Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

Yuji Yamamoto, Junji Matsui, Tomohiro Matsushima, Hiroshi Obaishi, Kazuki Miyazaki, Katsuji Nakamura, Osamu Tohyama, Taro Semba, Atsumi Yamaguchi, Sachi Suzuki Hoshi, Fusayo Mimura, Toru Haneda, Yoshio Fukuda, Jun-Ichi Kamata, Keiko Takahashi, Masayuki Matsukura, Toshiaki Wakabayashi, Makoto Asada, Ken-Ichi Nomoto, Tatsuo Watanabe, Zoltan Dezso, Kentaro Yoshimatsu, Yasuhiro Funahashi, Akihiko Tsuruoka, Yuji Yamamoto, Junji Matsui, Tomohiro Matsushima, Hiroshi Obaishi, Kazuki Miyazaki, Katsuji Nakamura, Osamu Tohyama, Taro Semba, Atsumi Yamaguchi, Sachi Suzuki Hoshi, Fusayo Mimura, Toru Haneda, Yoshio Fukuda, Jun-Ichi Kamata, Keiko Takahashi, Masayuki Matsukura, Toshiaki Wakabayashi, Makoto Asada, Ken-Ichi Nomoto, Tatsuo Watanabe, Zoltan Dezso, Kentaro Yoshimatsu, Yasuhiro Funahashi, Akihiko Tsuruoka

Abstract

Background: Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.

Method: We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.

Result: Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.

Conclusion: These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Keywords: FGFR kinase inhibitor; Lenvatinib; Microvessel density; Pericyte coverage; VEGFR2 kinase inhibitor.

Figures

Figure 1
Figure 1
Kinase inhibitory activity and antiangiogenic activity of lenvatinib in vitro. A: Chemical structure of lenvatinib. B: Ki values of lenvatinib. C: Effects of lenvatinib on the VEGF-induced proliferation and tube formation of HUVEC. D: Effects on the FGF-induced proliferation and tube formation of HUVEC. sTF assay; sandwich tube formation assay.
Figure 2
Figure 2
Effects of lenvatinib on in vivo angiogenesis induced by KP-1/VEGF and KP-1/FGF transfectants. (A) In vivo angiogenesis in mouse DAS assay. Angiogenesis was induced by overexpressed human VEGF121 (KP-1/VEGF) or mouse FGF4 (KP-1/FGF) in human pancreatic cancer KP-1 cells at the mouse dorsal skin. Representative photographs are shown. (B) Effect of lenvatinib and sorafenib on the VEGF- and FGF-driven in vivo angiogenesis in mouse DAS assay. Compounds were administered orally once daily for 4 days at the indicated doses. Data are the mean ± std. *: p < 0.05 and **: p < 0.01 compared to vehicle.
Figure 3
Figure 3
Antitumor activity of lenvatinib against the KP-1/VEGF and KP-1/FGF transfectants in nude mice. Lenvatinib was administered orally twice daily, when tumor volumes reached approximately 200 mm3(A,C,D). Each group consisted of 5 mice. Data are the mean ± std. *p < 0.05 compared to vehicle. (A-C) the KP-1/VEGF xenograft model. (D) the KP-1/FGF xenograft model. (A) Antitumor activity of lenvatinib against KP-1/VEGF xenografts. Lenvatinib was administered at 1–100 mg/day for 14 days. Tumor tissues were resected on day 26 for IHC analysis. Tumor vessels were stained with anti-mouse CD31 antibody. Photographs were taken using a light microscope (x25) and representative images are shown. (B) Antitumor activity of lenvatinib in the advanced KP-1/VEGF xenograft model. Lenvatinib was administered at 100 mg/kg for either 14, 18 or 14 days, when the tumor size reached 150, 650 and 1000 mm3, respectively. (C) Antitumor activity of lenvatinib with an interval of treatments. Lenvatinib was administered at 100 mg/kg for 14 days in the 1st cycle and again given for 10 days in a 2nd cycle with 11 days interval between the 1st and 2nd cycles. (D) Antitumor activity of lenvatinib in the KP-1/FGF xenograft model. Lenvatinib was administered at 30 and 100 mg/kg for 14 days.
Figure 4
Figure 4
Association of antitumor activity of lenvatinib with tumor vasculature in 19 human tumor xenograft models. Lenvatinib was administered orally twice daily for 7 days, when tumor volumes reached approximately 100–300 mm3. Each group consisted of 5 mice. (A) Antitumor activity of lenvatinib in 19 human tumor xenograft models. The ΔT/C (%) was presented as a mean. □: The lenvatinib-sensitive group; ■: the lenvatinib-relatively resistant group. The relationship between the antitumor activity and MVD is shown in (B), and that between the antitumor activity and the % of pericyte coverage of vessels is shown in (C). Each symbol (○) indicates the mean of MVD or pericyte coverage in each tumor xenograft model. (D) Vascular score in the lenvatinib-sensitive and –relatively resistant groups. The vascular score was the sum of the MVD and pericyte coverage scores.
Figure 5
Figure 5
IHC analysis of tumor vasculature in 18 different types of human tumor specimens. Microvessel density (MVD), pericyte coverage and vascular scores were determined with IHC analysis by staining CD31 and αSMA among 18 types of human tumor specimens. Analysis was performed as described in materials and methods. Bars (red) indicated median values for MVD or % of pericyte coverage of each type of tumors. (A) MVD. (B) % of pericyte coverage. (C) Summary of MVD, pericyte coverage and vascular score.

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