Journal: bioRxiv
Article Title: Metastasis-associated wound repair promotes reciprocal activation of the lung epithelium and breast cancer metastases during outgrowth
doi: 10.1101/2025.07.23.666437
Figure Lengend Snippet: A, Model indicating the cellular interactions being interrogated: targeting AT2-BC interactions during metastatic outgrowth. B, The proportion of AT2 secreted factor genes enriched following BC interactions that are CREB-regulated for all three AT2 RNAseq datasets, percentage indicated. C, t-SNE visualizations of cells from metastatic mouse lungs clustered by gene expression and colored by Pde4 (phosphodiesterase 4) isoform gene expression. D, Percent cell viability was measured by crystal violet assay after a 3 day treatment with vehicle DMSO (0) or increasing concentrations of the PDE4 inhibitor roflumilast. Data was normalized to the mean absorbance of DMSO-treated cells; mean ± SEM. E, AT2 cell viability was measured by crystal violet assay following co-culture with TNBC cells and treatment with 2nM roflumilast (ROF): 5 days for A549 cells and 7 days for iAT2 cells. The percent difference in cell viability, between DMSO and 2nM ROF treated cells, was calculated for AT2 cells cultured alone (-) or co-culture with TNBC cells. Mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test); * p ≤0.05, *** p <0.001. F, TNBC cell viability was measured by crystal violet assay following co-culture with AT2 cells and treatment with 2nM ROF: 5 days for A549 cells and 7 days for iAT2 cells. The percent difference in cell viability, between DMSO and 2nM ROF treated cells, was calculated for TNBC cells cultured alone (-) or co-culture with AT2 cells. Mean ± SEM (unpaired t -tests); * p ≤0.05. G, Schematic of metastatic outgrowth experimental design using the late-stage Met-1 metastasis model ( n =5-8 mice per group). Met-1 cells were IV injected into female mice and oral treatment with 5mg/kg ROF began 3 days later. Mice were treated daily for 3 weeks and metastatic lung tissue was collected. H, Lungs were stained for cell turnover markers Ki67 and cleaved-caspase 3 (CC3) by IHC. Shown are representative images of lung metastases; scale bar = 100µm. The percentage of positively stained cells was scored per metastasis and averaged per mouse; mean (unpaired t -tests with Welch’s correction when appropriate). I, Metastatic lungs were stained for the Met-1 mammary-specific marker PyMT. Metastatic burden was quantified in serial sections as the area of PyMT-positive metastases, means per group are indicated as dotted lines (unpaired t -test). Representative data is shown from a single section indicating the number and size of metastases per mouse; each notch on the x-axis represents an individual mouse.
Article Snippet: Roflumilast (ROF) was purchased from Selleck Chemical LLC. (No. S2131), and cilomilast (CILO) was purchased from MedChemExpress (No. HY-10790).
Techniques: Gene Expression, Crystal Violet Assay, Co-Culture Assay, Cell Culture, Comparison, Injection, Staining, Marker
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