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. 2000 Dec;157(6):1893-903.
doi: 10.1016/S0002-9440(10)64828-6.

Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells

S Yano  1 , H Shinohara, R S Herbst, H Kuniyasu, C D Bucana, L M Ellis, I J Fidler
Affiliations

Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells

S Yano et al. Am J Pathol. 2000 Dec.

Abstract

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

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Figures

Figure 1.
Figure 1.
Expression of MMPs, uPA, and TIMPs by human NSCLC cells. A: Zymography. Tumor cells were cultured in serum-free medium to 50% confluence, and then the culture supernatants were harvested for gelatin zymography as described in Material and Methods. B: Northern blot analysis. Tumor cells cultured under sparse (S) and confluent (C) conditions were harvested and mRNA was extracted. Northern blot analysis was carried out as described in the Materials and Methods. Densitometric quantitation was derived from the ratio of the signal of the specific transcripts to that of the 1.3-kb GAPDH (internal control). N. D., not detected.
Figure 2.
Figure 2.
Angiogenic cytokine expression in NSCLC cells in vitro. A: Tumor cell lines were cultured under sparse (S) or confluent (C) conditions, mRNA was extracted, and a Northern blot was performed. Densitometric quantitation was derived from the ratio of the signal of the specific transcripts to that of the 1.3-kb GAPDH (internal control). B: Tumor cells were incubated for 24 hours under sparse (open columns) or confluent (solid columns) conditions, and culture supernatants were harvested. The concentration of angiogenic cytokines in the supernatants was determined by ELISA. At the end of the cell culture, the cells were counted and the number of cytokines produced by 10 cells was calculated. N. D., not detected.
Figure 3.
Figure 3.
VEGF/VPF expression in lung metastasis by NSCLC cells and vascular permeability. Note that lung metastases produced by PC14PE6 cells expressed higher levels of VEGF/VPF than those produced by H226 cells. Leaking of Evans blue dye from vessels was observed in the diaphragm of mice bearing PC14PE6 cells but not H226 cells. Bar, 200 μm.
Figure 4.
Figure 4.
Expression of VEGF/VPF isoforms by NSCLC cell lines and tumor cells transfected with the sense or antisense VEGF/VPF gene. A: Expression of VEGF/VPF isoforms determined by RT-PCR. RT-PCR products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. The PCR products for the VEGF165 and VEGF121 transcripts are 457 and 303 bp, respectively. B: VEGF/VPF mRNA expression in H226 cells transfected with the sense VEGF/VPF genes. C: VEGF/VPF mRNA expression in PC14PE6 cells transfected with the antisense VEGF/VPF gene. Total RNA was extracted from confluent cultures, and Northern blot analysis was performed. The numbers in C are densitometric quantitation of the ratio of the area between the specific endogenous VEGF/VPF transcripts and the GAPDH transcripts with the value for parental cells defined as 1.0.
Figure 5.
Figure 5.
Vascular permeability and PE formation by tumor cells transfected with the sense or antisense VEGF/VPF gene. A: Biological activity of VEGF/VPF secreted by tumor cells transfected with the sense or antisense VEGF/VPF gene on vascular permeability as measured by the Miles assay. Serum-free culture supernatants of tumor cells were harvested as test samples. Nude mice were injected i.v. with Evans blue dye. Ten minutes later, 50 μl of medium, rhVEGF165 (50 ng/ml), rhVEGF121 (50 ng/ml), or test samples were injected intradermally. Thirty minutes later, test sites were photographed. B: PE formation, VEGF/VPF production, and vascular permeability. White arrows indicate pleural fluid (black area) produced in the thoracic cavity. Note that i.t. injection with H226/V165 cells, but not H226/Neo cells, induced the formation of PE. Intravenous injection with PC14PE6/Neo cells, but not PC14PE6/AS39 cells, induced PE formation. Lung lesions by H226/V165 or PC14PE6/Neo cells produced higher levels of VEGF/VPF protein than those by H226/Neo or PC14PE6/AS39 cells, respectively. Leaking of Evans blue dye from vessels was observed in the diaphragm of mice bearing H226/V165 or PC14PE6/Neo cells, but not H226/Neo or PC14PE6/AS39 cells. Bar, 200 μm.
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