Figures & data
Figure 1. Aging is associated with increased lung metastases
(A-C) WT C57BL/6 mice, ages 3,5,7,9 and 12 months old were injected with 1 × 106 WT LLC cells into the hindflank. Per IACUC regulations of endpoints, tumor formation and size were followed until a tumor size of ≥15 mm in length or width was established. All mice were then sacrificed, and lungs harvested and processed for examination of metastases. (A) All mice developed tumors at the site of injection, with no differences in size observed at day 17. (B) Percentage of mice in each group where lung metastases were identified. (C) Number of lung metastases in each animal in each group. (D, E) Young (~3.5 months) and aged (12 months) WT C57BL/6 mice were injected intravenously with 1 × 105 WT LLC cells and sacrificed at day 13. (D) Percentage of mice in each group where lung metastases were identified. (E) Number of intravenous lung tumors in each animal in each group. Aged mice developed more tumors in the lung (6.3 ± 3.4) compared to young mice (1.6 ± 3.1), *P < .05.
Figure 2. Bleomycin treatment is associated with increased metastases, but only in aged lungs
Young (~3.5 months) and aged (~9.5 months) WT C57BL/6 mice were instilled intratracheally with PBS (young, n = 7; aged, n = 8) or bleomycin (Bleo; young, n = 7; aged, n = 7). At day 14, 1 × 106 LLC cells were injected subcutaneously into the hindflank. (A) Change in weight in aged mice is shown. Bleomycin significantly induced weight loss in all mice at day 14 and day 21 post-instillation. There were no statistically significant differences in weight loss between young and aged mice. Mice instilled with PBS did not experience weight loss. (B) Representative H&E images depict PBS and bleomycin treated aged mice at 4x magnification. Tumor metastases are observed in all images (indicated by arrows), while fibrosis is readily apparent in the bleomycin treated lungs. (C) Number of lung metastases at day 31 (17 days post LLC injection). Young mice failed to develop lung metastases in either group, while bleomycin instillation in aged mice increased the number of lung metastases compared to PBS (6.0 ± 2.5 vs 1.4 ± 1.2), ***P < .001.
Figure 3. Fibronectin EDA is dispensable for aged-related lung cancer progression
WT C57BL/6 and FN EDA KO mice were injected with 1 × 106 WT LLC cells into the hind flank or 1 × 105 LLC intravenously. (A) Tumor volume in young WT (~2.5 months, n = 5) and FN EDA KO (2 months, n = 3) mice and in aged WT (10 months, n = 5) and FN EDA KO (10 months, n = 3) mice. (B) Number of metastases. Young mice in both groups failed to develop metastases. There were no statistically significant differences in the number of metastases between aged WT (1.6 ± 1.1) and aged FN EDA KO (4.3 ± 4.2) mice. (C) Number of intravenous lung tumors in aged WT and FN EDA KO mice is shown. There were no statistically significant differences in the number of lung tumors between aged WT (3 ± 2.5) and aged FN EDA KO (6.5 ± 6.3) mice.
Figure 4. Fibronectin EDA is required for bleomycin-induced augmentation of metastasis
WT C57BL/6 (~10 months) and FN EDA KO (~9 months) mice were instilled intratracheally with PBS or bleomycin. At day 14, 1 × 106 LLC cells were injected subcutaneously into the hindflank. (A) Ashcroft scores of 5 blinded reviewers showing similar fibrosis. (B) Representative trichrome stained images at 4x magnification depict lung fibrosis of varying severity in both bleomycin treated WT and FN EDA KO mice. (C) Number of lung metastases in aged mice are shown at day 31 (17 days post LLC injection). Bleomycin increased the number of lung metastases in WT mice (12.1 ± 10.0 vs 33.7 ± 8.6, ***P < .001) but not in FN EDA KO mice (7.5 ± 7). (D) Tumor volume was unchanged at the time of sacrifice (day 31).
Figure 5. Lung metastases develop primarily in fibrotic areas
(A) Paraffin embedded sections from WT C57BL/6 mice from were stained with Masons trichrome. The percentage of LLC metastases in areas of lung fibrosis in each individual mouse is shown. (B) Representative trichrome stained images at 0.4x and 4x magnification depict prominent lung fibrosis and LLC metastases (green circles). (C) LLC migration on plastic and cellular fibronectin (cFN) coated 24-well plates. (D) After 24 hours 59.1 ± 3.2% of the initial wound area remained on plastic vs 26.9 ± 2.7% on cFN. *** P < .001. (E) LLC cells were grown in 48-well plates in DMEM or DMEM with 10 ug/mL cFN for up to 7 days. Cells were refed every 2–3 days. Cell proliferation was quantified using Cell Titer-Glo Luminescent Cell Viability Assay Kit. The presence of cFN increased LLC cell proliferation ***P < .001.
Figure 6. Effect of Immunity on Lung Cancer Progression
Young (2 months; 7 animals) and Aged (9 months; 9 animals) NSG mice were injected subcutaneously into the hindflank with 1 × 106 LLC cells, and followed for tumor formation. Mice were sacrificed at day 17 and lungs removed and processed for examination of metastasis. (A) There were no differences between groups in tumor volume at the site of injection, or (B) the number of metastases to the lung. Although the number of metastases to the lung between groups was statistically unchanged, aged NSG mice developed larger metastases (in size) to the lung. (C) Graph shows numbers of metastases in 5 groups of varying tumor size. (D) Representative H&E stained lung images showing lung metastases at 10X are shown.
