Periostin antisense oligonucleotide suppresses bleomycin‐induced formation of a lung premetastatic niche for melanoma

Figures

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  Figure 1. Up‐regulation of periostin expression associated with progression of lung fibrosis. A, Serial sections of the lung from non‐treated mice (Day 0) and mice killed 4, 8, 12, or 16 d after receiving intratracheal bleomycin were subjected to hematoxylin‐eosin (H&E) and Masson's trichrome staining as well as to immunohistochemical analysis with antibodies to periostin. Scale bars, 100 μm. B, Ashcroft score for lung fibrosis and C, percentage of periostin‐positive cells in the lungs of mice treated as in (A). D, Sections of the lung from mice treated as in A, were subjected to immunohistochemical analysis of α‐smooth muscle actin (α‐SMA) expression. Scale bars, 50 μm. E, Density of α‐SMA‐positive cells in fibrotic areas of the lung from mice treated as in (A). All quantitative data are means ± SD for 4 mice in each group. **P < .01, ***P < .001 (one‐way ANOVA)

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  Figure 2. Bleomycin‐induced lung fibrosis promotes lung metastasis of B16 melanoma cells. A, Schematic representation of the experimental protocol. Mice received bleomycin (BLM) or saline intratracheally (i.t.) 10 d before injection of B16 murine melanoma cells into the tail vein. Mice were killed for analysis 11 d after cell injection. B, Representative images of the lungs of mice at day 21 and quantification of the number of macroscopically detectable lung metastases. Scale bars, 5 mm. Quantitative data are for individual mice and means ± SD for each group. C, Hematoxylin‐eosin (H&E) staining and immunohistochemical analysis of MelanA as well as quantification of the percentage tumor area based on MelanA staining for the lungs of mice killed on day 21. The boxed area in the low‐magnification image of bleomycin‐treated lung tissue is shown at higher magnification in the images on the right. Scale bars represent 1 mm and 100 μm for low‐ and high‐magnification images, respectively. Quantitative data are means ± SD for 5 (bleomycin) or 6 (saline) mice. D, Representative immunohistofluorescence staining of MelanA and periostin in metastatic tumors from saline‐ or bleomycin‐treated mice on day…

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  Figure 3. Effects of giving periostin antisense oligonucleotides (ASO) on the bleomycin‐treated lung. A, Mice were injected intratracheally with a control or periostin ASO at 2 and 7 d after receiving bleomycin and were killed for analysis on day 8. Serial sections of the lung were stained with hematoxylin‐eosin (H&E) and subjected to immunohistochemical analysis of periostin expression. Scale bars, 100 μm. B, Ashcroft score for lung fibrosis in the mice on day 8. C, Quantification of the percentage of periostin‐positive cells in the lung on day 8. D, Lung sections from the mice on day 8 were subjected to immunohistochemical analysis for quantification of the density of α‐smooth muscle actin (α‐SMA)‐positive cells in fibrotic areas. Scale bars, 100 μm. E, Representative immunohistofluorescence staining of lung fibrotic lesions for α‐SMA and Ki‐67 at day 8. Nuclei were stained with DAPI. Scale bars, 100 and 10 μm for low‐magnification and high‐magnification (insets) images, respectively. All quantitative data are means ± SD for 8 and 6 mice injected with the control or periostin ASO, respectively. *P < .05 (Student's t test)

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  Figure 4. Inhibition of periostin expression suppresses metastasis of melanoma cells to the fibrotic lung. A, Schematic representation of the experimental protocol. Mice received bleomycin (BLM) intratracheally 10 d before tail vein injection of B16 murine melanoma cells. The mice were also injected intratracheally with control or periostin antisense oligonucleotides (ASO) 2 and 7 d after receiving bleomycin and were killed for analysis on day 21. B, Representative images of the lungs and quantification of the number of macroscopically detectable lung metastases at day 21. Scale bars, 5 mm. Quantitative data are for individual mice and means ± SD for each group. C, Hematoxylin‐eosin (H&E) staining of lung tissue and quantification of the percentage tumor area based on immunohistochemical staining for MelanA at day 21. Scale bars, 1 mm. Data are means ± SD for 4 (control ASO) or 3 (periostin ASO) mice. *P < .05 (Student's t test)