Fig. 3: AFs chemoattract and activate NF and promote MPM cell chemoresistance.
From: Crosstalk with lung fibroblasts shapes the growth and therapeutic response of mesothelioma cells
A MRC-5 cells were placed in the upper part of a Boyden chamber with condition media from the indicated AFs, NFMet or MRC-5 cultures placed in the bottom chambers for the indicated time. Data shown are the mean ± SD of 3 biological replicates normalised to results obtained with NFMet CM treatment. Two-way ANOVA with Dunnett’s multiple comparison tests were performed to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). B MRC-5 cells were seeded in CM from the indicated AFs or NFMet for the indicated time. Cell lysates were analysed by western blotting for the indicated proteins. Left panel: Data shown are representative of 4 biological replicates. Right panel: Optical densitometry quantification of 4 biological replicates. C MRC-5 cells seeded for 72 h in CM from indicated AFs, NFMet or MRC-5 cells were subjected to a Caspase 3/7 substrate-based activity assay in 3D cultures. Data are mean ± SEM from 3 biological repeats. D Comparison of growth of 3D spheroids composed of either MPM or Met-5A cells alone or in co-culture with the corresponding AFs or NFMet, respectively. Left: Representative brightfield pictures acquired at Day 1 of the assay (24 h post seeding). Scale bar: 1 mm. Right: ImageJ-based quantification of the size of coculture microspheres at the indicated timepoints. E Representative fluorescence microscopy images of 3D cocultures of differentially-labelled MPM and corresponding AF cell lines. Scale bar: 1 mm. F MPM cells were seeded for 72 h in CM from the corresponding AFs, NFMet or MRC-5 cells and subjected to a Caspase 3/7 substrate-based activity assay in 3D cultures. Data are mean ± SEM from 3 biological repeats. G 3D spheroids made of either Meso 8 cells alone or cocultures of Meso 8 and AF 8 were treated with cisplatin/pemetrexed or diluent (NT). Left panel: representative images of n = 3. Scale bar: 1 mm. Right panel: Cell viability was determined using the Cell TiterGlo 3D assay. Data are mean ± SEM of n = 4. Statistics: Two-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ****P < 0.001).
