Differential response of primary tumor versus lymphatic metastasis to VEGFR-2 and VEGFR-3 kinase inhibitors cediranib and vandetanib

Abstract

Blood vessels are required for a tumor to grow and functional lymphatic vessels are required for it to disseminate to lymph nodes. In an attempt to eradicate both the primary tumor and its lymphatic metastasis, we targeted both blood and lymphatic vessels using two different tyrosine kinase inhibitors (TKIs): cediranib and vandetanib, which block vascular endothelial growth factor receptor (VEGFR)-2 and -3 in enzymatic assays. We found that although both cediranib and vandetanib slowed the growth rate of primary tumors and reduced blood vessel density, neither agent was able to prevent lymphatic metastasis when given after tumor cells had seeded the lymph node. However, when given during tumor growth, cediranib reduced the diameters of the draining lymphatic vessels, the number of tumor cells arriving in the draining lymph node, and the incidence of lymphatic metastasis. On the other hand, vandetanib had minimal effect on any of these variables, suggesting that vandetanib did not effectively block VEGFR-3 on lymphatic endothelial cells in our animal model. Collectively, these data indicate that the response of lymphatic vessels to a TKI can determine the incidence of lymphatic metastasis, independent of the effect of the TKI on blood vessels.

Figure 1

Analysis of molecular targets of cediranib and vandetanib in T241 tumor cell lines. A) Analysis of exogenous VEGF-C gene expression and other endogenous gene expression in T241 transfectants. The amplified cDNA products generated by RT-PCR analysis of total RNA from T241-GFP cells, T241-VEGF-C-GFP cells and murine embryo cells (as positive control) by oligonucleotide primers that recognize the specific genes indicated. B) Immunostaining of VEGFR-2, VEGFR-3 and PDGFR-β in T241-VEGF-C-GFP ear tumor. While VEGFR-2, VEGFR-3 and PDGFR-β expression were not detected in tumor cells, VEGFR-2 expression was detected on most tumor blood vessels and VEGFR-3 and PDGFR-β on a fraction of tumor blood vessels.

Figure 2

Tyrosine kinase inhibitors cediranib and vandetanib reduce the proliferation of blood and lymphatic endothelial cells more potently than T241 tumor cell lines. A) Dose response curves of cediranib and vandetanib on LECs and BECs. B) Dose response curves of cediranib and vandetanib on T241-GFP and T241-VEGF-C-GFP tumor cells. Proliferation was determined by WST-1 assay.

Figure 3

Cediranib and vandetanib reduce tumor angiogenesis. A) Cediranib treatment reduced the area and number density of intratumor blood vessels identified by MECA-32 immunohistochemistry. B) Vandetanib treatment also reduced the area density of intratumor blood vessels identified by MECA-32 immunohistochemistry.

Figure 4

Cediranib reduced lymphatic hyperplasia and tumor cell arrival in lymph nodes. A) Tumor margin lymphatic hyperplasia induced by T241-VEGF-C-GFP tumors (at 40 mm³) was suppressed by daily treatment with cediranib (n = 12) compared with vehicle treated animals (n = 10). B) The number of T241-VEGF-C-GFP cells (green) in the lymph node when the tumor size was 40 mm³ was greatly reduced by daily treatment with cediranib (n = 12) compared with vehicle treated animals (n = 8). Red signal from TMR-dextran lymphangiography shows lymph entering the lymph node from the efferent lymphatic vessel.

Figure 5

Vandetanib did not reduce lymphatic hyperplasia and tumor cell arrival in lymph nodes. A) Tumor margin lymphatic hyperplasia induced by T241-VEGF-C-GFP tumors (at 40 mm³) was not influenced by daily treatment with vandetanib (n = 11) compared with vehicle treated animals (n = 9). B) The number of T241-VEGF-C-GFP cells (green) in the lymph node when the tumor size was 40 mm³ was not changed by daily treatment with vandetanib (n = 10) compared with vehicle treated animals (n = 9). Red signal from TMR-dextran lymphangiography shows lymph entering the lymph node from the efferent lymphatic vessel.