Fig 1.
Primary tumor and pathological slides.
(A). Photo-macrography of the resected specimen of PIS. B: Composite photograph of the specimen derived from the left main pulmonary artery stained by elastic-van Gieson staining. The specimen contained different tumor, fibrotic, and necrotic components. (C) Low magnification photomicrographs (×200) with hematoxylin-eosin (HE) staining; (D) High-magnification photomicrographs with HE staining (×400). E-J were photomicrographs with immunohistochemistry. (E) Vimentin, (F) Murine double minute 2 (MDM2), (G) Cyclin-dependent kinase 4 (CDK4), (H) CD31, (I) CD34, (J) α-smooth muscle actin. (K) S-100 protein. Tm: tumor component; Fib: fibrotic component; Nec: necrotic component. Scale bar shows 100 μm unless otherwise stated.
Fig 2.
Characteristics of PIS-1 cells in vitro.
(A) PIS-1 cells present as small and spindle-shaped cells microscopically. Scale bar, 200 μm. (B) Transformed foci of PIS-1. Scale bar, 300 μm. (C) Immunocytochemistry shows that PIS-1 cells were diffusely positive for vimentin, murine double minute 2 (MDM2), and CD44, and negative for α-smooth muscle actin (SMA), von Willebrand factor (vWF), CD31, and desmin. Scale bars, 50 μm. Nuclei stained with DAPI are represented by blue.
Fig 3.
Malignant potential of PIS cells.
(A) Representative multicolor fluorescence in situ hybridization (mFISH) karyogram from PIS-1 cells. (B) Starvation assay. Three cell groups (PIS-1, A549, EC-like) were incubated with each serum-free medium. *p < 0.05 vs. EC-like cells. (C) Colony formation assay. Cell suspensions containing 5 × 103 PIS-1, A549, or EC-like cells were seeded on the semisolid soft agar layer. After seven days of incubation, the formed colonies and soft agar were dissolved and stained with fluorescent dye followed by reading with a fluorescence plate reader. RFU: relative fluorescence units, NC: negative control, *p < 0.05, vs EC like cells; §p < 0.05, vs negative control of each group. (D) Invasion assay. To evaluate the invasion and migration potential of PIS-1 cells, Matrigel invasion chambers (24-well) and Biocoat control culture inserts were used. BALB/3T cells were used for negative control according to the datasheet. Cell suspension (2.5 × 104) and serum-free medium were placed into the upper wells, and the incubation medium with FBS was added into lower wells. After 22 hours of incubation, the cells that migrated or invaded through membranes were counted microscopically. The percent invasion was defined as the ratio of invaded to migrated cells. Error bars show standard deviation. *: p < 0.05.
Fig 4.
Tumorigenicity of PIS-1 cells.
PIS-1 cells (2 × 106) were injected subcutaneously or intravenously into a male C.B-17/lcr-scid/scidJcl (SCID) mouse. (A) Subcutaneous enlarged tumor 28 days after subcutaneous injection (arrow head). Skin around the tumor was removed. (B) Neo tumor vessels were connected with the subcutaneous tumor and the back of the mouse (arrow head). (C) Multiple lung tumors 49 days after intravenous injection (arrow heads). (D) PIS-1 cells labeled by PKH-26 were present at the central area of a subcutaneous tumor. Scale bars, 100 μm. (E) Lung tumors were composed of cells positive for PKH-26. Scale bars, 50 μm. Nuclei stained with DAPI are represented by blue.
Fig 5.
PCR array and western blot analysis for tyrosine kinase receptors.
(A) PCR array analysis showing expression of tyrosine kinase receptors in PIS-1 cells in comparison to A549 cells. Vertical axis is in log scale. (B) Western blot analysis for whole protein of PIS-1 and A549 cells. The signal strength of platelet-derived growth factor receptor (PDGFR)-α (C), phospho-PDGFR-α (D), vascular endothelial growth factor receptor 2 (VEGFR2) (E), and phospho-VEGFR2 (F) were expressed as a ratio to β-actin. *:p<0.05 vs A549 cells. (G) Western blot analysis for nuclear protein of PIS-1 and A549. The signal strength of PDGFR-α (H), phospho-PDGFR-α (I), VEGFR2 (J), and phospho-VEGFR2 (K) were expressed as a ratio to lamin. Error bars show standard deviation.*:p < 0.05 vs A549 cells, N.S.: not significant.
Fig 6.
Immunocytochemistry and immunohistochemistry for tyrosine kinase receptors in PIS-1 cells.
(A) Immunocytochemistry shows that PIS-1 cells were positive for platelet-derived growth factor receptor (PDGFR)-α, PDGFR-β, and vascular endothelial growth factor receptor (VEGFR)-2. (B) Immunohistochemistry shows that the tumor components of the surgical specimen, and subcutaneous and pulmonary tumors formed from PIS-1 cells and were diffusely positive for PDGFR-α, PDGFR-β, and VEGFR-2. All scale bars, 50 μm unless otherwise stated.
Table 1.
Expression of tyrosine kinase receptors of PIS-1 on PCR array.
Fig 7.
Suppressive effect of pazopanib on the proliferation of PIS-1 cells.
(A) Effect of pazopanib, which is a multikinase inhibitor, on the proliferation of PIS-1 cells 14 days after treatment. *p < 0.05, vs control; §p < 0.05, vs 0.1 μM; †p < 0.05, vs 1.0 μM group. (B) Tumor volumes of mice with 14-day administration of pazopanib compared to those of the control. *p < 0.05, vs control of each day. (C) The mean weights of subcutaneous tumors resected from mice of the pazopanib group compared to those of the control group. Error bars show standard deviation. *p < 0.05, vs control.