Since its isolation, the 468LN cell line was shown to be a lymph node metastasizing variant of the MDA-MB-468 human breast adenocarcinoma cell line with increased in vitro
proliferative capacity and overexpression of α9β1 integrin and osteopontin 
. While earlier studies revealed multiple mechanisms underlying their metastatic phenotype 
, the precise molecular mechanism(s) responsible for their increased lymphatic metastatic ability remained undefined. In the present study, utilizing multiple in vitro
and in vivo
assays, we have dissected the mechanisms underlying the capacity of the cells in promoting lymphangiogenesis, lympho-vascular invasion and lymphatic metastasis. We have excluded the differential role of COX-2 expression in VEGF-C or D upregulation and show that differential overexpression of α9β1 and its ligand VEGF-D by 468LN cells underlie these events. Screening of several human breast cancer cell lines revealed that 468LN cells express the highest level of both markers (Fig S9
). However, another cell line, MDA-MB-231, also known for its ability for lymphatic metastasis, expresses both these markers. This observation suggests that these markers may also contribute to lymphatic metastasis in addition to the earlier reported COX-2 mediated upregulation of VEGF-C 
in this cell line. Since a diversity of in vitro
derived cell lines may reflect the tumor cell heterogeneity with some degree of clonal dominance in situ in advanced cancers, we suggest that present results are clinically relevant. We show that dual over expression of α9β1 integrin and its ligand VEGF-D by 468LN cells lead to multiple mechanisms in promoting lymphatic metastasis. While VEGF-D production accounted for their robust ability to induce lymphangiogenesis in vitro
and in vivo
, its interaction with α9β1 equipped them with an autocrine stimulus for motility and invasiveness, which represent key steps in the process of metastasis by both vascular and lymphatic routes. Furthermore, tumor-derived VEGF-D served both as a paracrine as well as a juxtacrine stimulus for tumor-induced lymphangiogenesis. Finally, our in vivo
studies in nude mice confirmed the obligatory roles of both α9 integrin receptor and its ligand VEGF-D in promoting tumor-associated lymphangiogenesis and lymphatic metastasis. Present study, to our knowledge, is the first report of a dual role of this receptor-ligand interaction in human breast cancer, in events associated tumor growth and metastasis, in particular, with a lymphatic metastatic phenotype.
We have employed multiple in vitro
and in vivo
approaches, which complement one another for unveiling mechanisms. In vitro
approaches included proliferation, migration, invasion assays for tumor cells revealing autocrine stimulation by endogenous VEGF-D. To test and dissect the paracrine and juxtacrine roles of VEGF-D produced in the tumor micro-environment, we employed HMVEC-dLy cells in a number of in vitro
assays: tube formation, migration and invasion of the ECM, which are recognized as key steps for angiogenesis and lymphangiogenesis. In all cases the stimulation provided by tumor cell-conditioned medium or co-culture with tumor cells was attenuated by knocking down VEGF-D, and the stimulation could be restored with exogenous VEGF-D. In co-culture experiments the role of tumor cell-derived VEGF-D in accelerating tubulogenesis by HMVEC-dLy cells can best be interpreted as a juxtacrine regulation (). As noted in the present study, an alignment of two partners was also reported during tube formation by coculturing HUVEC cells with a brain cancer cell line 
. We suggest that this alignment demonstrated in vitro
is an indicator of close physical association of the partners allowing for a juxtacrine stimulation of lymphatic and blood capillary growth by recruitment of endothelial cell precursors.
Schematic representation of the cross talk between tumor cells and lymphatic endothelial cells:
The in vivo studies of lymphangiogenesis utilized three different approaches: Evans blue dye tracing, quantification of LYVE-1 marker at the mRNA and protein levels, and identification of lymphatic vessels by immunofluorescence. The in vivo studies on lymphatic metastasis utilized two different approaches: the presence of human mitochondrial marker MTCO2 and the GFP marker. Our in vivo results clearly indicate that VEGF-D is a key lymphangiogenic factor for lymphangiogenesis on its own and abrogation of expression of either VEGF-D or its receptor α9 integrin could completely eradicate lymphangiogenesis and lymphatic metastasis. A concomitant reduction in tumor growth by VEGF-D knock down can be attributed to the autocrine proliferation stimulating role of VEGF-D shown in vitro. The fact that α9 integrin knock down alone was equally efficient as VEGF-D knock down in abolishing lymphangiogenesis and lymphatic metastasis reinforces the juxtacrine role of α9β1 bound VEGF-D, which can additionally bind to VEGF-R3 expressed by lymphatic endothelial cells to stimulate the cellular steps in lymphangiogenesis ().
In our earlier studies with human breast cancer 
, we identified the potential roles of COX-2, VEGF-C and a diverse family of VEGF-C binding receptors for lymphangiogenesis and lympho-vascular invasion. The present study shows that COX-2 independent overexpression of VEGF-D can play an equally important role in these events, and α9β1 integrin represents an important lymphangiogenic receptor as well as a receptor for autocrine motility in cancer cells. The migration and invasion-stimulating role of endogenous VEGF-D in 468LN cells was demonstrated by silencing VEGF-D gene. The obligatory role of α9β1 integrin as the dominant receptor of this ligand for mediating these functions was demonstrated by using α9β1 blocking antibody as well as silencing of the α9 gene in cancer cells. Furthermore, silencing of α9 was shown to suppress both native and VEGF-D stimulated Erk1/2 phosphorylation, a key signaling event in α9-VEGF-D interaction 
. Autocrine actions of VEGF-C and VEGF-D including their ability to stimulate cellular migration has been documented in other cell types, for example, Kaposi's sarcoma cells 
and lung cancer cells 
in conjunction with receptors like VEGFR-2 and VEGFR-3.
Overexpression of α9β1 integrin in 468LN cells appears to provide a metastatic advantage by virtue of overexpression of at least two ligands, osteopontin and VEGF-D. This integrin (also known as VLA-9) is a key receptor involved in leukocyte migration 
, due to interaction with multiple ligands including osteopontin 
. Since osteopontin, a metastasis promoter protein 
and a marker of human breast cancer progression 
, is over-expressed in 468LN cells, this ligand may present an additional autocrine pathway for metastasis, at least in part by interaction with α9β1 via the RGD domain 
. The important role of α9β1 as a lymphangiogenic receptor because of its binding to VEGF-C and VEGF-D has been well established 
. This function explains why homozygous null mutation of the α9 gene causes congenital chylothorax in the murine fetus 
. Tumoral expression of α9β1 showed a significant association with reduced overall patient survival and with distant-metastasis-free survival 
. The ability of α9β1 to bind VEGF-A can also contribute to angiogenesis 
, as also documented by reduced tumor-associated angiogenesis by α9 knock down in our in vivo
studies. Interestingly, another α9β1 ligand tenascin, an ECM protein, was reported to be a stromal marker for malignancy in mammary epithelium 
. Thus expression of α9β1 by breast cancer cells and presence of any of the above ligands in the tumor microenvironent could increase their migratory or invasive capacity to promote metastasis by both vascular and lymphatic routes. For example, host macrophages, which are known to be a rich source of both VEGF-C and VEGF-D during inflammation 
as well as tumor growth 
, could have a paracrine effect on cancer cells in promoting metastastic phenotype. Indeed, we found that the conditioned medium from the VEGF-C and VEGF-D producing macrophage cell line RAW 264.7 strongly stimulated migration and invasiveness of 468LN cells, and this stimulation was largely abrogated in α9 silenced cells. Furthermore, genetic depletion of α9 completely abrogated tumor-associated lymphangiogenesis and lymphatic metastasis in vivo
affirming the key role of this receptor in these events possibly utilising multiple ligands produced by the tumor as well as host cells.
We have previously shown that COX-2 mediated VEGF-C upregulation in human breast cancer cell lines 
confers a prometastatic phenotype by two mechanisms: increased ability for inducing lymphangiogenesis in situ 
and an autocrine stimulation of motility by utilizing a diverse family of VEGF-C receptors 
. VEGF-D can now be added to VEGF-C as a partner in promoting lymphatic metastasis in human breast cancer by utilizing the same mechanisms. Peritumoral lymph vessel density in invasive lobular breast cancer was correlated with VEGF-D expression by cancer cells and lymph node metastasis 
. VEGF-D, which can bind to both VEGFR-2 and VEGFR-3, was shown to be capable of inducing both angiogenesis and lymphangogenesis leading to lymphatic metastasis in NOD/SCID mice transplanted with a tumor cell line forced to over-express VEGF-D, but not VEGF-A. The lymphatic spread by the VEGF-D expressing line could be blocked with a VEGF-D specific antibody 
. We could block every event in tumor progression by either VEGF-D or α9 knock down, such as tumor growth, tumor associated lymphangiogenesis, angiogenesis, and lymphatic metastasis, indicating the independent as well as combined roles of the ligand and the receptor in cancer progression in vivo
. The independent role of this receptor in the present cell line can be attributed to the paucity of expression of an alternate lymphangiogenic receptor such as VEGF-R3 (). The independent role of this ligand suggests the importance of the demonstrated autocrine, paracrine and proposed juxtacrine roles of VEGF-D (). Furthermore, angiogenic ability of VEGF-D by binding to VEGF-R2 on endothelial cell precursors 
may explain reduced tumor associated angiogenesis in vivo
following VEGF-D knockdown. While VEGF-D expression was reported to be associated with poor prognosis in human breast cancer 
, the prognostic role of the combined expression of α9 integrin and VEGF-D in human breast cancer remains to be investigated.
In conclusion, our combined in vitro and in vivo results with the MDA-MB-468LN breast cancer cell line revealed that key events such as lymphangiogenesis, lympho-vascular invasion and lymphatic metastasis can be triggered by highly aggressive breast cancer cells themselves, in addition to host cells within the tumor micro-environment, involving autocrine, paracrine and juxtacrine pathways.