Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling

Tingting Qin, Zhujun Liu, Jing Wang, Junling Xia, Shaochuan Liu, Yanan Jia, Hailin Liu, Kai Li, Tingting Qin, Zhujun Liu, Jing Wang, Junling Xia, Shaochuan Liu, Yanan Jia, Hailin Liu, Kai Li

Abstract

Objective: Lymphatic metastasis is one of the leading causes of malignancy dispersion in various types of cancer. However, few anti-lymphangiogenic drugs have been approved for clinical use to date. Therefore, new therapies to block lymphangiogenesis are urgently required. Methods: Immunohistochemistry, immunofluorescence, Western blot, migration assays, and lymphangiogenesis and lymphatic metastasis assays were used. Results: Anlotinib, a receptor tyrosine kinase inhibitor, suppressed the rate of new metastatic lesions (31.82% in the placebo arm and 18.18% in the anlotinib arm) in patients with advanced lung adenocarcinoma who were enrolled in our ALTER-0303 study. D2-40+-lymphatic vessel density was strongly correlated with disease stage, metastasis, and poor prognosis in 144 Chinese patients with lung adenocarcinoma. In mice bearing A549EGFP tumors, tumor lymphatic vessel density, tumor cell migration to lymph nodes, and the number of distant metastatic lesions were lower in the anlotinib group than in the controls. Anlotinib inhibited the growth and migration of human lymphatic endothelial cells (hLECs) and lymphangiogenesis in vitro and in vivo. Treatment of hLECs with anlotinib downregulated phosphorylated vascular endothelial growth factor receptor 3 (VEGFR-3). Conclusions: Anlotinib inhibits lymphangiogenesis and lymphatic metastasis, probably through inactivating VEGFR-3 phosphorylation. The results indicate that anlotinib may be beneficial for treatment in avoiding lymphangiogenesis and distant lymphatic metastasis in lung adenocarcinoma. (Trial registration: ALTER0303; NCT02388919; March 17, 2015.).

Keywords: lymph node metastasis; Anlotinib; VEGFR-3 dephosphorylation; lung adenocarcinoma; lymphangiogenesis.

Copyright: © 2020, Cancer Biology & Medicine.

Figures

Figure 1
Figure 1
Anlotinib inhibits new metastatic lesions in 44 patients with advanced lung adenocarcinoma. (A) Kaplan-Meier plots of the progression-free survival of patients with advanced lung adenocarcinoma with anlotinib (blue) or placebo (red) treatment (P = 0.0006, log-rank test). (B) Kaplan-Meier plots of overall survival in patients with advanced lung adenocarcinoma with anlotinib (blue) or placebo (red) treatment (P = 0.4171, log-rank test). (C) Percentages of total new metastatic lesions and new lymph node metastasis of patients with advanced lung adenocarcinoma in the placebo group or anlotinib group. Marks on bars indicate the number of patients in each group (n = 22 in the placebo group, n = 44 in the anlotinib group). Blue: patients without total new metastatic lesions, green: patients with total new metastatic lesions, red: patients without new lymph node metastasis, yellow: patients with new lymph node metastasis. Spearman rank correlation test, **P < 0.01. (D) Typical images of D2-40 (brown) immunostaining of lymphatic vessels on lung cancer specimens, on the basis of UICC staging. Magnification, 200×, scale bar, 100 μm. (E) Box plot analysis of the correlation between baseline lymphatic density (D2-40 positive vessels per 200× field) and the UICC stages of lung adenocarcinoma patients (n = 94 in UICC stages I and II group, n = 50 in the UICC stage III and IV group). Data are mean values ± SD. ***P < 0.001; Student’s t-test. (F) Percentages of lymph node (LN) metastasis in UICC stages I and II or UICC stages III and IV. Marks on bars indicate the number of patients in each group (n = 94 in the UICC stage I and II group, n = 50 in the UICC stage III and IV group). White: patients with cancer cell-negative lymph nodes, stripes: patients with cancer-cell positive lymph nodes. Spearman rank correlation test, ***P < 0.001.
Figure 2
Figure 2
Anlotinib inhibits primary tumor growth and tumor-associated lymphangiogenesis in A549EGFP xenograft tumors. (A) Human lung adenocarcinoma A549EGFP cells were inoculated in the subcutaneous space near the axillary lymph nodes in nude mice, and tumor volume was continuously monitored. (B) Typical images of the primary tumors that formed in mice grafted with human A549EGFP tumor cells on the right shoulder, which were treated with anlotinib or saline. Ruler unit, cm. (C) Scatter plot analysis of volumes of primary tumors treated with anlotinib or saline (n = 7 per group). (D) Scatter plot analysis of weights of primary tumors treated with anlotinib or saline (n = 7 per group). (E) Representative images of metastatic lesions in different groups of tumor-bearing mice, which were monitored with the IVIS spectrum imaging system. Color bar represents radiant efficiency [(p/s/cm2/sr)/(μW/cm2)]. (F) A scatter plot analysis showing nearly 3-fold fewer metastatic lesions in the anlotinib group than the control group. (G) Representative images (brown) of podoplanin immunostaining of lymphatic vessels in primary tumors from mice receiving anlotinib or saline treatment. Magnification, 200×, scale bar, 50 μm. (H) Average lymphatic vessel density, as quantified by assessment of the numbers of podoplanin-positively stained vessels in primary tumors of mice (n = 7 per group). Data are mean values ± SD. **P < 0.01, ****P < 0.0001; Student’s t-test. These experiments were performed 2 times.
Figure 3
Figure 3
Anlotinib decreases tumor colonization in draining lymph nodes. (A) Typical images of inguinal lymph nodes from different groups, which were treated with anlotinib or saline. Ruler unit, cm. (B) Scatter plot analysis of volumes of lymph nodes treated with anlotinib or saline (n = 7 per group). (C) Images of A549EGFP cells positively stained (brown) for human Ki67. Magnification, 400×, scale bar, 50 μm. (D) Number of Ki67-positive A549EGFP cells (n = 7 per group) in different groups. Data are mean values ± SD. ****P < 0.0001; Student’s t-test. These experiments were performed twice.
Figure 4
Figure 4
Anlotinib inhibits tumor growth and lymphangiogenesis in syngeneic LLC tumors. (A) Typical images of the primary tumors that formed in mice grafted with LLC tumor cells at the right shoulder, which were treated with anlotinib or saline. Ruler unit, cm. (B) Box plots of volumes of primary tumors (n = 6 per group). (C) Representative images (brown) of podoplanin immunostaining of lymphatic vessels in primary tumors in mice treated with anlotinib or saline. Line one: HE-stained cross-sections of the primary tumors from the control or anlotinib group. Magnification, 200×; line two: magnification, 200×, scale bar, 100 μm; line three: magnification, 400×, scale bar: 50 μm. (D) Average lymphatic vessel density, as quantified by assessment of the numbers of podoplanin-positively stained vessels in primary tumors of mice (n = 6 per group). (E) Computerized measurements of relative lymphatic vessel area in primary tumors in mice (n = 6 per group). Data are mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test. These experiments were performed twice.
Figure 5
Figure 5
Anlotinib impairs lymphangioma development via inhibiting neolymphangiogenesis. (A) Typical images of the benign lymphangiomas (red arrows) that formed in the control and anlotinib groups. Ruler unit, cm. (B) Fluorescence confocal microscopy images showing lymphatic vessels (LYVE1+ in tumor area) in frozen sections of lymphangioma tissues from the control and the anlotinib groups, as assessed by immunostaining. L = liver, T = tumor (benign lymphangioma). Red (liver sinusoids were also positive), LYVE1. Blue, DAPI. The dotted line indicates the border between liver and tumor tissues; scale bar, 75 μm. (C) Weights of lymphangioma tissues in the control and anlotinib groups (n = 6 per group). (D) Average lymphatic vessel densities, determined by computer-assisted image analysis, of lymphangioma tissues in the livers of anlotinib treated and control mice (n = 6 per group). (E) Western blot analysis showing the effects of anlotinib on VEGFR-2 and VEGFR-3 phosphorylation in lymphangioma. (F) Densitometry analysis of VEGFR-2 phosphorylation, VEGFR-2, VEGFR-3 phosphorylation and VEGFR-3 bands in E. Data are mean values ± SD (n = 6). *P < 0.05; ***P < 0.001; Student’s t-test. These experiments were performed 3 times.
Figure 6
Figure 6
Anlotinib blocks tube formation and migration of hLECs in vitro. (A) Typical images of capillary-like tubules formed by hLECs on Matrigel in the absence (control group) or presence (anlotinib group) of different anlotinib concentrations. Magnification, 100×, scale bar, 100 μm. (B) Tube length was significantly shorter in the presence of different anlotinib doses than in the control. (C) Tube area significantly decreased in the presence of different anlotinib doses, as compared with the control. (D) Representative images of the invaded hLECs across a Transwell chamber after 24 h under various experimental conditions. Magnification, 50×, scale bar, 100 μm. (E) Changes in the number of migrated hLECs. Data are mean values ± SD. **P < 0.01, ***P < 0.001; one-way ANOVA. These experiments were performed twice.
Figure 7
Figure 7
Anlotinib interferes with VEGF-C-mediated activation of VEGFR-3 and downstream mediators in hLECs. (A) Western blot analysis showing the effect of anlotinib on VEGFR-3, Akt and Erk phosphorylation after stimulation with VEGF-C (200 ng/mL). (B) Densitometry analysis of VEGFR-3 phosphorylation and VEGFR-3 bands in A. (C) Densitometry analysis of Akt phosphorylation and Akt bands in A. (D) Densitometry analysis of Erk phosphorylation and Erk bands in a. Data are mean values ± SD. **P < 0.01, ***P < 0.001; one-way ANOVA. These experiments were performed 3 times.
Figure 8
Figure 8
Schematic presentation of suppression of lymphatic metastasis by anlotinib. VEGF-C binds its receptor VEGFR-3 on hLECs (process a), thus causing the phosphorylation of Akt and Erk, then promoting lymphangiogenesis (process b), lymphatic metastasis (process c), and formation of new distant metastatic lesions (process d). These 4 processes are suppressed by anlotinib (steps 1, 2, 3 and 4, respectively).

References

    1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.
    1. Christofori G. New signals from the invasive front. Nature. 2006;441:444–50.
    1. Zhang XY, Lu WY. Recent advances in lymphatic targeted drug delivery system for tumor metastasis. Cancer Biol Med. 2014;11:247–54.
    1. El-Sherief AH, Lau CT, Carter BW, Wu CC. Staging lung cancer: regional lymph node classification. Radiol Clin North Am. 2018;56:399–409.
    1. Wang YN, Yao S, Wang CL, Li MS, Sun LN, Yan QN, et al. Clinical significance of 4l lymph node dissection in left lung cancer. J Clin Oncol. 2018;36:2935–42.
    1. Ikemura S, Aramaki N, Fujii S, Kirita K, Umemura S, Matsumoto S, et al. Changes in the tumor microenvironment during lymphatic metastasis of lung squamous cell carcinoma. Cancer Sci. 2017;108:136–42.
    1. Heinzelbecker J, Gropp T, Weiss C, Huettl K, Stroebel P, Haecker A, et al. The role of lymph vessel density and lymphangiogenesis in metastatic tumor spread of nonseminomatous testicular germ cell tumors. Urol Oncol. 2014;32:178–85.
    1. Stacker SA, Baldwin ME, Achen MG. The role of tumor lymphangiogenesis in metastatic spread. FASEB J. 2002;16:922–34.
    1. Qin T, Huang D, Liu Z, Zhang X, Jia Y, Xian CJ, et al. Tumor necrosis factor superfamily 15 promotes lymphatic metastasis via upregulation of vascular endothelial growth factor-C in a mouse model of lung cancer. Cancer Sci. 2018;109:2469–78.
    1. Garcia-Caballero M, Paupert J, Blacher S, Van de Velde M, Quesada AR, Medina MA, et al. Targeting VEGFR-3/-2 signaling pathways with AD0157: a potential strategy against tumor-associated lymphangiogenesis and lymphatic metastases. J Hematol Oncol. 2017;10:122.
    1. Eklund L, Bry M, Alitalo K. Mouse models for studying angiogenesis and lymphangiogenesis in cancer. Mol Oncol. 2013;7:259–82.
    1. Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vinals F, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15:220–31.
    1. Ribatti D. Antiangiogenic therapy accelerates tumor metastasis. Leuk Res. 2011;35:24–6.
    1. Saif MW, Knost JA, Chiorean EG, Kambhampati SR, Yu D, Pytowski B, et al. Phase 1 study of the anti-vascular endothelial growth factor receptor 3 monoclonal antibody LY3022856/IMC-3c5 in patients with advanced and refractory solid tumors and advanced colorectal cancer. Cancer Chemother Pharmacol. 2016;78:815–24.
    1. Vaahtomeri K, Karaman S, Makinen T, Alitalo K. Lymphangiogenesis guidance by paracrine and pericellular factors. Genes Dev. 2017;31:1615–34.
    1. Roskoski Jr R. Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas. Pharmacol Res. 2017;120:116–32.
    1. Sun Y, Niu W, Du F, Du C, Li S, Wang J, et al. Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors. J Hematol Oncol. 2016;9:105.
    1. Han BH, Li K, Wang QM, Zhang L, Shi JH, Wang ZH, et al. Effect of anlotinib as a third-line or further treatment on overall survival of patients with advanced non-small cell lung cancer the alter 0303 phase 3 randomized clinical trial. JAMA Oncol. 2018;4:1569–75.
    1. Han BH, Li K, Zhao YZ, Li BL, Cheng Y, Zhou JY, et al. Anlotinib as a third-line therapy in patients with refractory advanced non-small-cell lung cancer: a multicentre, randomised phase ii trial (ALTER0302). Br J Cancer. 2018;118:654–61.
    1. Xie C, Wan X, Quan H, Zheng M, Fu L, Li Y, et al. Preclinical characterization of anlotinib, a highly potent and selective vascular endothelial growth factor receptor-2 inhibitor. Cancer Sci. 2018;109:1207–19.
    1. Otrock ZK, Makarem JA, Shamseddine AI. Vascular endothelial growth factor family of ligands and receptors: review. Blood Cells Mol Dis. 2007;38:258–68.
    1. Lin B, Song X, Yang D, Bai D, Yao Y, Lu N. Anlotinib inhibits angiogenesis via suppressing the activation of VEGFR2, PDGFRbeta and FGFR1. Gene. 2018;654:77–86.
    1. Wang G, Sun M, Jiang Y, Zhang T, Sun W, Wang H, et al. Anlotinib, a novel small molecular tyrosine kinase inhibitor, suppresses growth and metastasis via dual blockade of VEGFR2 and met in osteosarcoma. Int J Cancer. 2019;145:979–93.
    1. Mancardi S, Stanta G, Dusetti N, Bestagno M, Jussila L, Zweyer M, et al. Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete freund’s adjuvant. Exp Cell Res. 1999;246:368–75.
    1. Qin TT, Xu GC, Qi JW, Yang GL, Zhang K, Liu HL, et al. Tumour necrosis factor superfamily member 15 (TNFSF15) facilitates lymphangiogenesis via up-regulation of VEGFR3 gene expression in lymphatic endothelial cells. J Pathol. 2015;237:307–18.
    1. Iljin K, Karkkainen MJ, Lawrence EC, Kimak MA, Uutela M, Taipale J, et al. VEGFR3 gene structure, regulatory region, and sequence polymorphisms. FASEB J. 2001;15:1028–36.
    1. Deng Y, Yang Y, Yao B, Ma L, Wu Q, Yang Z, et al. Paracrine signaling by VEGF-C promotes non-small cell lung cancer cell metastasis via recruitment of tumor-associated macrophages. Exp Cell Res. 2018;364:208–16.
    1. Hamrah P, Chen L, Cursiefen C, Zhang Q, Joyce NC, Dana MR. Expression of vascular endothelial growth factor receptor-3 (VEGFR-3) on monocytic bone marrow-derived cells in the conjunctiva. Exp Eye Res. 2004;79:553–61.
    1. Zheng W, Aspelund A, Alitalo K. Lymphangiogenic factors, mechanisms, and applications. J Clin Invest. 2014;124:878–87.
    1. Deng Y, Zhang X, Simons M. Molecular controls of lymphatic VEGFR3 signaling. Arterioscler Thromb Vasc Biol. 2015;35:421–9.
    1. Cheng P, Dai W, Wang F, Lu J, Shen M, Chen K, et al. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1-RAGE and AKT pathways. Biochem Biophys Res Commun. 2014;443:1162–8.

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