VEGFR2 regulates endothelial differentiation of colon cancer cells

Abstract

Background: Recent studies suggested that cancer stem-like cells contribute to tumor vasculogenesis by differentiating into endothelial cells. However, such process is governed by still undefined mechanism.

Methods: At varying differentiation levels, three representative colon cancer cells were cultured in endothelial-inducing conditioned medium: human colon cancer cells HCT116 (HCT116) (poorly differentiated), SW480 (moderately differentiated), and HT29 (well differentiated). We tested for expression of endothelial markers (cluster of differentiation (CD) 31, CD34, and vascular endothelial (VE)-cadherin and their ability to form tube-like structures in 3D culture. We also observed VEGF secretion and expressions of endothelial markers and VEGFRs in HCT116 cells under hypoxia to simulate physiological conditions. In in vitro and in xenotransplantation experiments, VE growth factor receptor 2 (VEGFR2) antagonist SKLB1002 was used to test effect of VEGFR2 in endothelial differentiation of HCT116 cells. Expression levels of VEGFR2 and VE-cadherin were assessed by immunohistochemistry of human colon cancer tissues to evaluate clinicopathological significance of VEGFR2.

Results: After culturing in endothelial-inducing conditioned medium, poorly differentiated HCT116 cells expressed endothelial markers and formed tube-like structure in vitro. HCT116 cells secreted more endogenous VEGF and expressed higher VEGFR2 under hypoxia. SKLB1002 impaired endothelial differentiation in vitro and xenotransplantation experiments, suggesting a VEGFR2-dependent mechanism. Increased expression of VEGFR2 correlated with differentiation, metastasis/recurrence, and poor prognosis in 203 human colon cancer samples. Positive correlation was observed between VEGFR2 and VE-cadherin expression.

Conclusions: VEGFR2 regulates endothelial differentiation of colon cancer cell and may be potential platform for anti-angiogenesis cancer therapy.

Keywords: Colon cancer; VE-cadherin; VEGFR2; Vasculogenesis.

Fig. 1

Endothelial-inducing conditioned medium induced expressions of CD31, CD34, and VE-cadherin and promoted HCT116 cells to form tube-like structures. a Protein expressions of CD31, CD34, and VE-cadherin in HT29, SW480 or HCT116 after cultured for 5, 10 or 15 days in endothelial-inducing conditioned medium were determined by western blotting analysis. Representative data of three experiments are shown. b The tube-like structures formed by HT29, SW480 or HCT116 cells in endothelial-inducing conditioned or normal 3D culture (left). Black arrows indicate the typical tube-like structures. Scale bar: 100 μm; Quantitative analysis of the mean number of tube-like structures and shown as mean ± SD (right)

Fig. 2

VEGF secretion and VEGFR2 expressions were elevated in HCT116 cells under hypoxia. a VEGFA secretion of HCT116 cells under hypoxia or normoxia were determined by ELISA analysis. The data are shown as mean ± SD, n = 3 in triplicate experiments. b Protein expressions of CD31, CD34, and VE-cadherin in HCT116 cells under hypoxia or normoxia were determined by western blotting (left) and immunofluorescent staining analysis (right). Representative data of three experiments are shown. c Protein expressions of VEGFR1, VEGFR2, and VEGFR3 in HCT116 cells under hypoxia or normoxia were determined by western blotting. Representative data of three experiments are shown

Fig. 3

SKLB1002 inhibited tube-like structures formation and VE-cadherin expression of HCT116 cells. a The tube-like structures formed by HCT116 cells with or without SKLB1002 treatment (left). Red arrows indicate the typical tube-like structures. Scale bar: 100 μm; Quantitative analysis of the mean number of tube-like structures and shown as mean ± SD (right). b Protein expressions of VEGFR2, p-VEGFR2, FAK, p-FAK, ERK, p-ERK and VE-cadherin in HCT116 cells with or without SKLB1002 treatment were determined by western blotting analysis. Representative data of three experiments are shown

Fig. 4

SKLB1002 inhibited in vivo tumor growth in HCT116 xenograft mouse model and decreased VE-cadherin expression in tumor tissues. a Photographs illustrating mice tumors derived from HCT116 cells with or without SKLB1002 treatment 30 days after inoculation. b The volume of the mice tumors derived from HCT116 cells was evaluated at 5-day intervals for 30 days. Data are shown as mean ± SD. c Hematoxylin & eosin staining photographs illustrating necrosis in mice tumor tissues derived from HCT116 cells with or without SKLB1002 treatment 30 days after inoculation (left). (×40). Quantitative analysis of the proportions of non-necrotic tissue in the largest section of tumor masses is shown as mean ± SD (right). d Expressions of VE-cadherin in mice tumor tissues derived from HCT116 cells with or without SKLB1002 treatment 30 days after inoculation were determined by immunohistochemical staining. (×400)

Fig. 5

Expressions of VEGFR2 in colon cancer samles and Kaplan–Meier survival analysis of colon cancer patients. a Representative immunohistochemical staining photographs of human colon cancer samples with VEGFR2 negative (−), weak(+), and strong (++) expression. (× 200). b Kaplan–Meier survival analysis illustrating overall survival status in VEGFR2 negative (−), weak(+), and strong (++) groups of colon cancer patients

Fig. 6

Expression of VEGFR2 is concomitant with VE-cadherin expression. Representative immunohistochemical VE-cadherin staining photographs of human colon cancer samples in VEGFR2 negative (−), weak(+), and strong (++) groups. (×200)