Figure 6. Conditioned medium from Pros1-ablated BMDMs enhances Lewis lung carcinoma cancer cell aggressiveness.

Lewis lung carcinoma (LLC) cells were educated (24 hours) by the indicated conditioned medium (CM) and subject to a battery of in vitro assays. (A and B) Colony survival after 10 days. (A) Representative images (number of cells plated/well is indicated) and (B) quantification of surviving colonies. The mean ± SEM; n = 6 mice/group. ****P < 0.0001; 2-way ANOVA. (C) Representative images and (D) quantification of soft agar colonies (arrows) after 21 days. Scale bars: 50 μm. Average ± SEM; n = 8 mice/group. **P = 0.002; t test. (E) Representative Western blots and (F) quantifications showing ERK and AKT activation from educated LLC cells. n = 8 mice/group; 3 independent experiments. The average ratio ± SEM is plotted. *P = 0.018, **P = 0.006; t test. Representative Western blot (G) and quantifications (H) for MMP9 protein in lungs of metastatic mice. n = 7–8 mice/group in 2 independent experiments. The average ratio ± SEM is plotted. ***P = 0.001; t test. A representative Western blot (I) and quantifications (J) showing E-cadherin, N-cadherin, and vimentin in educated LLC cells. n = 9–10 mice/group in 3 independent experiments. The average ratio ± SEM is plotted. *P = 0.04, **P = 0.009; t test. (K and L) Invasion assay: Representative images (K) and quantifications (L) of cells that invaded through Matrigel after education with the indicated CM. Scale bars: 50 μm. (L) The average cell number ± SEM of invaded cells is shown from 7–10 different fields, n = 7 mice/group in 3 independent experiments. ***P = 0.0007; t test. NS, nonsignificant.