Effects of VEGFR-3 phosphorylation inhibitor on lymph node metastasis in an orthotopic diffuse-type gastric carcinoma model

Abstract

Background: Vascular endothelial growth factor receptor-3 (VEGFR-3) signalling mediates lymphangiogenesis and lymphatic invasion; however, the effect of VEGFR-3 inhibition on the lymph node (LN) metastasis remains unclear. The aim of this study is to clarify the benefit of a VEGFR-3 inhibitor Ki23057 for LN metastasis.

Methods: Ki23057 was administered orally to gastric cancer models created by orthotopic inoculation of diffuse-type gastric cancer cells, OCUM-2MLN. The effects of Ki23057 on lymphatic vessel invasion, lymphatic vessel density, and VEGFR-3 phosphorylation were examined by immunostaining or immunoblotting.

Results: Ki23057 inhibited the autophosphorylation of VEGFR-3, with IC50 values of 4.3 nM in the cell-free kinase assay. Murine gastric cancer models created by the orthotopic inoculation of OCUM-2MLN cells showed the diffusely infiltrating growth and frequently developed LN metastasis. The oral administration of Ki23057 significantly (P<0.01) reduced the size of orthotopic tumours and the number of the metastatic LN in gastric cancer models. The degree of lymphatic invasion and lymphangiogenesis was significantly (P<0.05) lower in the gastric tumours treated by Ki23057. Ki23057 inhibited the phosphorylation of VEGFR-3 of lymphatic endothelial cells in gastric tumours.

Conclusion: The inhibition of lymphangiogenesis targeting VEGFR-3 phosphorylation is a therapeutic strategy for inhibiting LN metastasis of diffuse-type gastric cancer.

Figure 1

Effect of Ki23057 on VEGFR-3 phosphorylation. (A) The ability of Ki23057 to inhibit VEGFR-3 phosphorylation was evaluated using human umbilical vein endothelial cells (HUVECs) that express the RTK of VEGFR-3. Ki23057 inhibited the tyrosine phosphorylation of VEGFR-3 with IC₅₀ values of 4.3 nM. (B) For cellular assay, HUVECs were incubated with serial concentrations of Ki23057. Ki23057 inhibited the growth of HUVEC with VEGFR-3 activated by VEGF-C. Ki23057 inhibited the autophosphorylation of VEGFR-3 at a dose-dependent manner. IC₅₀ kinase value was estimated as 37 nM.

Figure 2

Effects of Ki23057 in diffuse-type gastric carcinoma with lymph node metastasis. (A) Macroscopic findings of gastric tumours with lymph node metastasis. The orthotopic inoculation showed diffuse-type gastric tumours (arrowheads). The area of the orthotopically transplanted tumours in Ki23057-treated mouse (right panel) was less than that of the control (left panel). Large LN metastases (arrows) in control mice were recognised around the primary gastric tumour. Lymph node metastases in Ki23057-treated mice were fewer in number and smaller than in controls. No toxicity or body weight loss was observed in any of the groups. (B) H&E staining and Masson trichrome staining. Orthotopic tumours of OCUM-2MLN cells showed extensive fibrosis with the occasional presence of poorly differentiated adenocarcinoma cells that resembled DGC. Extensive stromal fibrosis was also found in OCUM-2MLN gastric tumours treated by Ki23057. No remarkable difference was found in the histological findings of orthotopic carcinomas between the control and Ki23057-treated mice by H&E staining. (C) The mean areas of the orthotopically transplanted tumours in the control and Ki23057-treated mice were 15 and 6 mm², respectively. Primary gastric tumours in mice receiving Ki23057 (25 mg kg⁻¹ day⁻¹) were significantly (P=0.008) smaller than those in controls. (D and E) The mean weights of metastatic LN in the control and Ki23057-treated mice were 91 and 11 mg, respectively. The mean number of metastatic LN in the control and Ki23057-treated mice were 7.8 and 1.5, respectively. Ki23057 significantly decreased both the mean weight (P<0.0001) and the mean number (P<0.0001) of involved nodes.

Figure 3

Immunohistochemical expression of LYVE-1 and VEGF-C in orthotopic tumours. (A) Orthotopically planted OCUM-2MLN cells frequently invade into lymphatic vessels. The number of lymphatic vessels at the invasive edge of tumours was fewer in mice treated by Ki23057, compared with the control. The number of lymphatic invasion was fewer in the gastric tumours treated by Ki23057, compared with the control. Lymphatic vessels located at the invasive edge of tumours were often enlarged and dilated. Expression level of VEGF-C was not different between the two groups. The LYVE-1 antibody stains lymphatic vessels (arrowheads) and highlights lymphatic invasion (arrows). Many lymphatic vessels were found at the invasive edge of tumours. The LYVE-1 antibody-stained vessels devoid of red blood cells corresponded to lymph vessels, whereas blood vessels were not stained (asterisks). Single-stained endothelial cells were excluded. (B) Lymphatic vessel density (LVD) was determined by LYVE-1-positive vessels without cancer emboli. Respective mean LVD in control and Ki23057 mice respectively were 26.5 and 10.4. The mean LVD was significantly decreased by Ki23057. (C) Lymphatic vessel endothelial hyaluronan receptor-1 immunostaining highlighted the presence of lymphatic invasion. Lymphatic vessel invasion was identified as the presence of cancer emboli within the channels lined by LYVE-1-positive vessels. Respective mean LVI (lymphatic vessel invasion) in control and Ki23057 mice were 3.5 and 0.7. The LVI was significantly decreased by Ki23057. Lymphatic vessel invasion or LVD was determined by counting the number of LYVE-1-positive vessels with or without cancer emboli in four high-power fields ( × 100) in areas of the highest lymphatic vessel as ‘hot spots’.

Figure 4

Inhibitory effect of Ki23057 on VEGFR-3 phosphorylation. (A) The inhibitory effect of Ki23057 on the phosphorylation of VEGFR-3 in gastric tumour specimens. VEGFR-3 phosphorylation was suppressed in the tumours of the Ki23057-treated mice (mouse 2 and mouse 4), in comparison with that in the vehicle mice (mouse 1 and mouse 3). (B) The effects of Ki23057 on VEGFR-3 phosphorylation levels on lymphatic endothelial cells. To assess the phosphorylation of VEGFR-3 in tumour tissue, the anti-Flt-4 antibody reacts with mouse VEGFR-3 and phosphotyrosine was examined. Tumours treated by Ki23057 showed an apparent attenuation of phosphorylation (arrows) on the lymphatic endothelial cells of VEGFR-3 staining (arrowheads), in comparison with that in vehicle mice.