Cancer-derived VEGF plays no role in malignant ascites formation in the mouse

Abstract

Aim: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites.

Methods: VEGF expression was eliminated by knockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi). Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown.

Results: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically.

Conclusion: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues, such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.

Figure 1

Establishment of VEGF knockdown pancreatic cancer cell lines. A: Western blot showing marked reduction of VEGF expression in knockdown cells. Screening of the most effective targeting site for the VEGF gene. B: VEGF production of stable knockdown PancO2 cell clones. VEGF-WT and VEGF-KD clones (KD 1-10) were grown in 6-well plates, and the VEGF concentration in culture supernatant was measured using ELISA in triplicates. The growth medium was used as negative control (NC).

Figure 2

Effect of VEGF knockdown on cell proliferation. A: 3×10⁴ VEGF-KD7 or VEGF-WT cells were grown in 24-well plates, and the numbers of viable cells were assessed using the MTT assay at 24 (P = 0.082) and 48 h (P = 0.078) (n = 4, mean±SD); B: 2×10⁶ VEGF-KD7 or VEGF-WT cells were injected into C57BL/6 mice subicutaneous, the tumor size was measured, and the volume was calculated as [(length (mm)×width (mm)²)]/2 (n = 4, P = 0.18, mean±SD).

Figure 3

Effect of VEGF knockdown in a tumor grafted mouse ascites model. A: The ascites volume of mice inoculated with VEGF-KD7 or VEGF-WT cells (n = 5, P = 0.10, mean±SD); B: The VEGF concentration in ascites. VEGF in the ascites was measured using ELISA (n = 4, P = 0.054, mean±SD). C: 1106 VEGF-KD7 or VEGF-WT cells were inoculated into mice intraperitoneally and the mice were weighed (n = 8, P≥0.05 in each data point).