VEGF-D-induced draining lymphatic enlargement and tumor lymphangiogenesis promote lymph node metastasis in a xenograft model of ovarian carcinoma

Reproductive Biology and Endocrinology

Xia Zhao²  Yu-Quan Wei³  Bing Kan³  Xue-Mei Wang²  Chun-Ting Wang³  Yan-Jun Wen³  Dong Wang¹  Xian-Cheng Chen²  Li-Cheng Du¹ 

[1] Department of Surgery, Shandong Provincial Hospital affiliated to Shandong University, Jingwu Road, Jinan, China;Department of Gynecology and Obstetrics, Second West China Hospital, West China Medical School, Sichuan University, South Renmin Road, Chengdu, China;National Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, South Renmin Road, Chengdu, China

关键词: Xenograft;    Lymphangiogenesis;    Metastasis;    VEGF-D;    Ovarian carcinoma;   

Others  :  804701
DOI  :  10.1186/1477-7827-12-14

 received in 2013-06-20, accepted in 2013-12-29,  发布年份 2014

【 摘 要 】

Background

Vascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer.

Methods

Human ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. Tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan’s blue lymphangiography. Tumoral lymphatics were delineated with both Evan’s blue and LYVE-1 immunostaining. Tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. Expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. Tumor cell apoptosis was analyzed by Hoechst dyeing.

Results

Mice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls.

Conclusions

Our data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.

【 授权许可】

   
2014 Du et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]di Re F, Baiocchi G, Fontanelli R, Grosso G, Cobellis L, Raspagliesi F, di Re E: Systematic pelvic and paraaortic lymphadenectomy for advanced ovarian cancer: prognostic significance of node metastases. Gynecol Oncol 1996, 62(3):360-365.
• [2]Suzuki M, Ohwada M, Yamada T, Kohno T, Sekiguchi I, Sato I: Lymph node metastasis in stage I epithelial ovarian cancer. Gynecol Oncol 2000, 79(2):305-308.
• [3]Li L, Liu B, Li X, Yang S, Xiao J, Chen M, Zhang Y, Ma J: Vascular endothelial growth factor D and intratumoral lymphatics as independent prognostic factors in epithelial ovarian carcinoma. Anat Rec (Hoboken) 2009, 292(4):562-569.
• [4]Yokoyama Y, Charnock-Jones DS, Licence D, Yanaihara A, Hastings JM, Holland CM, Emoto M, Umemoto M, Sakamoto T, Sato S, Mizunuma H, Smith SK: Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma. Br J Cancer 2003, 88(2):237-244.
• [5]Morice P, Joulie F, Camatte S, Atallah D, Rouzier R, Pautier P, Pomel C, Lhommé C, Duvillard P, Castaigne D: Lymph node involvement in epithelial ovarian cancer: analysis of 276 pelvic and paraaortic lymphadenectomies and surgical implications. J Am Coll Surg 2003, 197(2):198-205.
• [6]Christiansen A, Detmar M: Lymphangiogenesis and cancer. Genes Cancer 2011, 2(12):1146-1158.
• [7]de Poncheville L, Perrotin F, Lefrancq T, Lansac J, Body G: Does paraaortic lymphadenectomy have a benefit in the treatment of ovarian cancer that is apparently confined to the ovaries? Eur J Cancer 2001, 37(2):210-215.
• [8]Shirai Y, Wakai T, Hatakeyama K: Radical lymph node dissection for gallbladder cancer: indications and limitations. Surg Oncol Clin N Am 2007, 16(1):221-232.
• [9]Stacker SA, Stenvers K, Caesar C, Vitali A, Domagala T, Nice E, Roufail S, Simpson RJ, Moritz R, Karpanen T, Alitalo K, Achen MG: Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem 1999, 274(45):32127-32136.
• [10]Harris NC, Paavonen K, Davydova N, Roufail S, Sato T, Zhang YF, Karnezis T, Stacker SA, Achen MG: Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer. FASEB J 2011, 25(8):2615-2625.
• [11]Kärpänen T, Heckman CA, Keskitalo S, Jeltsch M, Ollila H, Neufeld G, Tamagnone L, Alitalo K: Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. FASEB J 2006, 20(9):1462-1472.
• [12]Thelen A, Scholz A, Benckert C, von Marschall Z, Schröder M, Wiedenmann B, Neuhaus P, Rosewicz S, Jonas S: VEGF-D promotes tumor growth and lymphatic spread in a mouse model of hepatocellular carcinoma. Int J Cancer 2008, 122(11):2471-2481.
• [13]Lin W, Jiang L, Chen Y, She F, Han S, Zhu J, Zhou L, Tang N, Wang X, Li X: Vascular endothelial growth factor-D promotes growth, lymphangiogenesis and lymphatic metastasis in gallbladder cancer. Cancer Lett 2012, 314(2):127-136.
• [14]Kozlowski M, Naumnik W, Niklinski J, Milewski R, Dziegielewski P, Laudanski J: Vascular endothelial growth factor C and D expression correlates with lymph node metastasis and poor prognosis in patients with resected esophageal cancer. Neoplasma 2011, 58(4):311-319.
• [15]Herrmann E, Eltze E, Bierer S, Kopke T, Gorge T, Neumann J, Hertle L, Wulfing C: VEGF-C, VEGF-D and Flt-4 in transitional bladder cancer: relationships to clinicopathological parameters and long-term survival. Anticancer Res 2007, 27(5A):3127-3133.
• [16]Niki T, Iba S, Tokunou M, Yamada T, Matsuno Y, Hirohashi S: Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. Clin Cancer Res 2000, 6(6):2431-2439.
• [17]Girling JE, Donoghue JF, Lederman FL, Cann LM, Achen MG, Stacker SA, Rogers PA: Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer. Reprod Biol Endocrinol 2010, 8:84. BioMed Central Full Text
• [18]Mohammed RA, Green A, El-Shikh S, Paish EC, Ellis IO, Martin SG: Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio- and lymphangiogenesis. Br J Cancer 2007, 96(7):1092-1100.
• [19]Thériault C, Pinard M, Comamala M, Migneault M, Beaudin J, Matte I, Boivin M, Piché A, Rancourt C: MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis. Gynecol Oncol 2011, 121(3):434-443.
• [20]Hakkarainen T, Hemminki A, Pereboev A, Barker S, Asiedu C, Strong T, Kanerva A, Wahlfors J, Curiel D: CD40 is expressed on ovarian cancer cells and can be utilized for targeting adenoviruses. Clin Cancer Res 2003, 9(2):619-624.
• [21]Gallagher NJ, Eliopoulos AG, Agathangelo A, Oates J, Crocker J, Young LS: CD40 activation in epithelial ovarian carcinoma cells modulates growth, apoptosis, and cytokine secretion. Mol Pathol 2002, 55(2):110-120.
• [22]Wissmann C, Detmar M: Pathways targeting tumor lymphangiogenesis. Clin Cancer Res 2006, 12(23):6865-6868.
• [23]Tobler NE, Detmar M: Tumor and lymph node lymphangiogenesis–impact on cancer metastasis. J Leukoc Biol 2006, 80(4):691-696.
• [24]Zwaans BM, Bielenberg DR: Potential therapeutic strategies for lymphatic metastasis. Microvasc Res 2007, 74(2–3):145-158.
• [25]Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, Riccardi L, Alitalo K, Claffey K, Detmar M: Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001, 7(2):192-198.
• [26]Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S, Huarte J, Montesano R, Jackson DG, Orci L, Alitalo K, Christofori G, Pepper MS: Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 2001, 20(4):672-682.
• [27]Chaudary N, Milosevic M, Hill RP: Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer. Gynecol Oncol 2011, 123(2):393-400.
• [28]Shimizu K, Kubo H, Yamaguchi K, Kawashima K, Ueda Y, Matsuo K, Awane M, Shimahara Y, Takabayashi A, Yamaoka Y, Satoh S: Suppression of VEGFR-3 signaling inhibits lymph node metastasis in gastric cancer. Cancer Sci 2004, 95(4):328-333.
• [29]Yasuoka H, Nakamura Y, Zuo H, Tang W, Takamura Y, Miyauchi A, Nakamura M, Mori I, Kakudo K: VEGF-D expression and lymph vessels play an important role for lymph node metastasis in papillary thyroid carcinoma. Mod Pathol 2005, 18(8):1127-1133.
• [30]Kurahara H, Takao S, Maemura K, Shinchi H, Natsugoe S, Aikou T: Impact of vascular endothelial growth factor-C and -D expression in human pancreatic cancer: its relationship to lymph node metastasis. Clin Cancer Res 2004, 10(24):8413-8420.
• [31]Nakamura Y, Yasuoka H, Tsujimoto M, Yang Q, Imabun S, Nakahara M, Nakao K, Nakamura M, Mori I, Kakudo K: Prognostic significance of vascular endothelial growth factor D in breast carcinoma with long-term follow-up. Clin Cancer Res 2003, 9(2):716-721.
• [32]Gisterek I, Matkowski R, Kozlak J, Dus D, Lacko A, Szelachowska J, Kornafel J: Evaluation of prognostic value of VEGF-C and VEGF-D in breast cancer–10 years follow-up analysis. Anticancer Res 2007, 27(4C):2797-2802.
• [33]Zhao YC, Ni XJ, Wang MH, Zha XM, Zhao Y, Wang S: Tumor-derived VEGF-C, but not VEGF-D, promotes sentinel lymph node lymphangiogenesis prior to metastasis in breast cancer patients. Med Oncol 2012, 29(4):2594-2600.
• [34]Rossochacka-Rostalska B, Gisterek I, Suder E, Matkowski R, Szelachowska J, Kornafel J: Vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor-D (VEGF-D) in ovarian carcinomas. Ginekol Pol 2006, 77(11):830-839.
• [35]Massi D, Puig S, Franchi A, Malvehy J, Vidal-Sicart S, González-Cao M, Baroni G, Ketabchi S, Palou J, Santucci M: Tumour lymphangiogenesis is a possible predictor of sentinel lymph node status in cutaneous melanoma: a case–control study. J Clin Pathol 2006, 59(2):166-173.
• [36]Hirakawa S, Brown LF, Kodama S, Paavonen K, Alitalo K, Detmar M: VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood 2007, 109(3):1010-1017.
• [37]Tilney NL: Patterns of lymphatic drainage in the adult laboratory rat. J Anat 1971, 109(Pt 3):369-383.
• [38]Harrell MI, Iritani BM, Ruddell A: Lymph node mapping in the mouse. J Immunol Methods 2008, 332(1–2):170-174.
• [39]Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, Dumont D, Breitman M, Alitalo K: Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A 1995, 92(8):3566-3570.
• [40]Fitz LJ, Morris JC, Towler P, Long A, Burgess P, Greco R, Wang J, Gassaway R, Nickbarg E, Kovacic S, Ciarletta A, Giannotti J, Finnerty H, Zollner R, Beier DR, Leak LV, Turner KJ, Wood CR: Characterization of murine Flt4 ligand/VEGF-C. Oncogene 1997, 15(5):613-618.
• [41]Karnezis T, Shayan R, Caesar C, Roufail S, Harris NC, Ardipradja K, Zhang YF, Williams SP, Farnsworth RH, Chai MG, Rupasinghe TW, Tull DL, Baldwin ME, Sloan EK, Fox SB, Achen MG, Stacker SA: VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium. Cancer Cell 2012, 21(2):181-195.
• [42]Yu M, Berk R, Kosir MA: CXCL7-Mediated stimulation of lymphangiogenic factors VEGF-C, VEGF-D in human breast cancer cells. J Oncol 2010, 2010:939407.
• [43]Yasuoka H, Kodama R, Hirokawa M, Takamura Y, Miyauchi A, Inagaki M, Sanke T, Nakamura Y: Neuropilin-2 expression in papillary thyroid carcinoma: correlation with VEGF-D expression, lymph node metastasis, and VEGF-D-induced aggressive cancer cell phenotype. J Clin Endocrinol Metab 2011, 96(11):E1857-E1861.
• [44]Reinartz S, Failer S, Schuell T, Wagner U: CA125 (MUC16) gene silencing suppresses growth properties of ovarian and breast cancer cells. Eur J Cancer 2012, 48(10):1558-1569.
• [45]Nagamoto N, Saito Y, Ohta S, Sato M, Kanma K, Sagawa M, Takahashi S, Usuda K, Nakada T, Hashimoto K: Relationship of lymph node metastasis to primary tumor size and microscopic appearance of roentgenographically occult lung cancer. Am J Surg Pathol 1989, 13(12):1009-1013.
• [46]Carter CL, Allen C, Henson DE: Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer 1989, 63(1):181-187.
• [47]Nathanson SD, Anaya P, Avery M, Hetzel FW, Sarantou T, Havstad S: Sentinel lymph node metastasis in experimental melanoma: relationships among primary tumor size, lymphatic vessel diameter and 99mTc-labeled human serum albumin clearance. Ann Surg Oncol 1997, 4(2):161-168.
• [48]Chung EJ, Rho YS, Baek SK, Woo JS, Kwon SY, Lee NJ, Chae YS, Jung KY: Does a growing tumour volume induce lymphangiogenesis? A study of oral/oropharyngeal cancer. J Otolaryngol Head Neck Surg 2011, 40(4):311-317.
• [49]Akahane M, Akahane T, Matheny SL, Shah A, Okajima E, Thorgeirsson UP: Vascular endothelial growth factor-D is a survival factor for human breast carcinoma cells. Int J Cancer 2006, 118(4):841-849.
• [50]Valtola R, Salven P, Heikkila P, Taipale J, Joensuu H, Rehn M, Pihlajaniemi T, Weich H, de Waal R, Alitalo K: VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am J Pathol 1999, 154(5):1381-1390.
• [51]Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo R, Jackson DG, Nishikawa S, Kubo H, Achen MG: VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 2001, 7(2):186-191.