Abstract B27: Lymphatic endothelium promotes angiogenesis, tumor growth, and metastasis in breast cancer
Authors: Lee Esak , Pandey Niranjan , Popel Aleksander 
Published: August 2014

Abstract
Background: Lymphatic endothelium is one of the elements of tumor microenvironment. However, lymphatic endothelium-mediated effects on cancer are not well understood, and the current prevailing view is that stromal lymphatics function as conduits for metastasis. Here we report that lymphatic endothelium promotes angiogenesis, tumor growth, and metastasis in breast cancer in a paracrine fashion.
Methods: Lymphatic endothelial cells (LEC) or HUVEC conditioned media was prepared in normal or cancerous condition. Those conditioned media were analyzed by reverse western assays, examining 55 angiogenesis-related factors, and 31 chemokine factors. Diverse activities of these conditioned media, including pro-angiogenic, tumorigenic, tumor cell recruiting potentials were tested, and the key paracrine factors were identified, using factor neutralization methods. LEC-embedded Matrigel plugs served as artificial organs containing LEC. Matrigel-implanted animals were subcutaneously treated with tumor-conditioned media (TCM) or serum-free media (SFM) then, the plugs were excised and analyzed after 10 days. Similarly, MDA-MB-231 tumors were orthotopically established with co-injection of LEC: tumors were analyzed in tumor growth, LEC paracrine signals, and angiogenesis. MDA-MB-231-luc-D3H2LN tumor xenografts were established on the inguinal mammary fat pad to observe anterior metastases to lymph nodes (LNs), lungs, liver, and brain. Before tumor inoculation, we pre-treated the mice by injecting TCM or SFM. The inhibitor of LEC paracrine signal for tumor recruitment was treated, then, the distal metastases were observed.
Results: Tumor-conditioned LEC overexpressed a multitude of pro-angiogenic factors and chemokine ligands, compared to normal LEC: the highest increase includes VEGFA (630%), PDGF-BB (300%), EGF (600%), CXCL7 (1,400%), and CCL5 (2,900%). In addition, 4 major anti-angiogenic factors of the normal LEC secretion, including Angiopoietin-2, Endostatin, Pentraxin-3 and TIMP-1 decreased by 80%, 48%, 40% and 54%. Tumor-conditioned LEC paracrine factors promoted EC migration, adhesion, proliferation, and tube formation. The LEC conditioned media showed MDA-MB-231 cell proliferation and migration activities as well. Factor neutralization methods revealed that VEGFA and PDGF-BB are crucial LEC paracrine factors for angiogenesis and pericyte coverage; EGF is central for tumor cell proliferation; CCL5-CCR5 is the key axis for tumor cell recruitment. Nevertheless, blood endothelial cells (HUVEC) conditioned media showed no significant change in paracrine factors as well as the phenotypes of EC and tumor cells. TCM-treated LEC-embedded Matrigel plugs showed enhanced angiogenesis and pericyte infiltration, compared to HUVEC-embedded ones. Moreover, TCM-treated animals showed more angiogenesis in axillary, brachial lymph nodes (LN), lungs and liver with enhanced lymphatic VEGF expression compared to those from serum-free media treated animals. MDA-MB-231 tumors co-injected with LEC showed faster tumor growth in early stage of the tumors with enhanced intratumoral angiogenesis and EGF-dependent tumor cell proliferation compared to normal xenograft models. Pre-treatment of mice with TCM accelerated metastasis to LNs, lungs, and liver, and these organs showed dramatic expression of CCL5 around the lymphatics. However, CCR5 inhibition with Maraviroc significantly blocked the metastases on LNs, lungs, and liver.
Conclusion: We showed LEC induced angiogenesis via VEGF, PDGF-BB; cancer cell proliferation via EGF; and recruitment of cancer cells into metastatic sites via CCL5-CCR5 axis. These results suggest stromal lymphatic endothelium is a novel inducer of angiogenesis, tumor growth and metastasis.
Citation Format: Esak Lee, Niranjan Pandey, Aleksander Popel. Lymphatic endothelium promotes angiogenesis, tumor growth, and metastasis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B27.