(A) GFP-positive tumor cells (2 × 10⁵) were delivered to mice by tail-vein injection. (B) Following humane euthanasia, the trachea was cannulated with 20-gauge intravenous catheter and attached to a gravity perfusion apparatus. The lungs were infused in the vertical position under a constant 20 cm H₂O hydrostatic pressure. (C) The lungs were allowed to cool at 4°C for 20 minutes to solidify the agarose medium solution. Complete transverse serial sections (1–2 mm in thickness) were gently sliced from each lobe with a scalpel, yielding 16–20 lung slices per pair of lungs. (D) 4–5 lung sections were placed on the sterile Gelfoam sections bathing in culture media. (E) Images were acquired and the area of GFP-positive cells in each lung was quantified. Scale bars: 200 μm.

(A) PuMA yields an intact lung microarchitecture. Day 0: bronchioles (B) were lined by epithelia that contact the basement membrane. Alveoli (A) were uniformly expanded throughout the lung, and the alveolar walls (AW, with arrows) were normal in diameter. The alveolar walls contained small numbers of migratory inflammatory cells, pneumocytes (type I and II), and endothelial cells. Blood vessels and alveolar capillaries were expanded by rbcs. Day 7: alveoli remained expanded. There were decreased numbers of migratory cells, pneumocytes, and endothelial cells in the alveolar walls, and many of those that remained contained pyknotic nuclei. Days 14 and 21: alveoli, airways, and large vessels (PA, pulmonary arteries; PV, pulmonary veins) remained expanded. Alveolar capillaries and rbcs were no longer discernible, and the alveolar walls contained fewer migratory cells and pneumocytes (loss of cellularity). Overall, lung microarchitecture was remarkably unchanged. (B) Movat stain was used to examine the connective tissue components of the lung culture. Black elastin fibers were present in large vessels, in the basement membrane supporting the airway epithelia, and within the alveolar interstitium. Black nuclei were scattered throughout the alveolar interstitium. Red muscle surrounded arteries and larger airways (B) and yellow collagen fibers were in the surrounding vascular submucosa and alveolar interstitium. Each of these components was identified at each time point. (C) TEM. Stromal elements composed of collagen microfibers (C) were evident from day 0 through day 21. Scale bars: 100 μm (A and B); 1 μm (C).

(A) Positive and negative control, (B) Ki-67 immunohistochemical staining, and (C) H&E staining of PuMA sections confirmed proliferative competency of metastatic osteosarcoma cells, most notably at days 14 and 21. Tissue from a primary osteosarcoma lesion was used as a positive control for Ki-67. Scale bar: 100 μm.

(A and B) Serial imaging of fluorescently labeled high- and low-metastatic human and murine osteosarcoma cells. Similar numbers of cells were seen at day 0; however, a difference between high- and low-metastatic cells was seen at day 7. Representative fields from lung are shown. Scale bar: 200 μm. (C and D) Quantification of metastatic burden (mean normalized fluorescent area) from A and B reveals a significantly lower sum of GFP-positive cell area in the lung section following injection of low-metastatic human and murine osteosarcoma cells. Similar results were generated using high- and low-metastatic pairs of human and murine breast cancer cells and murine melanoma cells (see Table and Supplemental Figure 4). Quantification of metastatic progression in PuMA was validated using both mean fluorescent area and enumeration of surface metastatic colony count (Supplemental Figure 2). Plotted data represent the mean ± SD.

A direct comparison of the metastatic phenotype of human osteosarcoma cell lines in vivo (experimental metastasis) and in the PuMA was conducted. (A and B) Serial imaging of fluorescently labeled high- and low-metastatic human osteosarcoma cells was conducted in the PuMA. At the identical time points, lungs from mice that had received tail-vein injection of tumor cells were collected and imaged as in the PuMA. Patterns of pulmonary metastatic progression were similar in both in vivo and PuMA. Representative fields from lung are shown. Scale bars: 200 μm. (C and D) Quantification of metastatic burden (mean normalized fluorescent area) from A and B. Identical results demonstrating the similarities in pulmonary metastatic progression for murine osteosarcoma cells was seen in vivo and in the PuMA. Plotted data represent the mean ± SD.

(A) Serial imaging of fluorescently labeled low-metastatic murine breast cells (DB7) in high- (AKR/J) and low-metastatic (DBA/2J) murine host microenvironments. Similar numbers of metastatic cells are seen at day 0 in both lung environments; however, a difference in metastatic progression between high- and low-metastatic microenvironments can be seen at day 7. Representative fields from lung are shown. Scale bar: 200 μm. (B) Reduced metastatic burden (quantified by total normalized fluorescent lung area) was seen in the low-metastatic phenotype of DAB/2J lung sections. Total metastatic burden in each lung section was normalized to day 0 and followed over time. Plotted data represent the mean ± SD.