently added to the three-dimensional structures that were then collected and transferred into Tissue-Tek®Cryomolds® (Sakura Finetek UK Ltd., Thatcham, UK). Solidified blocks were transferred into 50% ethanol for 1 h and then 80% ethanol before embedding in paraffin. Sections of 4 μm were cut using a semiautomated microtome HM 350 S (Microm International GmbH, Walldorf, Germany); the tissue sections were deparaffinized and rehydrated in water. Immunohistochemistry was performed as previously described  and sections were stained for Ki67 (monoclonal antibody, clone MIB1, Dako UK Ltd., Ely, UK), GLUT-1 (no. 07-1401, Millipore UK Ltd., London, UK) and CD34 (monoclonal antibody, clone MEC14.7, Abcam, Cambridge, UK). Slides were counterstained with hematoxylin and eosin and coverslipped with DPX mountant for microscopy (VWR Int., Lutterworth, UK).
The Student t test with Welch's correction was performed where indicated using Prism5 (Graphpad Software, La Jolla, CA, USA; http://www.graphpad.com/welcome.htm). Values are expressed as means ± SD. P values ≤ 0.05 were considered statistically significant. For correlations between spheroid size (volume) and luminescent signal as well as for spheroid size and number of viable cells, Spearman correlation analysis was performed.