Therapeutic effects of recombinant human endostatin adenovirus in a mouse model of malignant pleural effusion

Abstract

Purpose: Malignant pleural effusion (MPE) is a common clinical problem in patients with advanced cancer. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the pleural wall are due to high levels of vascular endothelial growth factor (VEGF), which plays an important role in the pathogenesis of MPE. The present study was designed to test whether the recombinant adenovirus-mediated delivery of human endostatin (Ad-hEndo), one of the potent inhibitors of angiogenesis, would inhibit the formation and progression of MPE.

Methods: We developed a novel mouse model of MPE by injecting Lewis lung carcinoma (LLC) cells directly into pleural cavity of C57BL/6 mice. To evaluate the therapeutic effects of endostatin in this MPE model, we injected the Ad-hEndo into the pleural cavity of MPE-bearing mice three times with the 3-day interval.

Results: We found that this treatment resulted in significant reduction in pleural effusion volume, the number of pleural tumor foci, microvessel density, and vascular permeability, while it significantly prolonged the survival time. In addition, VEGF level of MPE in the group administered with the Ad-hEndo was obviously decreased as compared with that in the two control groups administered with null-adenovirus (Ad-null) or normal saline.

Conclusions: Our work provides a rationale for future studies toward evaluating the effectiveness of the adenovirus-based endostatin therapy for MPE.

Fig. 1

CT images of malignant pleural effusions. Three days after LLC cells inoculation, MPE-bearing mice were intrapleurally administered with Ad-hEndo at 1.5 × 10⁹ pfu per mouse three times with the 3-day interval, or Ad-null at 1.5 × 10⁹ pfu, or equal volume of NS on the same schedule as above. On day 14 after LLC cells inoculation, transverse CT images of the three groups mice showed that Free-floating bilateral pleural effusions (mpe) were more clearly visible on CT scans in the mice of the two control groups administered with Ad-null (b) or NS (a) compared with that of the Ad-hEndo group (c)

Fig. 2

Malignant pleural tumors in C57B/6 mice after intrapleural administration. On day 4 after the completion of treatment as described in “Materials and methods” section, mice (n = 10 mice/group) were sacrificed by cervical dislocation, MPEs were gently aspirated and chest walls were open. Lung and chest wall explants showing multiple tumor foci (arrows) on the parietal and visceral pleura. Significantly decreased number of pleural tumor in Ad-hEndo treated group (c) compared with the NS (a), Ad-null (b) groups were shown (h heart). Section (×200) through a small visceral pleural Lewis lung cancer implantation stained with hematoxylin and eosin shows that pleural tumors consisted of adenocarcinomatous cells (d) and tumors grew on the pleural surface (e)

Fig. 3

The physical, cellular characterization of the MPE obtained from three groups. On day 4 after the completion of treatment, mice (n = 10 mice/group) were sacrificed by cervical dislocation and blood was drawn from the retro-orbital veins. Then, the abdominal walls of mice were cut down, the diaphragms were exposed and MPEs were gently aspirated using a 1-ml syringe. The volume of MPE was measured with the syringe. The number of red blood cells in MPE of the Ad-hEndo group was lower than that in the two control groups (a). And the mean MPE volume was clearly reduced in the Ad-hEndo group as compared with the control groups (b). Tumor implantations on the pleura were counted by two independent readers under a dissecting microscope, and the average number was used for data analyses. The number of pleural tumor foci and tumor cells in MPE of the Ad-hEndo group were significantly reduced as compared with the control groups (c). Columns mean value of each group, bars ±SD. *P < 0.05 compared with Ad-null or NS group

Fig. 4

The biochemical characterization of the MPE and pleural permeability in mice. On day 4 after the completion of treatment, mice (n = 10 mice/group) were sacrificed by cervical dislocation, blood was drawn from the retro-orbital veins and the MPE was gently aspirated using a 1-ml syringe. Serum and pleural fluid samples were assayed for total protein content, LDH and VEGF. The levels of total proteins (a) and LDH (b) in MPE of the two control groups were significantly higher than those in the Ad-hEndo group. In addition, VEGF protein level (c) in MPE of the Ad-hEndo group was significantly reduced by 84.3% as compared with the Ad-null group. To evaluate the pleural permeability of the mice, on day 4 after the completion of treatment, each mice (n = 8 mice/group) received 200 μl of 5 mg/ml Evans’ blue solution (total dose 1 mg) intravenously and were killed 2 h later. Pleural fluid and serum Evans’ blue concentration were determined by measuring absorbance at a wavelength of 630 nm in a spectrophotometer. The absorbance value in the MPE of the Ad-hEndo group was obviously lower than that in the two control groups (d). Columns mean value of each group, bars ±SD. *P < 0.05 compared with Ad-null or NS group

Fig. 5

Inhibition of angiogenesis assayed by immunohistochemistry with CD31. Pleural tumor angiogenesis was assessed by immunohistochemical staining with anti-CD31 antibody (brown) on paraffin-embedded sections of tumors resected 4 days after the completion of treatment. Microvessel counting was done at ×200. Tumors of the Ad-hEndo group revealed only occasional, isolated microvessels (c). At the same magnification, the section of representative images with well-formed capillaries surrounding nests of tumor cells in Ad-null (b) or NS control treated group (a). Ad-hEndo treatment group displayed decreased microvessel as compared with the two control groups in pleural tumor tissues (d). Columns mean of microvessel per high-power field, bars ±SD. *P < 0.05 compared with Ad-null or NS group

Fig. 6

Survival advantage in mice Mice (10 mice per group) were administered intrapleurally with the Ad-hEndo at 1.5 × 10⁹ pfu per mouse three times with the 3-day interval, or Ad-null at 1.5 × 10⁹ pfu, or equal volume of NS on the same schedule as above. Life span analysis showed that neither Ad-null group nor NS group survived more than 21 days on average. A significant increase in survival in the Ad-hEndo group compared with the two control groups (P < 0.01, by log-rank test) was found in this mouse model