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Supplementary Information
Supplementary text - RANKL mediates directed osteoclast migration
The establishment and expansion of breast cancer metastasis in bone involves osteoclastic
resorption2.
Therefore, we considered the possibility that RANKL, in addition to its well
established roles in stimulating osteoclast formation and activity7,8, directly recruits osteoclasts.
RANKL was found to induce chemotaxis of isolated rat osteoclasts (supplementary Fig. 4a).
The migration of osteoclasts towards a source of RANKL was significantly oriented, in contrast
to the random movement of control osteoclasts (supplementary Fig. 4b). Moreover, osteoclasts
migrated significantly further towards RANKL compared to vehicle (supplementary Fig. 4c),
with mean rate of migration to RANKL of 24 μm/h. This mean migration rate to RANKL is
comparable to that elicited by other known chemotaxins for osteoclasts such as TGF-β (27
m/h)ref. 23. Osteoclast chemotaxis to RANKL was blocked by OPG (supplementary Fig. 4c). In
contrast to the effects of RANKL on osteoclasts, there was no chemotactic response by stromal
cells. Thus, RANKL may regulate the recruitment of both osteoclasts and tumor cells to sites of
bone metastases.
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Methods
Tumor cell lines and primary cell cultures
B16F10 murine melanoma cells, MCF-7 human breast cancer, Hs578T human breast cancer,
Colo205 human colon cancer, SW480 human colon cancer, LNCaP human prostate cancer,
Du145 human prostate cancer, and T47D human epithelial breast tumor (all from ATCC) were
maintained in culture in DMEM plus 10% fetal bovine serum (FBS; Invitrogen, Canada) at 37 oC
within humidified 5% CO2 air. MDA-MB-231 human breast cancer cells were grown at 37oC in
Leibovitz’s media (Invitrogen) plus 10% FBS without CO2 supplementation in air. Nontransformed MCF10A mammary gland epithelial cells and primary mouse mammary gland
epithelial cells freshly isolated from non-pregnant C57Bl6 mouse mammary glands were
cultured as described24. Osteoclasts were isolated from the long bones of neonatal Wistar rats as
described23. Osteoclasts stained positive for tartrate-resistant acid phosphatase (TRAP) and
retracted in response to calcitonin. Animal care was carried out in accordance with the guidelines
of the Council on Animal Care at the University of Toronto and at The University of Western
Ontario.
RANKL and RANK expression analysis
Total RNA was isolated from cell lines and mouse tissues using Trizol (Invitrogen) and reverse
transcribed into cDNA (Clonetech). PCR analysis of transcript expression was performed using
primers for murine RANK (5’-GCAACCTCCAGTCAGCA-3’and 5’-GAAGTCACAGCCCTCAGAATC3’),
murine
RANKL
ATCTAGGACATCCATGCTAATGTTC-3’),
and
(5’-ATCAGAAGACAGCACTCACT-3’
and
5’-
human RANK (5’-GGGAAAGCACTCACAGCTAATTTG-3’
5’-GCACTGGCTTAAACTGTCATTCTCC-3’),
and
human
RANKL
(5’-
3
TGGATCACAGCACATCAGAGCAG-3’
and 5’-TGGGGCTCAATCTATATCTCGAAC-3’). Amplification
of RANK and RANKL from mouse tissues involved an initial denaturation for 1 minute at 94 oC,
followed by 35 cycles of denaturation at 94oC for 30 seconds, annealing at 56oC for 30 sec and
extension for 30 seconds at 72oC. Human RANK and RANKL expression was detected as above,
except for a change in annealing temperature to 57oC for RANK and 55oC for RANKL. -actin
was used as internal control. In some experiments, RANK (TNFRSF11A, GenBank accession
number NM_003839) transcripts were confirmed by quantitative real-time RT-PCR using a
primer/TaqMan probe set specific for hRANK on RNA isolated from MDA-MB-231, MCF7,
and LnCap cell lines, and mRANK on RNA isolated from RAW 264.7 and MC3T3 cell lines and
the 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). RANK mRNA
levels were normalized to the -actin levels (endogenous control, human for MDA-MB-231,
MCF7, and LnCap and mouse for RAW 264.7, and MC3T3) and expressed relative to levels in
MC3T3 cell line. Detection of cell surface expression of RANK protein via FACS utilized FITCconjugated human RANKL (aa159-317; Amgen, Thousand Oaks).
RANK signaling, proliferation, and cell death assays
Cancer cells were serum starved for 12 hours and then stimulated with recombinant murine
RANKL (recombinant aa158 to aa316) in the presence or absence of recombinant murine OPGFC protein (aa22-401; rOPG, both from Amgen, Thousand Oaks)11, SDF-1 (R&D Systems), or
recombinant prolactin (Sigma). In addition, commercially available RANKL (R&D Systems)
was used with similar results in osteoclastogenesis indicating that the observed effects were not
due to secondary effects of recombinant RANKLaa158-aa316. Cell lysates (10 mM Tris pH 7.6,
5 mM EDTA, 50 mM NaCl, 1% triton-X, 30 mM tetra-sodium pyrophosphate, 200 M sodium
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orthovanadate, 1 mM PMSF, 5 g/ml aprotinin, 1 g/pepstatin, 2g/ml leupeptin) were resolved
on 8-12% polyacrylamide gels under reducing conditions and proteins transferred to Hybond
ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Primary
antibodies reactive to ERK1/ERK2, active ERK1/ERK2 (phospho-Thr202/Tyr204), PKB/AKT,
active PKB/AKT (phospho-PKB/AKT-Ser473), STAT5A/B, phospho-Stat5A/B (phosphorTyr694) (Cell Signaling and Transduction Lab), and actin (Sigma) were used. For actin
polymerization studies, tumor cells were stimulated with RANKL or SDF-1 and actin
polymerization determined using phalloidin-FITC. Tumor cell proliferation was determined
using H3-thymidine uptake. Cell death was detected via FACS using propidium
iodide/AnnexinV-FITC double staining
Tumor cell chemotaxis
Migration of cancer cells was assessed using a 96-well chemotaxis chamber (Neuro Probe Inc.
MD) with fibronectin (Sigma) coated polycarbonate filters (8 and 12 m pore size). All cells
were starved for 12 h in DMEM (10 mM Hepes, 0.1% BSA) media, detached using 5 mM EDTA
in Ca2+/Mg2+-free Hank’s buffer, counted and re-suspended for each assay. DMEM media
(10mM Hepes, 0.1% BSA) containing rRANKL, rOPG, or the chemokines SDF-1, 6Ckine, and
CTACK (all chemokines were purchased form R&D Systems) was placed in the lower wells and
5 X 105 B16F10 or 2 X 105 human breast, prostate or colon cancer cells were placed in the upper
wells. Migration of cells was determined at 37oC for 16 h (B16F10 cells) or 6 h (human cancer
cells), fixed, and stained as previously described for chemokine assays6. Migration was
quantified using a plate reading spectrophotometer (Beckman). It should be noted that tumor
cells migrated in a dose response starting from doses as low as 100ng/ml of rRANKL and that
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two different sources of RANKL (Amgen and R&D) gave similar results. Inhibition of
chemotaxis with the PI3K inhibitor wortmannin, the PLC blocker U73122, the PKC inhibitor
GF109203X and the MEK1 inhibitor PD98059 (Calbiochem) was performed as previously
described25-27.
RANK detection on human breast cancer tissue arrays
Paraffin embedded specimens of tumors, lymph node metastasis, and adjacent normal tissue
were collected from 59 female breast cancer patients who underwent surgery in 1988-1994, and
analyzed retrospectively under protocols approved by the institutional review board of the
Medical University of Vienna. Triplicate core biopsies of 0.6mm were taken from each donor
paraffin block and arrayed into a recipient block with a MTA1 manual tissue puncher/arrayer
(Beecher Instruments, Silver Spring, MD) as described28. 5 μm thick paraffin sections were
treated in xylene and rehydrated in a gradient of ethanol. After antigen retrieval by 10 mM
sodium citrate (pH6.0), endogenous peroxidase activity was blocked with 3% H2O2. The
sections were then incubated with 20g/ml of a goat polyclonal anti-RANK antibody (M-20;
Santa Cruz Biotechnology, Santa Cruz, CA). Goat and rabbit IgG antibody were used as a
negative control. After one hour incubation at RT and washing, the sections were incubated with
biotinylated anti-goat/rabbit IgG antibodies for 30 minutes, followed by incubation with
streptavidin–peroxidase for 15 minutes and 3,3’-diaminobenzidine. Sections were counterstained
with hematoxylin. Immunostaining was scored on triplicate tissues by two independent observers
(T.N. and R.S.) using the following arbitrary scale: 0, no staining; 1, weak staining; 2, medium
staining; 3, strong staining. It should be noted that all of the cancer tissues showed staining in
greater than 50% of the total tumor area.
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Osteoclast chemotaxis
Osteoclasts were plated on 35-mm culture dishes or 12-mm glass coverslips in medium 199 (25
mM HEPES, 26 mM HCO3-, supplemented with 15% FBS) and incubated for 1 hour (37°C, 5%
CO2). Dishes and coverslips were washed to remove non-adherent cells and osteoclasts were
cultured as described. Because mature osteoclasts are not amenable to Boyden chamber assays,
chemotaxis was assessed in a single-cell chemotaxis assay23 using an inverted phase contrast
microscope coupled to a time-lapse video recorder (AG-6720, 1 frame/2 sec; Panasonic,
Secaucus, NJ, USA). A glass micropipette containing soluble rRANKL (1 μg/ml), rRANKL
premixed with OPG (1 g/ml and 2.5 g/ml) or vehicle in supplemented media was positioned
200-400 µm from the cell under study. The contents of the pipette flowed passively into the bath
and migration was monitored for 6-12 h. Images of osteoclasts were digitized and centroids
determined using Optimas 3.1 (Bioscan, Inc., Edmonds, WA, USA). Polar data were analyzed
using Rayleigh's and modified Rayleigh's tests.
In vivo tumor metastasis
Murine B16F10 melanoma cells and human colon cancer cells that do not express RANK were
injected into the left cardiac ventricle of 7-10 wk old female C57BL/6 mice (Harlan Sprague
Dawley, Houston, TX) or immunodeficient nude mice, respectively, under avertin anesthesia as
described9. Simultaneously, mice were daily treated with either vehicle (PBS), 20 g/day rOPG
or zolendronic acid (s.c. g/mouse per day) as previously described18. After final treatment,
mice were sacrificed and bones (femur, tibia, humerus, and lumbar vertebrae) and organs (brain,
ovary, spleen, kidney and adrenal glands) collected for histological analysis. Serum was
collected for calcium (SeCa), phosphorous (SeP), alkaline phosphatase (ALP) and tartrate-
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resistant acid phosphatase (TRAP) analysis. Bones were fixed in either 70% ethanol or were
processed through 10% formalin before being rinsed thoroughly and decalcified in formic acid
solution or decalcified using 10% EDTA in PBS (pH 7.5) for TRAP staining. Radiographic and
histomorphometric analysis of all bones was as previously described7,21. Briefly, tissues were
fixed in 10% formalin, sectioned and stained with hematoxylin and eosin (H&E) to determine
the presence or absence of tumor metastases. Midline longitudinal sections of long bones were
stained for TRAP activity. Two non-serial sections of each bone were assessed. The total tissue
section area and the tissue area occupied by tumor cells were measured using an Osteomeasure
bone analysis program (Osteometrics Inc., Decatur, GA). All mice were kept at the Ontario
Cancer Institute Animal Care Facility according to institutional guidelines.
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Supplementary references
23. Pilkington, M.F., Sims, S.M.& Dixon, S.J. Transforming growth factor-β induces osteoclast
ruffling and chemotaxis: potential role in osteoclast recruitment. J. Bone Miner. Res. 16,12371247 (2001).
24. Medina, D. & Kittrell, F. in Methods in Mammary Gland Biology and Breast Cancer
Research (Kluwer Academic/Plenum Publishers, New York, 2000).
25. Curnock, A.P., Logan, M.K. & Ward, S.G. Chemokine signaling: pivoting around multiple
phosphoinositide 3-kinases. Immunology 105(2), 125-136 (2002).
26. Wells, A., Ware, M.F., Allen, F.D. & Lauffenburger, D.A. Shaping up for shipping out:
PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration. Cell
Motil. Cytoskeleton 44(4), 227-233 (1999).
27. Rabinovitz, I., Toker, A. & Mercurio, A.M. Protein kinase C-dependant mobilization of the
64 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive
the chemotactic migration of carcinoma cells. J. Cell Biol. 146(5), 1147-1160 (1999).
28. Kononen, J., Bubendorf, L., Kallioniemi, A., Barlund, M., Schraml, P., Leighton, S.,
Torhorst, J., Mihatsch, M.J., Sauter, G. & Kallioniemi, O.P. (1998) Tissue microarrays for highthroughput molecular profiling of tumor specimens. Nat. Med, 4, 844-847 (1998).
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Supplementary Figures
Supplementary Figure 1. RANK expression on primary and metastatic breast
cancer cells and epithelial cancer cell lines
a) Human breast and lymph node tissue-array slides were staining for RANK expression (see
methods). Staining was assessed in normal breast tissue biopsies from a patient subset (n=10),
primary breast cancers (n=59), and breast cancer cells that have metastasized to the local
draining lymph nodes (n=30). Staining intensity was scored in arbitrary units: 0 (no staining), 1
(weak staining), 2 (medium staining), and 3 (strong staining). Statistical significance of increased
RANK expression as compared to normal mammary gland epithelial cells was determined using
an ANOVA-Tukey test. ns = none significant; * p<0.001. b) Expression of RANK in (1)
Colo205, (2) SW480, (3) LNCaP, (4) Du145, (5) MDA-MB-231, (6) Hs578T, (7) MCF-7 cancer
cell lines, and primary human breast tumor samples (8-11). Expression was detected by RT-PCR.
Supplementary Figure 2. Expression of functional RANK on epithelial cancer
cells
a) Expression of RANK on T47D breast cancer cells (solid line). Background is shown as a
dotted line. RANK expression was detected using RANKL-FITC. b,c) RANKL induces
ERK1/ERK2 phosphorylation on MDA-MB-231 (b) and PKB/Akt and ERK1/ERK2, but not
STAT5 activation, in T47D (c) human breast cancer cells. Serum-starved cells were stimulated
with rRANKL [1g/ml] or prolactin [5g/ml] for the indicated time periods. PKB/AKT
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activation (Ser473 phosphorylation; p-AKT), ERK1/ERK2 activation
(Thr202/Tyr204
phosphorylation; p-ERK), and STAT5A/B (Y694 phosphorylation; p-STAT5) were detected by
Western blotting. Total PKB/AKT, STAT5A/B, and control -actin protein levels are shown.
Similar to MDA-MB-231 cells, the human epithelial breast tumor cell line T47D expresses high
levels of the prolactin-receptor and RANK but does not express detectable levels of RANKL as
determined by RT-PCR and protein analyses. Prolactin-induced activation of downstream
signaling pathways is shown for specificity. One experiment representative of 5 different
experiments using different stimulation conditions is shown. d,e) RANKL has no apparent role
in proliferation (d) or cell death (e) of human MDA-MB-231 breast cancer cells. Cells were
serum starved and proliferation measured on day 1 and day 3 following rRANKL [2.5 g/ml]
addition using H3-thymidine uptake. For cell death, RANKL-activated [2.5 g/ml] MDA-MB231 breast cancer cells were left untreated (control) or treated with anisomycin [50 M], sorbitol
[0.4M], or UV irradiation [240mJ/cm2]. % survival was detected by FACS using propidium
iodide/Annexin5.
Supplementary Figure 3. RANKL triggers chemotaxis of MDA-MB-231
breast cancer cells
Migration of MDA-MB-231 human breast cancer cells in response to different doses of
rRANKL, SDF-1, or both rRANKL plus SDF-1. Percentage migration (mean values of
triplicate cultures +/- SD) compared to non-stimulated control cells is shown.
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Supplementary Figure 4. RANKL triggers chemotaxis of mature osteoclasts
a-c) Migration of osteoclasts monitored by time-lapse microscopy. a) Videomicrographs
illustrate fields containing 3 different osteoclasts (lower left of each frame) and micropipette tips
outlined in black (upper right) at time 0 (time of introduction of micropipette containing vehicle
(Control) or RANKL (1 μg/ml)). White outlines represent positions of the osteoclasts at the
times indicated. Video files are available as supplementary material. b) Polar plots in which
origin represents the position of the centroids of all cells at time 0 and axes are oriented to set the
tip of the micropipette on the positive x-axis (90°). The positions of the centroid of each cell at 6
h are illustrated by closed symbols for vehicle (left panel, 7 cells) and open symbols for
rRANKL (right panel, 7 cells). Arrow illustrates the mean vector. The migration of control
osteoclasts was not significantly oriented. In contrast, the mean direction of migration induced
by RANKL was significantly oriented towards the tip of the micropipette (p<0.004). c) Bars
illustrate mean distances traveled by osteoclasts from 0 to 6 h in the direction of the micropipette
tip (determined as the projection on the x-axis). Osteoclasts migrated significantly further
towards the RANKL-containing micropipette (RANKL) than towards the micropipette
containing vehicle (control) as assessed by Student's t-test (* p<0.03). OPG blocked RANKLinduced chemotaxis of isolated osteoclasts (RANKL + OPG). Data are representative means 
SEM for 8 osteoclasts for vehicle, 16 osteoclasts for RANKL and 6 osteoclasts for
RANKL+OPG from 5 different cell preparations for each condition.
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Supplementary Figure 5. RANKL induces chemotaxis of B16F10 melanoma
cells
a) Expression of RANK and -actin mRNA in (1) untreated (2) RANKL [2.5 g/ml] treated
B16F10 melanoma cells. Expression was detected using RT-PCR. b) Additive effects of
chemokines and rRANKL in tumor cell migration. Migration of B16F10 melanoma cells in
response to rRANKL (2.5 g/ml) and the chemokines 6Ckine (120 ng/ml), SDF-1 (80 ng/ml),
and CTACK (100 ng/ml). Percentage migration (mean values of triplicate cultures +/- SD)
compared to non-stimulated control cells is shown. Data are representative of 3 different
experiments. Treatment of cells with 6Ckine plus RANKL or CTACK plus RANKL
significantly increased migration above treatment with chemokine alone (* p<0.05). c) Migration
of B16F10 melanoma cells in response to RANKL (2.5 g/ml) or SDF-1 [80 ng/ml] in the
absence or presence of rOPG [10 g/ml] or the CXCR4 blocking Ab [25 g/ml, clone 4471].
Percentage migration (mean values of triplicate cultures +/- SD) compared to non-stimulated
control cells is shown. Note that OPG does not block chemokine-induced cell migration and that
anti-CXCR4 does not block RANKL-induced migration. RANKL-induced migration of cells is
blocked with rOPG (* p<0.05) and SDF-1-induced migration of cells is inhibited by antiCXCR4 (* p<0.01). Results are representative of 3 different experiments. d) Stimulation of
B16F10 melanoma cells with RANKL (2.5 g/mL) or SDF-1 (1g/ml) triggers
phosphorylation of ERK1/2 (Thr202/Tyr204 phosphorylation; p-ERK) and PKB/AKT (Ser473
phosphorylation; p-AKT). Total ERK1/2 and PKB/AKT expression are shown as controls.
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Supplementary Figure 6. B16F10 melanoma cells do not activate osteoclasts
a) In line with previous results16, B16F10 melanoma cells (asterix) that have metastasized into
the long bones and vertebrae are not associated with osteoclasts. Bone sections were stained with
TRAP to detect osteoclasts in situ (arrows). b,c) B16F10 melanoma tumors did not decrease
bone density as determined by radiography (a typical femur radiogram is shown) (b) nor changed
total, trabecular, and cortical bone densities as determined by pQCT measurements (c). There
was also no increase in TRAP activity, and no evidence of altered serum calcium, phosphorous
or alkaline phosphatase activity in mice that carried confirmed B16F10 tumor metastases in the
bones as compared to non-injected controls, confirming that B16F10 melanoma tumors are nonosteoclastogenic.
Supplementary Figure 7. Inhibition of RANKL/RANK signaling results in
selective abrogation of tumor metastasis into bones, but not ovaries or adrenal
glands
a-f) Histology of vertebrae (a-c), ovaries (d-f), and adrenal glands (g-i) in control mice (a,d,g;
control), after injection of B16F10 melanoma cells (b,e,h; tumor), and after injection of B16F10
melanoma cells into female mice treated with OPG (c,f,i; tumor + OPG). Typical examples of
metastases are shown for each treatment. Arrows indicate B16F10 tumor cell foci. * indicate
infiltration of tumor cells into the spinal cord that ultimately results in bleeding and paralysis.
Arrowheads = spinal cords. Note the complete destruction of normal tissue in ovaries.