Anti-VEGF-A therapy reduces lymphatic vessel density and expression of VEGFR-3 in an orthotopic breast tumor model Brandt Whitehurst 1 , Michael J. Flister 1 , Juhi Bagaitkar 1 , Lisa Volk 1 , Christopher M. Bivens 1 , Brent Pickett 1 , Emely Castro-Rivera 2 , Rolf A. Brekken 2 , Robert D. Gerard 3 and Sophia Ran 1 * 1 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 2 Hamon Center for Therapeutic Oncology, University of Texas Southwestern Medical Center, Dallas, TX 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX Because metastasis contributes significantly to cancer mortality, understanding its mechanisms is crucial to developing effective therapy. Metastasis is facilitated by lymphangiogenesis, the growth of new intratumoral or peritumoral lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor A (VEGF-A) is a well-known angiogenic factor. Increasing evidence implicates VEGF-A in lymphangiogenesis, although the mecha- nism of its pro-lymphangiogenic effect is poorly understood. We examined the effect of the anti-VEGF-A neutralizing antibody 2C3 on tumor lymphangiogenesis and metastasis in an orthotopic breast carcinoma model using MDA-MB-231 cells and its lucifer- ase-tagged derivative, 231-Luc 1 cells. Anti-VEGF-A antibody therapy reduced blood and lymphatic vessel densities by 70% and 80%, respectively, compared with the control antibody. Treat- ment with 2C3 antibody also decreased incidence of lymphatic and pulmonary metastases by 3.2- and 4.5-fold, respectively. Mac- rophage infiltration was reduced in 2C3-treated tumors by 32%, but VEGF-C expression was unchanged. In contrast, neoplastic cells and blood vessels in tumors from 2C3-treated mice expressed significantly less angiopoietin-2 (Ang-2) than tumors from control mice. The reduction in Ang-2 was associated with inhibition of VEGFR-3 expression in intratumoral lymphatic endothelial cells. Both VEGF-A and Ang-2 upregulated the expression of VEGFR-3 in cultured lymphatic endothelial cells. VEGF-A induced prolifer- ation of lymphatic endothelial cells was reduced by 50% by solu- ble Tie-2, suggesting that Ang-2 is an intermediary of the pro-lym- phangiogenic VEGF-A effect. These results suggest a novel mecha- nism by which anti-VEGF-A therapy may suppress tumor lymphangiogenesis and subsequent metastasis supporting the use of anti-VEGF-A therapy to control metastasis clinically. ' 2007 Wiley-Liss, Inc. Key words: VEGF-A; VEGFR-3; lymphangiogenesis; breast cancer; metastasis Metastasis, the dissemination of tumor cells from the primary tumor site, is a major cause of mortality from solid tumors. Meta- static behavior can be facilitated by lymphangiogenesis, the growth of new lymphatic vessels from pre-existing lymphatic ves- sels. 1,2 Several types of malignancies, including breast, 2,3 mela- noma, 4 and head and neck, 5 predominantly utilize the lymphatic vasculature as the route of dissemination. Tumor lymphatic vessel density (LVD) correlates with increased incidence of intravascular invasion, 5,6 lymph node metastasis, 7 cancer recurrence 8 and decreased patient survival. 3 Thus, understanding the mechanisms of tumor lymphangiogenesis is essential for designing effective anti-metastatic therapies and improving patient survival. Lymphangiogenesis is thought to be induced primarily by 1 of the 2 well-characterized lymphangiogenic factors: vascular endo- thelial growth factor C (VEGF-C) or VEGF-D. 9,10 Of these 2 fac- tors, VEGF-C appears to be more clinically significant because its expression is frequently increased in tumors, whereas VEGF-D is highly expressed in normal tissues and often down-regulated in malignancies. 11 VEGF-C expression correlates with increased lymphatic vessel invasion, 8 metastasis 6,12 and poor survival 8,13 in many human cancers. In experimental models, forced expression of VEGF-C induced intratumoral 2,14,15 and peritumoral 16 lym- phangiogenesis associated with increased lymphatic metasta- sis, 2,16–19 whereas neutralization of VEGF-C inhibited both proc- esses. 15,20 The lymphangiogenic activity of VEGF-C is mediated by the VEGFR-3 receptor 21 which, in adulthood, is primarily expressed on lymphatic endothelial cells (LEC). 22,23 VEGFR-3 activation appears to be critical for tumor dissemination through lymphatics because antibody-mediated blockade of VEGFR-3 has been shown to inhibit both the formation of new tumor lymphatic vessels and lymphatic metastasis. 20,24,25 Angiogenesis in solid tumors is thought to be primarily driven by vascular endothelial growth factor A (VEGF-A). 26,27 Several recent studies, however, provided extensive evidence demonstrat- ing that VEGF-A can also regulate lymphangiogenesis. 25,28–31 Nagy et al. were first to show that over-expression of VEGF-A induced new lymphatic vessel formation in the ear and peritoneal lining of nude mice. 28 These neo-lymphatics were structurally and functionally abnormal, resembling hyperplasic vessels found in lymphatic malformations, thus suggesting that elevated VEGF-A at malignant and chronically inflamed sites might contribute to pathological lymphangiogenesis. This hypothesis was subse- quently supported by studies demonstrating induction of a strong lymphangiogenic response in the rat 32 and mouse 33 models of cor- neal injury, chronic inflammation 34 and VEGF-A-induced skin tumorigenesis. 29 Additionally, VEGF-A mediated lymphangio- genesis has been also shown in T241 fibrosarcoma 31 and MDA- MB-435 breast carcinoma cells 25 that have been engineered to over-express this factor. In all tumor experimental models studied to date, induction of VEGF-A dependent intratumoral or peritu- moral 31 lymphatic vessels correlated with lymphatic invasion, 29 lymph node and distant metastasis. 25,29,31 Inhibition of VEGF-A in these models suppressed both new lymphatic vessels and malig- nant spread, thus supporting a long-suspected causality between tumor lymphangiogenesis and metastasis. VEGF-A can promote lymphangiogenesis by direct and indirect mechanisms. The indirect mechanism involves VEGF-A mediated recruitment of inflammatory cells that subsequently supply the Grant sponsor: American Cancer Society, Illinois Division; Grant num- ber: 04-07; Grant sponsor: Department of Defense Breast Cancer Program; Grant number: BC045160; Grant sponsor: Southern Illinois University School of Medicine. Brandt Whitehurst and Michael J. Flister contributed equally to this work. *Correspondence to: Department of Medical Microbiology, Immunol- ogy, and Cell Biology, Southern Illinois University School of Medicine, 801 N. Rutledge, Springfield, IL 62794-9678, USA. Fax: 1217-545-3227 or 1217-545-7333. E-mail: sran@siumed.edu Received 19 July 2006; Accepted after revision 15 May 2007 DOI 10.1002/ijc.22937 Published online 27 June 2007 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: Ang-2, angiopoietin-2; BEC, blood endothelial cells; CCLR, cell culture lysis reagent; LEC, lymphatic endothelial cells; LVD, lymphatic vessel density; MECA32, anti-mouse pan endothelial cell anti- body; MFP, mammary fat pad; RLEC, rat lymphatic endothelial cells; Tie- 2, tyrosine kinase with immunoglobulin-like and EGF-like domains 2; VEGF-A, vascular endothelial growth factor-A; VEGF-C, vascular endo- thelial growth factor-C; VEGFR-2, vascular endothelial growth factor re- ceptor 2; VEGFR-3, vascular endothelial growth factor receptor 3. Int. J. Cancer: 121, 2181–2191 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer