Figure 3.

Effect of α-AnxA1 neutralizing antibody (nAb) on spheroids and EMT of cE1 cells. A, Addition of α-AnxA1 nAb to cE1 SC^hi and SC^med cells reduced the number of spheroids grown in the presence of CAF CM. B, Unsorted cE1 cells showed increase in expression of Snail, Slug, Twist, and Oct4 after treatment with CAF CM, AnxA1 enriched CAF CM AS fraction from two CAF cultures (CAF1 and CAF2 in parallel western blot analyses) and Ac2-26. cE1 cells treated with α-AnxA1 nAb in the presence of CAF CM, AnxA1 enriched CAF CM AS fractions and Ac2-26 had reduced expression of Snail, Slug, Twist and Oct4. All conditions were analyzed using untreated media control and compared to the experimental control condition of media + IgG. CAF1 and CAF2 were used for two independent repeats, and CAF3 was analyzed in two additional independent repeats. C–D, Analysis of pathways associated with AnxA1. C, α-TGFβ western blots revealed close to two-fold increase in TGF-β1 after treatment of cE1 spheroids with increasing concentrations of Ac2-26, compared to expression in media control and media + IgG. TGF-β1 expression was reduced after α-AnxA1 nAb was combined with Ac2-26 treatment. D, qRT-PCR indicated activation of MAPK and TGFβ pathways, as shown by significant increase in Mapk and Tgfbr2 expression after addition of Ac2-26 to cE1 cells grown in 3-D culture. Control is media with IgG. In all panels, statistical significance is indicated by •, P < 0.05; *, P < 0.01; **, P < 0.001.