Extended Data Fig. 4: Pdgfc/PDGFC expression in the aged microenvironment and its role in release from dormancy.
a, Transcriptional profiling of C57BL/6 mouse lungs treated with vehicle or bleomycin (from GSE40151). Analysis of Pdgfa-d and Plat expression in lungs 7 and 14 days post-treatment, n = 8 mice per group except n = 7 mice for vehicle 14 days. b, Associated with Fig. 2i. Transcriptional profiling of human non-cancerous lung tissues (GSE23546). Correlation of PDGF expression and expression of the age-associated genes EDA2R, ITGBL1, FRZB and SFRP115,17 in lung samples from patients aged 4 − 85 years of age (n = 1197 patients). c, Expression of Pdgfc in the aged (18-month) C57BL/6 mouse lung (n = 6 mice, 4 females and 2 males). Lung tissue was dissociated and FACSorted for the following populations: macrophages (CD45+ F4/80+), other immune cells (CD45+ F4/80-), endothelial (CD45- CD31+), epithelial (CD45-, CD31-, EpCAM+) and PDGFRα- (cells negative for all markers) and PDGFRα+ fibroblasts. d, Pdgfc expression in GFP+ mouse CAFs compared to 10T1/2 fibroblasts and ER+ mouse tumour cells (RT-qPCR). e, GFP+ mouse CAFs were transduced with two non-targeting control shRNAs (shNTC1, shNTC2) or two independent shRNAs targeting Pdgfc (shPdgfc1, shPdgfc5). RT-qPCR to assess Pdgfc expression. f, TSAE1-mChLuc2 cells were cultured in BME assays (see Extended Data Fig. 2f) for 9 days with or without shNTC or shPdgfc CAFs and then imaged on an EVOS microscope; tumour cells (mCherry+) and CAFs (GFP+). Scale bar, 1 mm (phase-contrast images) and 100 µm (fluorescent images). g, TSAE1-mChLuc2 or HRM1-mChLuc2 cells were cultured in BME assays for 9 days (see Extended Data Fig. 2f) with or without immortalised young or aged mouse lung fibroblasts. Cells were imaged on an EVOS microscope: tumour cells (mCherry+). Scale bar, 1 mm (phase-contrast images) and 200 µm (fluorescent images). a,c-e, Data are presented as mean values; a,c, ±SEM; a, two-way ANOVA; b, two-tailed Pearson’s correlation (line shows linear regression). f,g, Representative of two-independent repeats. g, Two independent young and old fibroblast populations were used with equivalent results.
