Fig 1: Three-dimensional cell culture potently influences the expression of enzymes involved in the LT cascade in HT-29 and HCT-116 cells.(A) Representative pictures of the cell monolayers and MCTS grown for 4 or 7 d (seeding density 0.05 × 106 cells per well, respectively). Pictures were taken employing a light microscope (monolayers, scale bar: 100 µm; MCTS, scale bar: 200 µm). One representative picture of three is shown. (B) Average diameters of MCTS grown for 4 or 7 d (4 d, n = 4; 7 d, n = 3). (C) Cell cycle distribution of cells grown as monolayers for 4 or 7 d compared with monolayer controls (co), which were harvested before confluency after 48 h of culture receiving daily medium changes. Cells were analyzed via flow cytometry. (D) Protein expression of 5-LO, cPLA2a, LTA4H, and FLAP. The growth medium in the subconfluent monolayer controls (co) was changed daily, and cells were harvested after 48 h of culture to prevent cell stress. One representative Western blot of three is shown. (E) mRNA expression of 5-LO (ALOX5) and LTC4 synthase (LTC4S). Expression was normalized to the housekeeping gene ACTB and the respective monolayer control (2-??CT method). (F) 5-HETE release from intact and digested HT-29 and HCT-116 MCTS and respective monolayer controls. Intact and digested MCTS, and monolayer controls were stimulated with 2.5 µM Ca2+ ionophore (A23187) in the presence of 1 mM CaCl2 (10 min, 37°C). All incubations received exogenous ARA (20 µM). 5-HETE formation was determined by LC-MS/MS analysis and normalized to the total protein content for each sample. (G) VEGF, IL-8, and TGF-ß1 release from MCTS in relation to the respective monolayer (in %) grown for 7 d. Mediators were determined via Cytometric Bead Array (CBA; VEGF, IL-8) or ELISA (TGF-ß1). Mean release in cell monolayers: HT-29 (VEGF, 5,388 pg/106 cells; IL-8, 272 pg/106 cells; and TGF-ß1, 51,222 pg/106 cells); and HCT-116 (VEGF, 3,830 pg/106 cells; IL-8, 50 pg/106 cells; and TGF-ß1, 149,397 pg/106 cells). Data information: results shown in (B, C, E, F, G) are depicted as the mean + SEM from three independent experiments if not stated otherwise. Squares indicate single data points. (C, E, F, G) Asterisks indicate significant changes versus co determined by two-way ANOVA coupled with Dunnett’s post-test for multiple comparisons (C) or by a t test with Welch’s correction (E, F, G). *P < 0.05, **P < 0.01, and ***P < 0.001. d, days; co, subconfluent monolayer control; MCTS, multicellular tumor spheroids.