Identification of tumour-reactive lymphatic endothelial cells capable of inducing progression of gastric cancer

Abstract

Background: Tumour cells and stromal cells interact in the tumour microenvironment; moreover, stromal cells can acquire abnormalities that contribute to tumour progression. However, interactions between lymphatic endothelial cells (LECs) and tumour cells are largely unexamined. In this study, we aimed to determine whether tumour-specific LECs inhabit the tumour microenvironment and examine their influence on this microenvironment.

Methods: We isolated normal LECs (NLECs) from a non-metastatic lymph node and tumour-associated LECs (TLECs) from cancerous lymph nodes. We examined proliferative and migratory potency, growth factor production, and gene expression of each type of LEC. Moreover, we developed a co-culture system to investigate the interactions between gastric cancer cells and LECs.

Results: When compared with NLEC, TLECs had an abnormal shape, high proliferative and migratory abilities, and elevated expression of genes associated with inflammation, cell growth, and cell migration. NLECs co-cultured with gastric cancer cells from the OCUM12 cell line acquired TLEC-like phenotypes. Also, OCUM12 cells co-cultured with TLECs expressed high levels of genes responsible for metastasis.

Conclusions: Our results demonstrated that LECs interacted with tumour cells and obtained abnormal phenotypes that could have important roles in tumour progression.

Figure 1

Isolation and identification of normal and tumour-associated lymphatic endothelial cells (LECs). CD31-positive, podoplanin-positive cells were isolated from two different lymph nodes, one normal and one containing metastatic gastric cancer cells. (A) Normal LECs (NLECs) isolated from the normal lymph node; NLECs had a cobblestone-like appearance (at × 40 magnification). (B) Tumour-associated LECs (TLECs) isolated from a lymph node with metastatic gastric cancer cells. TLECs were spindle-shaped cells, like fibroblasts (at × 40 magnification). (C) Both types of LECs expressed the pan-endothelial cell marker CD31 and the lymphatic marker podoplanin.

Figure 2

Comparison between NLECs and TLECs with regard to proliferation and migration. (A) TLECs showed markedly higher proliferative ability than NLECs (46.5±7.67-fold). The data represent means of triplicate measurement±the standard deviation (s.d.). ***P<0.001. (B) Microscope images depicting cell density and morphology in wounds at multiple time points. Cells present at the beginning of each experiment are shown in grey, and migrating cells are shown in purple. (C) Relative LEC density in wounds cut through NLEC or TLEC monolayers was monitored for 24 h. TLECs showed significantly greater wound closure than did NLECs. Each value represents the mean density of five wells±s.d. **P<0.01, ***P<0.001. A full color version of this figure is available at the British Journal of Cancer journal online.

Figure 3

Comparison between NLECs and TLECs with regard to cell characteristics. (A) Differences in mRNA expressions between NLECs and TLECs. TLECs showed significantly higher expression of mRNAs encoding cytokines, chemokines, adhesion molecules, and growth factors than did NLECs. The relative expression levels of mRNA are shown on a logarithmic scale, and the values represent means of quintuplet measurements±s.d. ***P<0.001. (B) Differences in production of growth factors and cytokines between NLECs and TLECs. TLECs secreted significantly higher concentrations of VEGF-A, VEGF-C, and IL-1β (355.61±22.13 pg ml⁻¹, 3057.04±45.87 pg ml⁻¹, and 4304.32±112.14 pg ml⁻¹, respectively) than did NLECs. The concentrations are shown as pg ml⁻¹ per 1 × 10⁶ cells, and the values represent means of triplicate measurements ±s.d. ***P<0.001, ND, not detected.

Figure 4

Phenotypic changes in NLECs co-cultured with OCUM12 cells as assessed with MTT assays, qRT-PCR, and ELISA. (A) The proliferation activity of NLECs was stimulated when NLECs were cultured in tumour-conditioned medium (TCM) (1.81±0.02-fold). The values represent means of triplicate measurements ±s.d. ***P<0.001. (B) Changes in gene expression in NLECs co-cultured with OCUM12 cells. Expression of mRNAs encoding cytokines and chemokines in NLECs co-cultured with OCUM12 cells was significantly higher than those in NLECs cultured alone, but not mRNAs encoding CXCL6, COLA1, MMP2, or VEGF-C were not upregulated. Values represent means of quintuplet measurements±s.d. **P<0.01, ***P<0.001. (C) Production of IL-1β, VEGF-A, and VEGF-C was significantly higher in NLEC co-cultured with OCUM12 cells (842.28±0.95 pg ml⁻¹, 246.23±3.90 pg ml⁻¹, and 314.78±9.81 pg ml⁻¹, respectively) than in those cultured alone. Values represent means of triplicate measurements±s.d. **P<0.01, ***P<0.001, ND, not detective.

Figure 5

Differences in expression of lymphatic endothelial markers between NLECs, TLECs, and NLECs co-cultured with OCUM12 cells. (A) Expression of VEGFR3, LYVE-1, and Prox1 was downregulated in TLECs and NLECs co-cultured with OCUM12 cells. (B) NLECs expressed VEGFR3 and LYVE-1. The expression of VEGFR3 was significantly downregulated in TLECs and NLECs co-cultured with OCUM12 cells. The expression of LYVE-1 was reduced in TLECs, but no significant difference was observed in NLECs co-cultured with OCUM12 cells. Values represent means of triplicate measurements±s.d. ***P<0.001. MFI, mean fluorescent intensity.

Figure 6

Alterations in mRNA expression in OCUM12 cells co-cultured with TLECs. TLECs induced increases in expression of mRNAs encoding CXCR2, SNAIL, or TWIST in OCUM12 cells co-cultured with TLECs. The values represent means of quintuplet measurements±s.d. ***P<0.001.