Fig. 3.

LPA2 inhibition disrupts spheroid formation in 4T1 mouse mammary carcinoma cells. 4T1 cells were plated in triplicate in ultra-low adherence 24-well plates at a density of 7.5 × 10³ cells per well in spheroid formation medium (RPMI with 200 μM L-glutamine, 100 U penicillin, 100 μg/ml streptomycin, 1× N-2 supplement, 20 ng/ml mouse EGF and 20 ng/ml mouse FGF). Cells were incubated at 37 °C with 5% CO₂ for 4 days to allow spheroid formation, then treated with a range of 0–3 μM Amgen 35 (LPA₂ antagonist), BMP-22 (ATX inhibitor) or BESA-3 (ATX/LPA₁ inhibitor) and incubated an additional 3 days. Cells were surveyed under 100× magnification (microscopy panels), and spheroids were manually counted, plotted versus Amgen 35 concentration and GraphPad Prism v. 5.0a was used to perform non-linear regression in a variable slope model to determine the IC₅₀ of Amgen 35 for spheroid formation (dose-response panel). Finally, cell viability was determined using Promega CellTiter Blue reagent as per manufacturer’s instructions. Briefly, 50 μl CellTiter Blue were added to each well, and plates were incubated at 37 °C for 3 h prior to measuring fluorescence at excitation/emission wavelengths of 560/590 nm, respectively. Relative fluorescence was then background-corrected and normalized to percentage vehicle signal for each compound tested (bar graph panel). GraphPad Prism was used to perform one-factor ANOVA with Bonferroni’s post-test to determine whether cell viability differed significantly between vehicle and drug treatment (** = p < 0.01, *** = p < 0.001; n = 3).