Tumor-Induced Activation of Lymphatic Endothelial Cells via
Vascular Endothelial Growth Factor Receptor-2 Is Critical
for Prostate Cancer Lymphatic Metastasis
Yiping Zeng,1,4 Kenneth Opeskin,2,5 Jeremy Goad,3 and Elizabeth D. Williams1,4,6
1Bernard O’Brien Institute of Microsurgery; Departments of 2Anatomical Pathology and 3Urology, St. Vincent’s Hospital, Fitzroy,
Victoria; Departments of 4Surgery and 5Pathology, University of Melbourne, Parkville, Victoria;
and 6Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Abstract
Prostate cancer disseminates initially and primarily to
regional lymph nodes. However, the nature of interactions
between tumor cells and lymphatic endothelial cells (LEC) is
poorly understood. In the current study, we have isolated
prostate LECs and developed a series of two-dimensional and
three-dimensional in vitro coculture systems and in vivo
orthotopic prostate cancer models to investigate the inter-
actions of prostate cancer cells with prostate LECs. In vitro ,
highly lymph node metastatic prostate cancer cell lines (PC-3
and LNCaP) and their conditioned medium enhanced prostate
LEC tube formation and migration, whereas poorly lymph
node metastatic prostate cancer cells (DU145) or normal
prostate epithelial cells (RWPE-1) or their conditioned
medium had no effect. In vivo , the occurrence of lymphatic
invasion and lymph node metastasis was observed in PC-3 and
LNCaP xenografts but not in DU145 xenografts. Furthermore,
vascular endothelial growth factor (VEGF) receptor (VEGFR)-2
is expressed by prostate LECs, and its ligands VEGF-A,
VEGF-C, and VEGF-D are up-regulated in highly lymph node
metastatic prostate cancer cells. Recombinant VEGF-A and
VEGF-C, but not VEGF-C156S, potently promoted prostate
LEC tube formation, migration, and proliferation in vitro ,
indicating that signaling via VEGFR-2 rather than VEGFR-3 is
involved in these responses. Consistent with this, blockade of
VEGFR-2 significantly reduced tumor-induced activation of
LECs. These results show that the interaction of prostate
tumor cells with LECs via VEGFR-2 modulates LEC behavior
and is related to the ability of tumor cells to form lymph node
metastases. (Cancer Res 2006; 66(19): 9566-75)
Introduction
Lymphatic vessels are one of the major anatomic pathways for
prostate cancer dissemination (1). Although the importance of
intratumoral lymphatic vessels in mediating lymphatic metastasis
is controversial (2, 3), peritumoral preexisting lymphatic vessels
have been proven to be sufficient for lymphatic metastasis in
several studies (2, 4, 5). However, because peritumoral lymphatic
vessels exist before tumor development in nearly all tissues, why
then do tumors of some origins preferentially metastasize to lymph
nodes? Studies on metastasis patterns and lymphatic mapping of
human cancers have shown that the dissemination of tumor cells
to lymph nodes is nonrandom (1, 6, 7), suggesting that active
molecular interactions between tumor and lymphatic endothelium
are crucial for lymphatic metastasis.
The nature of interactions of tumor cells with lymphatic
endothelium is poorly understood. Vascular endothelial growth
factor (VEGF)-C and VEGF-D have been reported to activate
lymphatic endothelium and promote lymph node metastasis via
the VEGF receptor (VEGFR)-3 pathway (8–11). However, targeting
the VEGF-C/VEGF-D/VEGFR-3 pathway to inhibit tumor lymph
node metastasis has not been successful in some tumor models,
including prostate cancer (2). In addition, the expression of
VEGF-C or VEGF-D does not correlate with lymph node metastasis
in all human tumors (12), implying that other factors participate in
tumor lymphatic metastasis.
There remains much to be discovered about the mechanisms
underlying tumor lymphatic metastasis. In the current study, we
have used two-dimensional and three-dimensional in vitro
coculture systems and in vivo orthotopic xenograft models to
investigate the interactions of prostate cancer cells with prostate
lymphatic endothelial cells (LEC) and show that the activation of
LECs by cancer cells is associated with lymph node metastasis.
Materials and Methods
Reagents. The following antibodies were used: mouse a-human
podoplanin (D2-40; Signet Laboratories, Dedham, MA), mouse a-human
CD31 (DAKO, Glostrup, Denmark), mouse a-human CD34 (NeoMarkers,
Fremont, CA), mouse a-human N-cadherin (Invitrogen, Carlsbad, CA),
mouse a-human fibroblast antigen (Oncogene, San Diego, CA), mouse a-
human cytokeratin (AE1/AE3, DAKO), mouse a-human pan-actin (Ab-5,
NeoMarkers), mouse a-human mitochondria (Chemicon, Temecula, CA),
mouse a-human VEGF-A (NeoMarkers), rabbit a-human VEGF-A (Genen-
tech, San Francisco, CA), rabbit a-human Prox1 (Research Diagnostics,
Flanders, NJ), goat a-mouse LYVE-1 (Santa Cruz Biotechnology, Santa Cruz,
CA), rat a-mouse Ki-67 (DAKO), rabbit a-green fluorescent protein (a-GFP;
Molecular Probes, Eugene, OR), Alexa Fluor 488–, Alexa Fluor 568–, and
Alexa Fluor 680–conjugated secondary antibodies (Molecular Probes), 800
CW IRDye–conjugated secondary antibodies (Rockland, Gilbertsville, PA),
and Dynabeads M-450 sheep a-mouse IgG (Invitrogen). Goat a-human
VEGF-C, mouse a-human VEGF-D, goat a-mouse VEGFR-2, mouse a-
human VEGFR-2 (used in neutralizing experiments), and goat a-human
VEGFR-3 antibodies and recombinant human VEGF-A, VEGF-C, VEGF-C
(Cys156Ser), and VEGF-D protein were purchased from R&D Systems
(Minneapolis, MN).
Isolation of lymphatic and vascular endothelial cells from human
prostate. Primary cultures were established using enzymatic digestion of
fresh prostate tissue obtained from patients undergoing radical prostatec-
tomy. The portions of tissue used for this study contained no prostate
carcinoma as determined by histologic evaluation of immediately adjacent
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Elizabeth D. Williams, Monash Institute of Medical
Research, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.
Phone: 61-3-9594-7164; Fax: 61-3-9594-7114; E-mail: Elizabeth.Williams@med.
monash.edu.au.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-1488
Cancer Res 2006; 66: (19). October 1, 2006 9566 www.aacrjournals.org
Research Article

serial sections. Finely minced tissues were incubated with PBS containing
0.25% collagenase II (Worthington, Lakewood, NJ) and 0.01% DNase I
(Worthington) for 30 to 45 minutes at 37jC followed by gently squeezing
endothelial cells into DMEM (Invitrogen) containing 10% fetal bovine serum
(FBS; JRH Biosciences, Brooklyn, Victoria, Australia). The resulting cell
suspension was passed through a 100-Am nylon cell strainer, pelleted, re-
suspended, and plated in EGM-2 MV medium (Cambrex, Walkersville, MD)
on fibronectin-coated (10 Ag/mL; Sigma, St. Louis, MO) flasks at 37jC,
5% CO2 in a humidified atmosphere. Cell selection was done as previously
described (13) in the subconfluent primary culture that consists of endo-
thelial cells, fibroblasts, and epithelial cells. Briefly, prostate CD34-positive
blood vessel endothelial cells (BVEC) were isolated by immunomagnetic
purification with anti-human CD34 antibody-conjugated immunomagnetic
beads. Prostate LECs were then isolated by incubation of the remaining
CD34-negative cells with anti-human CD31 antibody-conjugated immuno-
magnetic beads. Prostate LECs were propagated in LEC growth medium
(EGM-2 MV medium supplemented with 50 ng/mL VEGF-C), and BVECs
were cultured in EGM-2 MV without VEGF-C. LECs between passages 3 and
6 were used for the experiments described herein. All studies were
conducted with the approval of the St. Vincent’s Hospital (SVH) Human
Research Ethics Committee and in accordance with Australian National
Health and Medical Research Council (NHMRC) guidelines.
Cell culture, transfection, and vital dye labeling. Human prostate
cancer cell lines LNCaP (UroCor, Oklahoma City, OK), PC-3, and DU145
[both from the American Type Culture Collection (ATCC), Rockville, MD]
were cultured in DMEM supplemented with 10% FBS. Human prostate
epithelial cell line RWPE-1 (ATCC) was cultured in keratinocyte serum-free
medium (Invitrogen). Primary human umbilical vascular endothelial cells
(HUVEC; Cambrex) were maintained in EGM medium (Cambrex). PC-3 and
DU145 cells were transfected with either pEGFP-N1 (BD Biosciences,
Bedford, MA) or pDsRed2-N1 plasmid (BD Biosciences) using Lipofect-
AMINE 2000 (Invitrogen) as per the manufacturer’s protocol, and stable
transfectants were selected by G418 (800 Ag/mL; Invitrogen) and sorted by
fluorescence-activated cell sorting to obtain the brightest population (top
10%). Prostate LECs, LNCaP, and RWPE-1 cells were labeled with CM-DiI
(CellTracker, Molecular Probes) as per the manufacturer’s protocol.
Immunofluorescence and immunohistochemistry. Immunofluores-
cence analyses were done on isolated and Matrigel-embedded (BD
Biosciences) cells as previously described (see Supplementary Data for
details; ref. 14). Immunohistochemistry of human prostate and mouse
xenograft tissues was done as described (15, 16). 4T1 mouse mammary
tumor, a kind gift from A/Prof. Robin Anderson (Peter MacCallum Cancer
Center, East Melbourne, Victoria, Australia; ref. 17), was used as a positive
control for a-mouse Ki-67 staining.
Western blot analyses. Western blotting was done as previously described
(see Supplementary Data for details; ref. 14). Signals were visualized using
an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Real-time quantitative reverse transcription-PCR. Quantitative
reverse transcription-PCR (RT-PCR) was done as previously described
(14). The primer sequences used were as follows: Prox1, 5¶-CTGAAGA-
GCTGTCTATAACCAG-3¶ ( forward) and 5¶-GGATCAACATCTTTGCCTGCG-
3¶ (reverse; 40 cycles); podoplanin, 5¶-CGAGGATCTGCCAACTTCAGAAA-3¶
( forward) and 5¶-CAACCAGGGTCACTGTTGACAAA-3¶ (reverse; 30 cycles);
CEA-CAM, 5¶-GTAGCAAAGCCCCAAATCAA-3¶ ( forward) and 5¶-AACG-
GATGGAGATTCCAGTG-3¶ (reverse; 30 cycles); VEGFR-2, 5¶-GTCAAGG-
GAAAGACTACGTTGG-3¶ ( forward) and 5¶-AGCAGTCCAGCATGGTCTG-3¶
(reverse; 50 cycles); and VEGFR-3, 5¶-CACTCCCGCCATACGCCACATCAT-3¶
( forward) and 5¶-CTGCTCTCTATCTGCTCAAACTCC-3¶ (reverse; 30 cycles).
The relative expression level of each target gene was normalized to the
ribosomal housekeeping gene L32 as previously described (14). All
quantitative PCR products were visualized on 2% agarose gel containing
1 Ag/mL ethidium bromide (Bio-Rad, Hercules, CA).
Tube formation assay. To assess the tube-forming ability of isolated
endothelial cells, standard Matrigel was allowed to polymerize in a 96-well
plate and prostate LECs or BVECs were seeded at a density of 3
104 per
well in EGM-2 MV medium. For coculture experiments, a mixture of DiI-
labeled LECs (1.5
104 per well) with each prostate cell line (7.5
103 per
well) was seeded on growth factor–reduced (GFR) Matrigel in serum-free
DMEM. In control cultures, prostate LECs were plated at two cell densities:
low density (same as the LEC density in coculture) and high density (same
as the total cell density in coculture). For VEGFR-2 blocking experiments,
the mixed cells were resuspended in DMEM serum-free medium with
20 Ag/mL of a-human VEGFR-2 or control IgG (R&D Systems) before
seeding on GFR Matrigel. To assess growth factor effects, prostate LECs
were seeded on GFR Matrigel at a density of 1.5
104 per well in serum-free
DMEM with or without specified growth factors. The concentrations of
recombinant human growth factors used were as follows: VEGF-A, 100 ng/
mL; VEGF-C, 100 ng/mL; VEGF-C156S, 500 ng/mL; and VEGF-D, 500 ng/mL.
All tube formation experiments were observed using inverted fluorescence
microscopy (Olympus, Tokyo, Japan), and images were digitally captured
(Olympus) at 0, 3, 6, 9, 12, 24, and 48 hours after plating. The total length,
area, and number of tube-like structures formed by LECs in each well were
measured using Axiovision Rel 4.2 software (Carl Zeiss AG, Jena, Germany).
All experiments were done using three different prostate LEC cell lines.
Wound-healing assay. DiI-labeled prostate LECs (104 per well) were
mixed with prostate cell lines (2.5
103 per well), seeded in 96-well plates,
and grown in EGM-2 MV medium overnight. A wound was made by scraping
the confluent monolayer, and the cells were grown in EGM basal medium
supplemented with 2% FBS. To assess conditioned medium (collected from
subconfluent prostate cell cultures in serum-free DMEM for 48 hours) and
growth factor effects, prostate LECs (104 per well) were grown in a 96-well
plate overnight. After wounding, cells were incubated with EGM basal
medium/2% FBS with or without VEGFs or with 30% conditioned medium.
The concentrations of recombinant human VEGFs used were same as those
used in the tube formation assays. For the VEGFR-2 inhibition assay, cells
were incubated with the corresponding growth medium containing 20 Ag/mL
of a-human VEGFR-2 or control IgG immediately after wounding. The
migration of the cells was digitally recorded (Olympus) at 0, 9, 16, 20, 25, 32,
45, 54, 71, 96, 100, and 106 hours after wounding. The experiment was
terminated when LEC wound was closed. The area of the uncovered wound
gap was measured using Axiovision Rel 4.2 software, and the percentage of
wound closure was calculated at each time point. All experiments were done
using duplicates of three different prostate LEC cell lines.
In vitro growth. For coculture assays, prostate LECs (200 per well) were
mixed with specified prostate cell lines (200 per well) and seeded in 384-well
plates in EGM-2 MV medium. For conditioned medium and growth factor
experiments, prostate LECs (200 cells well) were seeded in 384-well plates in
EGM basal medium/2% FBS with or without VEGFs or with 30%
conditioned medium. The concentrations of human recombinant growth
factors used were same as those used in the tube formation assays. At days
0 to 4, cells were formalin fixed and immunostained with a-human CD31.
The total number of LECs (CD31-positive cells) in each well was counted.
All experiments were done in triplicate.
Xenotransplantation and in vivo imaging tumor metastasis. Five-
week-old severe combined immunodeficient mice (Animal Resources
Centre, Perth, Western Australia, Australia) were anesthetized, and 106
prostate cancer cells (PC-3-DsRed, DU145-DsRed, or LNCaP) were
inoculated into the prostate. Tumor growth was monitored weekly using
in vivo fluorescence imaging from 24 days after inoculation. Mice were
anesthetized, abdominal hair was removed, and fluorescence was emitted
from the tumors and/or metastases were detected using the Kodak (Kodak,
New Haven, CT) or LAS3000 (Fuji, Tokyo, Japan) Imaging Systems. Images
were digitally captured and overlaid onto the X-ray reference image (Kodak
Imaging System). Mice were sacrificed 7 to 8 weeks after tumor cell
inoculation. At harvest, primary tumors and regional lymph nodes were
removed, imaged ex vivo , measured using digital calipers, snap frozen (half),
and formalin fixed (half) for further analysis. Primary tumor volume was
calculated as length
width2
0.5 as previously described (18). All animal
studies were conducted with the approval of the SVH Animal Ethics
Committee and in accordance with NHMRC guidelines.
Quantification of tumor lymphatic vasculature. Lymphatic vessels
were identified using LYVE-1 immunostaining, and lymphatic vessel density
(LVD) was evaluated using absolute counting (16). Briefly, lymphatic vessels
were counted within each tissue section in consecutive intermediate power
Tumor-Lymphatic Endothelium Interactions in Metastasis
www.aacrjournals.org 9567 Cancer Res 2006; 66: (19). October 1, 2006