Figure 6 from Metastasis-Associated Wound Repair Promotes Reciprocal Lung Epithelium Activation and Breast Cancer Metastatic Outgrowth

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Targeting CREB-regulated AT2 secreted factors to block AT2–breast cancer (BC) interactions and metastatic outgrowth. 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 RNA-seq datasets, percentage indicated. C, t-distributed stochastic neighbor embedding visualizations of cells from metastatic mouse lungs clustered by gene expression and colored by Pde4 isoform gene expression. D, Percent cell viability was measured by crystal violet assay after a 3 days of treatment with vehicle DMSO (0) or increasing concentrations of the PDE4 inhibitor (PDEi) ROF. Data were 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 2 nmol/L ROF: 5 days for A549 cells and 7 days for iAT2 cells. The percent difference in cell viability, between DMSO and 2 nmol/L ROF treated cells, was calculated for AT2 cells cultured alone (−) or co-culture with TNBC cells. Mean ± SEM (one-way ANOVA with Tukey 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 2 nmol/L ROF: 5 days for A549 cells and 7 days for iAT2 cells. The percent difference in cell viability, between DMSO and 2 nmol/L 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 intravenously injected into female mice, and oral (PO) treatment with 5 mg/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 CC3 by IHC. The percentage of positively stained cells was scored per metastasis and averaged per mouse; mean (unpaired t tests with Welch correction). I, Metastatic lungs were stained for the Met-1 mammary-specific marker PyMT. Average metastatic burden was quantified in serial sections as the number of PyMT+ metastases or the size of PyMT+ metastases as measure by area in μm2. The average of three serial sections was calculated per mouse. Mean (n = 5–8 mice per group, unpaired t tests); *, P ≤ 0.05. J, Metastasis-adjacent and -distant lung tissue was stained for cell-specific markers of wound repair (adjacent: n = 16 metastases from five mice per group; distant: n = 64 regions of interest from 5–8 mice per group). The number of positively stained cells, normalized to the total number of cells, was quantified in the 300 μm surrounding metastases for the adjacent lung and the full region of interest in the distant lung. Mean ± SEM (multiple unpaired t tests with Welch correction); *, P ≤ 0.05; **, P < 0.01. K, Lungs adjacent and distant to large metastases were stained for AT2 cell activation. Shown are representative images of large metastases from control (Ctrl) and ROF-treated mice indicating proSP-C and Ki67 double-positive proliferating AT2 cells (white arrows); scale bar, 5 μm, inset zoom 3x. The percentage of proSP-C–positive cells that are Ki67-positive was quantified in the lung (adjacent: n = 21 metastases from 3–4 mice per group; distant: n = 50 regions of interest from 4–6 mice per group). Mean ± SEM (unpaired t tests with Welch correction); **, P < 0.01. L, The mean fluorescent intensity (MFI) of proSP-C in metastasis-adjacent proSP-C+ AT2 cells was quantified in Ki67+ and Ki67− cells (n = 21 metastases from 3–4 mice per group). Mean ± SEM (t tests with Welch correction); ****, P < 0.0001. Shown are representative distributions demonstrating the number of cells per mouse expressing increasing levels of proSP-C.

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