terGlo reagent was added to each well and was mixed by re-suspension. The plates were incubated at room temperature on a shaker for 30 min and re-suspended again. The relative luminescence units were measured using a microplate reader. Measurement of secreted growth factors/cytokines Supernatants were collected from the 5-day co-cultures were collected and were either used immediately or were stored at -80C until further use. The Human cytokine/chemokine 96-well plate assay was used to measure 42 different PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666584 cytokines in the supernatants. Specific analytes that were not included in the 42-plex ) were purchased and used to measure additional growth factors. This assay was performed according to the manufacturer’s instructions. Briefly, 2.5 x 105 tumor cells or fibroblasts per well were seeded as monocultures or for co-cultures 1×105 tumor cells were combined and 1.5 x 105 fibroblasts per well and were seeded as co-cultures in 2 ml of DMEM supplemented with 5% Panexin NTA on polyHEMA polyHEMA-coated 6-well plates 
as described for the cell viability assay. Undiluted supernatants were incubated with capture beads or a bead mix overnight at 4C in the provided 96-well filter plates. Then, the beads were washed and incubated with the detection antibody for one hour at RT in the dark, followed by incubation with Phycoerythrin-labeled streptavidin for 30 minutes at RT in the dark. Next, the beads were washed twice, and the mean fluorescence get BHI-1 intensity was measured using a Bioplex 2000 instrument. The analysis was performed using the 5-parameter logistic regression tool in Bioplex manager software. Microscopy The cells were cultured as a mono-culture or a co-culture as indicated for the cell viability assay, and images were captured on day 5 using an inverted microscope at a 20x magnification. For confocal imaging, the cells were trypsinized and washed once with warm PBS followed by a wash with warm serum-free DMEM. The tumor cells were incubated in 10 M Cell Tracker Green 5-chloromethylfluorescein diacetate, and the fibroblasts were incubated in 10 M Cell Tracker Red CMTPX in serum-free medium for 15 min. Then, the cells were washed twice with warm PBS. The labeled tumor cells were cultured either alone or in co-culture with the labeled MRC5 fibroblasts for 5 days in polyHEMA-coated 6-well plates. On day 5, the spheroids were washed three times with warm PBS and then fixed using 4% PFA in PBS for 20 min at RT. After fixation, the spheroids were washed once with PBS and mounted in mounting medium before PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667083 imaging. Z-stack sections of the spheroids were captured using a confocal laser scanning microscope. Statistical analysis Data analysis was performed using GraphPad Prism Software version 6.0. Cell proliferation in the mono-cultures and co-cultures and the responses of the mono-cultures and the co-cultures to treatment with therapeutics agents were compared using two-way ANOVA, followed by posttest analysis using the Holm-Sidak method. P<0.05 was considered to be significant. Results Three dimensional co-culture of cancer cells with fibroblasts induces differential survival We tested different ratios of tumor cells and MRC5 fibroblasts at various time points to understand the growth kinetics of the co-cultures. Although increased survival was observed at all of the tested ratios, the ratio of 1 tumor cell to 1.5 MRC5 fibroblasts resulted 4 / 18 Influence of Fibroblasts on Tumor Cell Growth in the highest cell survival. We further observed that cell s
