Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

Abstract

Introduction and ObjectivesLymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.ResultsA comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.ConclusionDifferential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.

A practical and sensitive method of quantitating lymphangiogenesis in vivo

“…In an earlier study, utilizing numerous other in vitro assays and tumor xenotransplants in nude mice, we have defined the multiplicity of mechanisms underlying the capacity of the 468LN cells in promoting lymphangiogenesis, lymphovascular invasion and lymphatic metastasis. 8 We showed that they resulted from the dual overexpression of a9b1-integrin and its ligand VEGF-D by 468LN cells. The limitation of the above-mentioned tumor model is that it is time consuming for the xenograft to produce tumors of appreciable sizes to measure intratumoral lymphangiogenesis.…”

“…VEGF-D secretion (24 h, in serum-free medium) by these cells were, respectively, 229.50 pg/ml for 468LN and 48.33 pg/ml for DVEGF-D/468LN, measured by ELISA. 8 In the illustrative study, tubes containing BME alone provided the negative control; those with recombinant Using these five types of implants, we quantified both lymphangiogenesis and angiogenesis with indirect and direct measurements in nude mice as outlined in Figure 1 and detailed in methodology. Figure 3 Lymphatic ingrowth assay and mRNA expression.…”

“…We established stable VEGF-D knocked down 468LN cells using shRNA plasmids. 8 Both 468LN-and VEGF-D-silenced 468LN cells (named DVEGF-D/468LNs) were grown as monolayers in a-MEM supplemented with 10% FBS, 50 U/ml penicillin and 50 mg/ml streptomycin (all materials from GIBCO/ Invitrogen, ON, Canada).…”

“…LECs, when placed on collagen gel or matrigel, align themselves to form tube-like structures under appropriate stimuli. 8 However, most of these systems suffer from limitations including the short life or limited number of primary cells, or possible non-physiological nature of immortalized cells. In a more physiological, organotypic model of thoracic duct rings explanted in collagen gels allows lymphatic capillary-like sprouting under stimulated conditions, which can be quantified by computerassisted imaging.…”

“…9 At the microscopic level, immunohistochemical staining for specific markers such as lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1), vascular endothelial growth factor receptor 3 (VEGFR3) or Podoplanin (Pdpn) or PROX-1 (Prox1) provides an opportunity for visualization of lymphatic microvessels. [10][11][12][13] Examples of in vivo models manipulating lymphangiogenesis include (a) induction of tumor-associated lymphangiogenesis by VEGF-C or -D overexpression in tumor cells 8,14,15 or in transgenic mice, 16,17 (b) lymphatic hyperplasia induced by intraperitoneal injection of incomplete Freund's adjuvant, 18 (c) implantation of growth factor-containing pellets into corneal micropockets 19,20 and (d) the use of avian chorioallantoic membrane (CAM) to stimulate lymphangiogenesis. 21 Each assay has its own advantages and limitations depending on the objectives.…”