Role of tumor-associated lymphatic endothelial cells in metastasis: a study of epithelial ovarian tumor in vitro

Abstract

Tumor-associated lymphatic endothelial cells (TLEC) could play a key role in the process of tumor metastasis. The aim of this study was to investigate the effect of TLECs that were isolated from human epithelial ovarian tumor (EOT) on ovarian cancer cell line CAOV-3 in vitro. First, TLECs in EOT were detected by immunochemistry and flow cytometry, then marked by lymphatic endothelial cell (LEC) marker LYVE-1, isolated by magnetic beads, and cultured in vitro. The cells were identified by immunostaining of LEC markers LYVE-1, Prox-1, Podoplanin, VEGFR-3, and pan-endothelial cell marker CD31. TLECs from EOT can be detected, cultured, and identified in vitro successfully. The effects of TLECs on invasion and migration of CAOV-3 cells were investigated by 12-well Boyden chamber; the proliferation effect was studied by counting the Trypan blue exclusion cell number. Furthermore, changes in MMP-2/9 secreted by CAOV-3 cells treated with TLEC were shown using real-time PCR and zymography, and TIMP-1/2 was detected by real-time PCR. In vitro, TLECs can enhance invasion and migration of CAOV-3 cells, but have no significant effect on proliferation. It was clear that the expression of MMP-9 increased and TIMP-2 decreased in CAOV-3 cells treated by TLECs, and the increasing of MMP-9 was confirmed by zymography. TLECs from EOT can enhance migration and invasion of human ovarian carcinoma cell line in vitro, and the possible mechanism was through activation of MMP-9/TIMP-2.

Figure 1

Tumor‐associated lymphatic vessels in epithelial ovarian tumor (EOT). (A) Analysis of lymphatic endothelial cell in EOT by immunohistochemistry (magnification, ×400). Several lymphatic vessels (yellow) under the papillae in serous ovarian tumor (b, c) or surrounded by tumor cells (a), lymphatic vascular invasion was popular in EOT (d–f). D2‐40 was expressed on the membrane only. Double immunstaining of Podoplanin (black) and Ki‐67 (red) showed they did not coexist in any cell. (e, f) Black and yellow arrows point to lymphatic vessels. (B) Analysis of LYVE‐1⁺ cells in a sample of EOT single cell suspension by flow cytometry. Blank control, 0.37%; negative control, 1.02%; sample, 3.57%.

Figure 2

The culture and identification of LYVE‐1⁺ cells isolated from epithelial ovarian tumor. LYVE‐1⁺ cell growth was recorded under phase‐contrast microscope. Identification of second‐generation LYVE‐1⁺ cells by lymphatic endothelial cell marker Prox‐1 (nuclear) and LYVE‐1 (membrane). Immunofluorescence shows LEC markers Podoplanin and VEGFR‐3, and pan‐endothelial cell marker CD31 (all located on membrane) were all expressed by these cells.

Figure 3

Effects of tumor‐associated microvascular lymphatic endothelial cells (TLECs) on CAOV‐3 cell invasion, migration, and proliferation. The different treatment groups for migration and invasion are shown at the top of this figure. (A) Migration assay (without Matrigel on the filter) was detected 24 h after incubation. (B) Invasion assay (with Matrigel) was detected 48 h after incubation. As the error bar shows, the influence of tumor‐associated microvascular lymphatic endothelial cells on CAOV‐3 cell migration and invasion was evident. Groups b and d had more cells than group a or c on the filter. *P < 0.05. The difference between group a and c or group b and d was not significant (P > 0.05). The chemoattractant of TLEC conditioned medium (TLM) to CAOV‐3 was not found. (C) Proliferation assay showing that no statistical difference was seen among CAOV‐3 cell treated by lymphatic endothelial cell medium (LEM), normal lymphatic microvascular endothelial cell conditioned medium (NLM), or TLM (P > 0.05).

Figure 4

Changes in gene expression in CAOV‐3 cells treated by tumor‐associated microvascular lymphatic endothelial cell conditioned medium (TLM). (A) The expression of MMP‐2, MMP‐9, TIMP‐1, TIMP‐2 and CXCR4 mRNA in CAOV‐3 cells after treatment with normal lymphatic microvascular endothelial cell conditioned medium (NLM) or TLM. The real‐time PCR data, shown by the column graph, suggested that the level of MMP‐9 clearly increased and TIMP‐2 clearly decreased by TLM at the mRNA level. (B) Electrophoretogram of gelatin zymography confirmed the changes in MMP‐2 and MMP‐9 at the protein level. In the error bar map, the level of MMP‐2/9 secreted by CAOV‐3 cells treated with NLM were defined as 1 compared to CAOV‐3 cells treated with TLM. Only the expression of MMP‐9 secreted by TLM treated CAOV‐3 cells increased significantly. *P < 0.05.