JOURNAL OF BONE AND MINERAL RESEARCH

Volume 20, Number 2, 2005

Published online on November 16, 2004; doi: 10.1359/JBMR.041109
© 2005 American Society for Bone and Mineral Research

Skeletal Localization and Neutralization of the SDF-1(CXCL12)/

CXCR4 Axis Blocks Prostate Cancer Metastasis and Growth in
Osseous Sites In Vivo
Yan-Xi Sun,1 Abraham Schneider,1 Younghun Jung,1 Jianhua Wang,1 Jinlu Dai,2 Jingcheng Wang,1 Kevin Cook,3
Nadir I Osman,4 Amy J Koh-Paige,1 Hyusuk Shim,5,6 Kenneth J Pienta,7 Evan T Keller,2 Laurie K McCauley,1,8 and
Russell S Taichman1

ABSTRACT: To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive
correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited
the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide
critical support that SDF-1/CXCR4 plays a role in skeletal metastasis.
Introduction: Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is
activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we
evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or
intraosseous growth of CaPs.
Materials and Methods: SDF-1 levels were established in various mouse tissues by ELISA, immunohisto-
chemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were
established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence
of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined
using intratibial injections and anti-CXCR4 antibodies and peptides.
Results: There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP
lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys com-
pared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or
undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis
of the long bones nearest to the growth plate where intense expression was observed near the endosteal
surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic
load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neu-
tralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors
compared with controls.
Conclusions: These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Impor-
tantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate
cancer.
J Bone Miner Res 2005;20:318–329. Published online on November 16, 2004; doi: 10.1359/JBMR.041109

Key words: metastasis, chemokine, marrow, prostate cancer, bone

INTRODUCTION
P ROSTATE CANCER (CaP) frequently metastasizes to the
bone marrow, resulting in significant morbidity and
mortality. Nearly 90% of patients with advanced prostate
The authors have no conflict of interest.
cancer are at high risk for pathologic fractures, spinal cord
compression, and pain due in part to dysregulated cycles of
osteoblastic and osteolytic resorption/formation driven by
the growing tumor mass.(1) What defines the molecular and
cellular predilection for prostate cancers to metastasize to
bone is not well known. Clearly, multiple factors, including
the acquisition of metastatic abilities by the cancer cell,

1
Department of Periodontics, Prevention, Geriatrics, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA; 2De-
partment of Urology, University of Michigan Medical School, Ann Arbor, Michigan, USA; 3University of Michigan Undergraduate
Research Opportunity Program, Ann Arbor, Michigan, USA; 4Department of Anesthesiology, University of Michigan, Ann Arbor,
Michigan, USA; 5Department of Hematology/Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA; 6Depart-
ment of Radiology, School of Medicine, Emory University, Atlanta, Georgia, USA; 7Division of Internal Medicine and Urology,
University of Michigan School of Medicine, Ann Arbor, Michigan, USA; 8Department of Pathology, University of Michigan School of
Medicine, Ann Arbor, Michigan, USA.

318
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER
chemotactic responses to bone-derived factors, preferential
adhesion to bone marrow endothelium, and the interaction
of CaP cells with the bone microenvironment leading to
tumor in the bone marrow, play considerable roles.(2)
Chemokines are a group of molecules known to play
significant roles as activators and chemoattractants for
many blood cell types as well as an increasing array of
non-hematopoietic cell types (e.g., epithelial cells, fibro-
blasts, and endothelial cells). Chemokines are 8- to 10-kDa
proteins subdivided into four structural groups on the basis
of the relative position of the first two cysteine residues in
the mature protein (CXC, ML, C3XC, and C). At present,
>50 chemokines have been identified, most of which are
thought to activate cell migration by binding to G-protein–
coupled cell surface receptors. Currently, there are at least
18 known chemokine receptors.(3) Activation of chemokine
receptors triggers activation of many downstream pathways
including nonreceptor tyrosine kinases, inositol trisphos-
phate, MAPK, protein kinase C, and calcium mobilization.
Previously, we determined that the stromal-derived
factor-1 (SDF-1)/CXCR4 chemokine axis is activated in
CaP metastasis to bone.(4,5) Specifically, CXCR4 expres-
sion is related to increasing tumor grade.(5) Moreover, we
have shown that SDF-1 signaling through CXCR4 triggers
the adhesion of CaPs to bone marrow endothelial cells.
Similar demonstrations have also been made suggesting
that the SDF-1/CXCR4 axis may play parallel roles in other
tumors that also metastasize to the marrow.(6,7) For ex-
ample, Muller et al.(7) reported that CXCR4 and SDF-1 are
central players in regulating metastasis by showing that nor-
mal breast tissues express little CXCR4, whereas breast
neoplasms express high levels of CXCR4. Furthermore, an-
tibody to CXCR4 blocks the metastatic spread of the tu-
mors to the lung and lymph nodes. Similarly, mRNA levels
for CXCR4 are elevated in regions of angiogenesis and
degeneration but decreased in areas of rapid cell prolifera-
tion in glioblastoma multiforme that may prime the cells for
metastasis.(8) Results consistent with these have also been
reported for human melanoma cell lines, melanoma cells
that had macroscopically infiltrated draining lymph
nodes,(9) and for pancreatic, neuroblastoma, and renal
carcinomas.(10,11)
Common to most of these reports is the analogy with
hematopoietic stem cell homing where gene knockout ap-
proaches in mice have shown that SDF-1 and CXCR4 play
an important role in embryologic development.(12–14) Spe-
cifically, SDF-1–deficient mice die in utero with severe car-
diac septum defects and poorly developed marrow. CXCR4
receptor deletion is similarly lethal, resulting from circula-
tory, CNS, immune, and hematopoietic defects.(13,15) The
phenotype of the SDF-1−/− mouse so closely followed the
CXCR4−/− that these investigations showed that the recep-
tor-ligand pair play important roles in cardiac, CNS, and
hematopoietic stem cell homing.(16) In addition, CXCR4
expression levels correlate with the ability of human pro-
genitors to engraft into the marrow in nude mice, and an-
tibody to CXCR4 prevented the engraftment of progenitors
in the bone marrow.(14) Finally, osteoblasts and marrow
endothelial cells express SDF-1, which may thereby localize
hematopoietic progenitor cells in the marrow.(14,17,18)
319
In this report, the goal was to delineate the role of SDF-
1/CXCR4 receptor in CaP disease. Two independent stud-
ies were performed. First, to establish a positive correlation
between the SDF-1 gene and protein expression with tumor
metastasis, SDF-1 levels were characterized for a variety of
murine tissues by ELISA and in situ hybridization. In an in
vivo metastasis model, the role of CXCR4 in metastasis of
prostate cancer to the bone was examined. In this model,
neutralizing antibody to CXCR4 limited the extent of bone
metastases. Finally, antibody and blocking peptide to
CXCR4 limited the growth of intraosseous CaPs after CaP
cell intratibial injections. Together, these in vivo metastasis
data provide critical support that SDF-1/CXCR4 plays a
role in skeletal metastasis. Most importantly, these novel
data show that SDF-1/CXCR4 participates in localizing tu-
mors to the bone marrow in CaP and may help in design of
therapeutic treatments to prevent the spread and growth of
metastasis.
MATERIALS AND METHODS
CaP cell line
PC3 prostate cancer cells originally isolated from a ver-
tebral metastasis of a human prostate cancer patient were
obtained from American Type Culture Collection (Rock-
ville, MD, USA) and were maintained in RPMI containing
10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.
PC3 cells infected with the pLazarus retroviral construct
expressing luciferase were selected for stable transfectants
in G418.(19,20)
SDF-1 ELISA
Bone marrow and tissue extracts were derived from
C57BL6 mice in PBS with protease inhibitors (Sigma). In
some cases, the femur and tibia were dissected free of soft
tissues, and 2-mm sections of the bones were generated on
ice using a 0.5-mm diamond mandrill set at low speed in a
dental drill. Harvested tissues were subjected to sonication
and spun, and the resulting supernatants were stored at
−80°C until assayed for cytokine SDF-1 levels by double-
antibody sandwich method assembled with commercially
available components according to the directions of the
manufacturer (sensitivity, 31.25 pg/ml; range, 62.5–5000 pg/
ml; R&D Systems). SDF-1 levels are presented as mean ±
SE picograms per milliliter for duplicate determinations.
Conditioned media from human osteoblasts (HOBs) and
the human osteosarcoma cell line MG-63 (ATCC
CRL1424) were used as positive controls for SDF-1.
SDF-1 bioactivity in tissues
Bone and liver tissue extracts were generated from 20-
day-old C57BL6 mice in RPMI. In vitro invasion assays
were performed using a reconstituted extracellular matrix
membrane (BD BioCoat Matrigel Invasion Chamber; BD
Biosciences, San Jose, CA, USA) using PC3 cells after de-
termining the levels of SDF-1 in the extract by ELISA. For
these investigations, test cells were placed in the upper
chamber (1 × 105 cells/well) in RPMI medium with 1%
serum and 0–200 ng/ml SDF-1 or 50% tissue extract: RPMI
(vol/vol) was added to the lower chamber. Spontaneous
320
invasion was compared with invasion supported by a SDF-1
gradient. Invasion into the matrix was assayed after 24 h by
quantification with MTT. The effect of 30 ␮g/ml CXCR4
blocking antibody (MAB173; R&D Systems), IgG control,
or peptides designed to block SDF-1 binding to the CXCR4
receptor (TC14012 or TN14003(21,22)) synthesized by the
Emory Microchemical Facility served as positive controls.
They were added to the top chamber of the Transwell to
provide additional proof that observed responses are de-
pendent on CXCR4 receptor binding.
Histological evaluation
Femurs and tibia were trimmed of musculature, fixed in
10% formalin at 4°C, decalcified in 10% EDTA (pH
7.4) for 10 days, and embedded in paraffin. Longitudinal
sections of tibias were cut and stained with H&E for histo-
logical evaluation. Where indicated, histomorphometric
analyses of the femurs/tibias were performed using a
computer-assisted bone histomorphometric analyzing sys-
tem (Image-Pro Plus version 4.0; Media Cybernetics, Silver
Spring, MD, USA).
Digoxigenin in situ hybridization
and immunohistochemistry
Normal murine bones were harvested and fixed in 10%
buffered formalin and decalcified in EDTA, and 2- to 3-␮M
paraffin embedded slides were prepared. Human bone was
obtained from patients undergoing orthopedic surgery in
accordance with the University of Michigan’s Investiga-
tional Review Board. Each slide was dewaxed and rehy-
drated and subsequently treated with proteinase K, 10%
formalin, and washed in SSC. Hybridization was performed
with a digoxin-labeled SDF-1 sense or antisense SDF-1
riboprobe at 55°C overnight. After incubation, the slides
were washed in 4× SSC, RNase A, 2× SSC-10 mM DTT, 1×
SSC-10 mM DTT, and 0.5× SSC-10 mM DTT.(23) The slides
were washed, blocked in 2% blocking buffer for 1 h, 1:500
antibody (anti-digoxigenin), and color developed using
nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-
chloro-3⬘-indolyphosphate p-toluidine salt (BCIP). In some
cases, antibody to SDF-1 (MAb Clone 79018; R&D Sys-
tems) or IgG control (Sigma) were used in conjunction with
a DAB Chromogen kit (LSAB + Peroxidase Kit; Dako,
Carpinterina, CA, USA) to identify the protein in tissues.
SDF-1 binding assay
To show the validity of the antibody and peptide re-
agents, PC3 cells were plated in 96-well plates at an initial
density of 2 × 104 cells/well. After 24 h, antibody to SDF-1
(polyclonal and monoclonal), unlabeled SDF-1 (0–100 ␮g),
anti-CXCR4 (clone 12G4 or MAB 171 [clone 44708.111]),
and anti-CXCR4 peptide or IgG control (0–100 ␮g/ml)
were added to select wells (all reagents from R&D Sys-
tems). Similarly, peptides designed to block SDF-1 binding
to the CXCR4 receptor (TC14012 or TN14003(21,22)) were
synthesized by the Emory Microchemical Facility and
added to several wells. Biotinylated SDF-1 was added to
the wells, and binding was allowed to proceed for 1 h at 4°C.
Thereafter, the cells were washed, streptavidin horseradish
peroxidase (HRP) was added to wells for 0.5 h at 4°C, and
SUN ET AL.
O-phenylenediamine dihydrochloride (OPD; Sigma) in ci-
trate buffer was added for development.
In vivo metastasis assay
For these studies, 2 × 105 PC3luc cells that were stably
transfected with luciferase before the experiment were
used. Two experimental groups were established; sodium
azide-free monoclonal antibodies, anti-CXCR4 (clones
MAB171 from R&D and 12G5 from BD PharMingen, San
Diego, CA, USA) and polyclonal anti-SDF-1 (R&D), were
used to neutralize the CXCR4-CXCL12/SDF-1 circuitry.
Appropriate irrelevant antibodies (sodium azide-free
44708.111 clone [MBA171] monoclonal antibody [IgG2a];
R&D Systems) or IgG matched controls were used. Before
inoculation, the tumor cells were preincubated with anti-
body to CXCR4 or control at 5 ␮g per 1 × 104 injected cells.
Our choice of reagents and dosing was based on the re-
ported use in blocking human hematopoietic CD34+ cell
engraftment into the bone marrows of NOD/SCID ani-
mals,(14) the ability to block invasion of LNCaP, C4-2B cells
and PC3 invasion in vitro,(4) and the ability to block SDF-1
binding to PC3 cells. Moreover, these reagents were used
by Muller et al.(7) to block metastasis of breast cancer cell
lines mediated by SDF-1 in vivo. Inbred HSD:athymic male
mice were purchased from Harlan Bioscience (Indianapo-
lis, IN, USA) and were housed under constant humidity
and temperature, with 12-h light and 12-h dark cycles. The
animals were anesthetized with a mixture of xylazine (100–
200 mg/kg) and ketamine HCL (5–16 mg/kg; Ketaset; Fort
Dodge Laboratories, Fort Dodge, IA, USA), and the left
cardiac ventricle was punctured percutaneously and in-
jected using a 25-gauge needle attached to a 1-ml syringe as
previously described.(20) The animals were subsequently in-
jected 2 and 24 h afterward with equal doses of the same
antibody.
After 4 weeks, bioluminescence imaging was used to fol-
low prostate-derived bone metastases as a primary out-
come. The mice were injected intraperitoneally with lu-
ciferin (100 ␮l at 40 mg/ml in PBS) before imaging. This
dose and route of administration have been shown to be
optimal for rodent studies with a 10- to 20-minute after
luciferin injection.(24) Mice were anesthetized with 1.5%
isoflurane/air, and the Xenogen IVIS cryogenically cooled
imaging system was used as described.(20) Selected mice
were imaged weekly after tumor injection to monitor tumor
development. Bioluminescence generated by the luciferin/
luciferase reaction served as a locator for cancer growth and
was used for quantification using the LivingImage software
on a red (high intensity/cell number) to blue (low intensity/
cell number) visual scale. A digital grayscale animal image
was acquired followed by acquisition and overlay of a pseu-
docolor image representing the spatial distribution of de-
tected photon counts emerging from active luciferase within
the animal. Signal intensity was quantified as the sum of all
detected photons within the region of interest during a
1-minute luminescent integration time.
Intratibial injections
PC3Luc cells were inoculated intratibially to measure the
effect of blocking CXCR4 on tumor growth.(25) Intratibial
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER
injection was performed as previously described.(25) Briefly,
animals were anesthetized, and both legs were cleaned with
betadine and 70% ethanol. PC3luc cells were injected
through the cortex of the anterior tuberosity of the tibia
with a drill-like motion to prevent cortical fracture using a
25-␮l syringe fitted with a 25-gauge needle. Ten microliters
of cell suspension was injected (107 cells/ml) to the tibia.
Growth of the intraosseous tumor was examined using the
a CXCR4 antagonist synthesized by the Microchemical
Core Facility at Emory University. A control peptide was
produced by randomly scrambling the amino acid sequence
of CXCR4 antagonist while maintaining the disulfide bond
to maintain the U-type structure of the antagonist (NH2-
KY-Nal-YR-DK-Cit-RCRRP-Cit-C-amide).(26)
Calcium and pyridinoline cross-links levels in serum
Serum calcium and pyridinoline (PYD) levels were used
as measures of bone turnover. Blood samples were ob-
tained from the experimental mice after death, serum was
separated by centrifugation, and total calcium was deter-
mined by colorimetric assay with the cresolphthalein com-
plexone method (Sigma). Serum PYD levels were assayed
using an EIA kit (Quidel Corp., San Diego, CA, USA)
after filtration by adding ∼200 ␮L of each serum to a 30-k
molecular weight cut-off (MWCO) Spinfilter and centrifu-
gation at 10,000g for 30 minutes (Quidel Corp.).
Statistical analysis
Numerical data are expressed as means ± SD. Statistical
differences between the means for the different groups
were evaluated with Instat 4.0 (GraphPAD software) using
one-way ANOVA, with the level of significance at p < 0.05.
RESULTS
SDF-1 location
There is now substantial evidence to suggest that chemo-
kines and their receptors are involved in the pathogenesis
of many diseases, ranging from infectious to autoimmune
disorders. Recently, chemokine receptors have been impli-
cated in the homing of tumors to the marrow microenvi-
ronment, and our own data suggest that SDF-1 and CXCR4
may play a critical role in these phenomena based on the
expression of CXCR4 by CaPs and SDF-1 secretion by hu-
man osteoblasts in vitro. To further characterize the role
that SDF-1 may play in CaP metastasis, a variety of murine
tissues were examined for the level of expression of SDF-1
protein by ELISA. As shown in Fig. 1, high SDF-1 levels
were identified in tissues to which CaPs frequently metas-
tasize to including the femur, tibia, and pelvis. Surprisingly
SDF-1 levels were also elevated in the optic nerve region
and in cardiac muscle. In contrast, tissues that are rarely
sites of CaP metastasis in humans had lower levels of SDF-1
(eye, tongue, maxilla, mandible). To localize the sites of
SDF-1 production in bone, bone marrow extracts derived
from the tibia were examined by ELISA after sectioning
the bones into 2- to 3-mm lengths. The highest levels of
321
FIG. 1. Tissue distribution of SDF-1. Bone and other tissues
were collected on ice from C57BL6 mice in cold PBS with prote-
ase inhibitors. Harvested tissues were subjected to sonication,
spun, and SDF-1 levels were determined by ELISA. Data are
presented as nanograms per milligrams SDF-1 of total tissue pro-
tein. Optic N., optic nerve; Kid/Ad, kidney/adrenal gland.
SDF-1 in the medium fraction of the marrow were localized
to the distal and proximal extremities of the long bones
(data not shown).
Further studies were performed to localize the sites of
SDF-1 production in the marrow. SDF-1 mRNA expression
as determined in situ hybridization was predominantly lo-
calized to the endosteal surfaces, particularly near the me-
taphysis in both the human and murine bone samples ex-
amined. SDF-1 staining was generally low or undetectable
in the center of the marrow and in the diaphysis. Impor-
tantly, SDF-1 mRNA was localized to the metaphysis of the
long bones examined nearest to the growth plate. At higher
magnification, intense SDF-1 expression was frequently ob-
served near the endosteal surfaces covered by osteoblastic
and lining cells (Figs. 2A–2F). Immunohistochemistry for
SDF-1 protein further verified that SDF-1 expression was
generally low or undetectable in the diaphysis regions of
long bones (Figs. 3A–3F). Peak SDF-1 protein expression
was observed at the growth plates (Figs. 3E and 3F).
SDF-1 bioactivity in tissues
The preceding ELISA, in situ hybridization, and immu-
nohistochemistry results show that SDF-1 is expressed in
tissues that are frequent sites of metastasis. To determine if
SDF-1 is biologically active, bone and liver tissue extracts
were generated from 20-day-old C57BL6 mice. In vitro in-
vasion assays were performed using the PC3 prostate can-
cer cell line to evaluate bioactivity. In the presence of SDF-
1, significantly more PC3 cells invaded into the invasion
chambers (Fig. 4). Function blocking antibody and specific
peptides that block SDF-1 binding to CXCR4 significantly
reduced invasion of the PC3 cells mediated by tissue ex-
tracts (Fig. 4). As SDF-1 is the only known ligand for
CXCR4, these data show that SDF-1 in tissue extracts is
biologically active.
322
Inhibition of SDF-1 binding by antibodies
to CXCR4
Previous reports suggest that metastasis is frequently
found at sites of high bone turnover, including the region
near the growth plate. Our findings that SDF-1 levels were
elevated in the growth plate region and that it is bioactive
suggest that sites of elevated SDF-1 production may be
particularly prone to CaP metastasis. To test this hypoth-
esis, agents that blocked the binding of SDF-1 to CXCR4
on CaP cells are desired. To validate the employment of
several commercial reagents for use in our investigations,
we first evaluated whether anti-CXCR4 or anti-SDF-1 an-
tibodies would block the binding of biotinylated SDF-1 to
PC3 prostate cancer cells. For these investigations PC3 cul-
tures were established in 96-well plates, where unlabeled
SDF-1 (0–100 ␮g), antibody to SDF-1 (polyclonal and
monoclonal), anti-CXCR4 antibodies (clone 12G4 or clone
44708.111), antibody IgG controls (0–100 ␮g/ml), or an
anti-CXCR4 peptide designed to block SDF-1 (TC14012)
binding to the CXCR4 receptor were added to the wells.
Subsequently biotinylated SDF-1 was added to the wells,
and binding was allowed to proceed for 1 h at 4°C. There-
after, the cells were washed, and streptavidin HRP and
OPD were added for detection.
The data showed that either unlabeled SDF-1, which
competes for binding of the CXCR4 receptor with biotinyl-
SUN ET AL.
FIG. 2. Expression of SDF-1 mRNA and
protein in murine and human bone. Mouse
and human bone were fixed in 10% formalin
at 4°C and decalcified in 10% EDTA (pH
7.4) for paraffin embedding. Localization of
SDF-1 mRNA was performed by digoxin in
situ hybridization and counterstained with
H&E (arrows). (A) Antisense and (B) sense
murine riboprobe detection of SDF-1 in the
diaphysis and epiphysis/diaphysis junction of
murine femur (2× and 10×). Antisense ribo-
probe detection of SDF-1 mRNA in epiph-
yseal region of the mouse femur (C) 10× and
(D) 20× views. Antisense riboprobe detec-
tion of SDF-1 mRNA in epiphyseal region of
the (E) mouse femur 40× and (F) human
femur 40× views. The data show that osteo-
blastic expression of SDF-1 mRNA is great-
est in the epiphyseal regions of mouse and
human bone.
ated SDF-1, antibody to CXCR4, which binds to the ligand-
binding domain of CXCR4, neutralizing polyclonal or
monoclonal antibody to SDF-1, or the peptide TC14012
reduced binding of labeled SDF-1 to PC3 cells (Fig. 5).
These data confirmed that these reagents were appropriate
for further investigation.
In vivo metastasis investigations
To test our hypothesis that the SDF-1/CXCR4 pathway is
critical for the development of bone metastases, in vivo we
established two experimental groups (Fig. 6A). In each
case, PC3 cells tagged with luciferase were preincubated
with antibody to CXCR4 or an isotype-matched nonspecific
antibody control group on ice immediately before intracar-
diac injection. The antibody that was chosen to block
CXCR4 (MBA171 monoclonal antibody; R&D Systems)
was used at 5 ␮g/1 × 104 injected cells. The animals were
subsequently injected intraperitoneally twice at 2 and 24 h.
After 4 weeks, bioluminescence imaging to follow the CaP
cell-derived metastases was used as our primary out-
come.(24) As shown in Fig. 6A, we observed that all of the
animals that received the IgG control antibody established
osseous metastases. Antibody to CXCR4 significantly re-
duced the total metastatic load of the animals compared
with IgG control, regardless of whether the animal was im-
aged from the dorsal or ventral surface (Fig. 6C). Exami-
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER
nation of individual sites of bone metastasis also showed
that administration of antibody to CXCR4 significantly re-
duced the total luminescent signal (i.e., total tumor bur-
den), further showing that the SDF-1/CXCR4 chemokine
axis in part regulates osseous metastasis, including femur/
tibia regions, spine, and maxilla/mandible areas (Fig. 6D).
Radiographic analysis confirmed these findings, suggesting
that the antibody to CXCR4 had a significant effect on
reducing osseous metastases.
Because antibody to CXCR4 decreased the metastatic
load, we tested if this also affected tumor-induced bone
resorption. For these evaluations, both serum calcium and
serum pyridinoline cross-links levels were evaluated as
markers of bone turnover. There was no difference in total
serum calcium concentration in the experimental group in-
jected with the IgG antibody compared with those animals
receiving the CXCR4 antibody (Fig. 7A). The PYD levels
were lower in the CXCR4 antibody group compared with
those of the control group, indicating that there was an
overall decrease in osteoclastic activity in the CXCR4 ex-
perimental groups (Fig. 7A). In animals with bone lesions,
however, there were no statistical differences in the tumor
size/bone area in anti-CXCR4 antibody treated versus IgG
control-treated animals (41.8 ± 9.3 versus 45.5 ± 5.6 mm), in
number of osteoclasts/mm of tumor adjacent to the trabec-
ular or cortical bone surfaces (1.8 ± 1.0 versus 1.0 ± 0.8
mm), or for the percentage of the growth plate that was
associated with tumor (38.7 ± 13.1% versus 57.69 ± 19.3%;
323
FIG. 3. Expression of SDF-1 protein in mu-
rine long bones. Mouse tibias were fixed in
10% formalin at 4°C and decalcified in 10%
EDTA (pH 7.4) for paraffin embedding. Lo-
calization of SDF-1 protein was performed
using a DAB chromogen kit and IgG control
antibody or monoclonal antibody to SDF-1
(Dako LSAB + Peroxidase Kit). (A and B)
Epiphysis (10×) and diaphysis (5×) stained
with control antibody. (C) Diaphysis (10×)
and (D) epiphysis (5×). (E and F) Growth
plate (40×) stained with anti-SDF-1 antibod-
ies. Detection of SDF-1 in the epiphyseal re-
gion of the (E) mouse femur 40× and (F)
human femur 40× views. The data show that
osteoblastic expression of SDF-1 is greatest
in the epiphyseal regions of mouse and hu-
man bone.
Figs. 7B and 7C). We interpret these data to suggest that,
whereas the tibia/femur markers were not significant on a
per animal basis with many metastatic lesions in the IgG
control group, the systemic PYD levels were elevated, re-
flecting the total metastatic burden. These data suggest that
if CaP cells localize to the skeleton, the early treatment to
block CXCR4 had no influence on tumor growth under
these conditions.
Growth of intraosseous tumors in vivo
The observation that antibody to CXCR4 blocked me-
tastasis induced by PC3 and our prior observation that an-
tibody to SDF-1 decreased PC3 proliferation in vitro(5)
prompted us to determine what effect blocking CXCR4
would have on the growth of prostate cancers in bone. Ac-
cordingly, PC3 cells were injected intratibially into nude
mice. One day later, anti-CXCR4 antibody, anti-CXCR4
peptide (TC14012), or control antibody was administered
daily for 4 weeks intraperitoneally. Skeletal lesions identi-
fied by bioluminescence imaging were used as our primary
outcome. Tumors were identified in all of the control
groups. The majority of the animals treated with either the
anti-CXCR4 antibody or TC14012 also had tumors; how-
ever, the tumors were significantly smaller (Fig. 8A). His-
tological analyses revealed that tumor infiltration in all the
control-treated animals was greater than the anti-CXCR4
antibody or TC14012-treated mice (Fig. 8B). The levels of
324
FIG. 4. SDF-1 is bioactive in bone and liver tissues. PC3s were
placed in the upper chamber (1 × 105 cells/well) of invasion cham-
bers in serum-free RPMI medium, and 0–200 ng/ml SDF-1 or 50%
tissue extract in RPMI (vol/vol) (BM, bone marrow or liver) was
added to the lower chamber. Invasion was determined at 24 h by
XTT staining. Antibody to CXCR4, IgG control, or peptides de-
signed to block SDF-1 binding to the CXCR4 receptor (TC14012)
was used to test the bioactivity of the tissue extract. *Significant
difference from control at p < 0.05. The data show that SDF-1 in
the extracted tissues is bioactive as determined by the ability of
specific antibody or peptide to block invasion of PC3 cells.
pyridinoline cross-links were lower in the CXCR4 antibody
and TC14012 peptide groups compared with those of the
control group, indicating that there was an overall decrease
in osteoclastic activity; however, serum calcium levels were
not altered (Fig. 8C). Radiographic analysis of tumor bur-
den of the two groups also showed smaller tumors in the
treatment groups versus control groups (data not shown).
Similarly, histologic analysis of osteoclast number, percent
tumor at the growth plate, and tumor area revealed a re-
duction in all parameters for the anti-CXCR4 antibody and
specific peptide-treated groups relative to the control-
treated animals (Fig. 8D). Taken together, these data show
that blockage of the SDF-1 receptor, CXCR4, inhibits the
development of PC3-derived tumors. Furthermore, the
data strongly suggest that the development of bone metas-
tases is dependent on SDF-1 for growth.
DISCUSSION
In this study, we examined the hypothesis that SDF-1/
CXCR4 plays a significant role in the skeletal metastasis of
CaP. In a first set of investigations, SDF-1 levels in several
murine tissues were examined. High levels of SDF-1 were
noted in many tissues to which CaP frequently metastasize.
In bone, SDF-1 expression was observed near the endosteal
surfaces covered by osteoblasts and lining cells in both hu-
man and murine tissues. Importantly, SDF-1 message was
localized to the metaphysis of the long bones nearest to the
SUN ET AL.
FIG. 5. Blockade of SDF-1 binding to PC3 cells in vitro. PC3
cells were plated for 24 h in 96-well plates. Where indicated, un-
labeled SDF-1 (0–100 ␮g), anti-SDF-1 antibody (polyclonal and
monoclonal), or an N-terminal SDF-1 peptide fragment designed
to block SDF-1 binding to the CXCR4 receptor (TC14012) served
as controls. In some cases, anti-CXCR4 monoclonal antibodies
(clones 12G4 or 44708.111 [MAB171]) or IgG control (0–100 ␮g/
ml) were employed. After the addition of the biotinylated SDF-1,
binding proceeded for 1 h at 4°C to minimize receptor turnover or
downregulation. Thereafter, the cells were washed, streptavidin
HRP was added to the wells for 0.5 h, and OPD in citrate buffer
was added for color development. *Significant difference from
control at p < 0.05. The data show anti-CXCR4 antibodies com-
pete with biotinylated SDF-1 for binding to the receptor.
growth plate. SDF-1 message and protein levels were gen-
erally low in the center of the marrow and in the diaphyseal
regions. These findings correlate with the sites of osseous
metastasis observed in our in vivo intracardiac model me-
tastasis model. Moreover, similar results have been pre-
sented by other investigators and other tumors using these
antibodies, suggesting their appropriateness for use in in-
vestigations of the SDF-1/CXCR4 axis in metastasis. Peled
et al.(14) showed that the engraftment of human hemato-
poietic CD34+ cells into the bone marrow of NOD/SCID
animals was could be blocked by CXCR4 neutralization.
Likewise, Muller et al.(7) showed that antibody to CXCR4
or SDF-1 blocked metastasis of breast cancer cell lines in
vivo, and neutralization of CXCR4 prevented homing of
human NHL cells and lung cancer cell lines to a number of
organs.(27,28) Further support for a participation of SDF-1/
CXCR4 in osseous metastasis was provided using antibody
to CXCR4 immediately before and at 2–24 h after tumor
inoculation. Overall, less tumor was observed and fewer
osseous lesions were identified by Xenogen imaging, radio-
graphic, and histologic examinations. These observations
were further substantiated in that pyridinoline cross-links in
serum were significantly lower in the anti-CXCR4 anti-
body–treated group relative to control IgG-treated groups.
It is therefore interesting to speculate why SDF-1 levels are
elevated in the cardiac muscle and the optic nerve area.
These are not frequently sites of CaP metastasis in humans;
however, CaP cell metastasis in the intracardiac model of-
ten are localized to these sites,(20,29) but neutralization of
CXCR4 decreased metastasis. Clearly, additional events
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER 325

FIG. 6. Blockade of CXCR4 prevents bone metastasis in vivo. Intracardiac injection of PC3Luc cells was performed in the presence
or absence of neutralizing antibody to CXCR4 or IgG control. Antibody treatments were repeated 2 and 24 h after PC3 injection.
Imaging was performed at 4 weeks. (A) Study outline. (B) Demonstration of the establishment of metastatic CaP cell tumors by
Xenogen IVIS system with radiographic and histologic confirmation of tumor in the left femur (R, right; L, left). (C) Dorsal and ventral
surface and (D) individual osseous site quantification of osseous tumor burden in the presence of antibody to CXCR4 or control.
*Significant difference from control at p < 0.05. The data show that blockage of CXCR4 resulted in less total tumor burden and fewer
metastatic bone lesions.

must participate in localizing metastatic tumors to bone in
addition to the SDF-1/CXCR4 axis in humans.
To evaluate what role SDF-1 has on intraosseous growth
of CaPs, intratibial injection of PC3 cells was performed.
Antibody or specific peptide against CXCR4 decreased the
growth of pre-established CaP cell lesions in bone. These
data correlated with our previous finding that showed that
human CaPs express elevated levels of SDF-1 mRNA rela-
tive to benign hyperplasia in situ and that CaP cell lines that
secrete biologically active SDF-1 protein regulate their ex-
pression of SDF-1 mRNA in response to exogenous SDF-
1.(5) Correlating with the in vivo data, neutralizing antibody
to SDF-1 decreased the proliferation metastatic tumor cells
that were isolated from bone, but not those isolated from
other tissues.(5) These findings, together with those from
our metastasis study, suggest that neutralization of the
SDF-1/CXCR4 axis may prevent osseous metastasis by
both inhibiting metastatic spread within the vasculature,
and once in bone, preventing tumor growth.
We recently reported that SDF-1 activates binding of
prostate cancer cells to marrow endothelial cells.(4) We de-
termined that part of the mechanism that facilitates CaP
cell binding to endothelium in response to SDF-1 may be
through activation of ␣v␤3 integrins already expressed on
CaP cancer cell lines (Y-X Sun and RS Taichman, unpub-
lished data, 2004). Clearly, binding to endothelium is a ma-
jor event regulating the establishment of metastasis of
blood-borne tumors. Recently, it has been reported that
SDF-1 is bound to bone marrow endothelial cells by endo-
thelial heparan sulfate and chondroitin/dermatan proteo-
glycans, where it would activate adhesion receptors on the
tumor cells to facilitate transendothelial migration.(30–32)
For hematopoietic cells, it is well established that efficient
rolling on endothelial cells requires P-selectin, E-selectin,
and the CD44 under physiologic flow conditions. On the
other hand, intercellular adhesion molecule-1 (ICAM-1)
alone does not seem to facilitate binding of hematopoietic
cells under flow conditions. In the presence of surface-
bound SDF-1, however, rolling on endothelial cells is rap-
idly developed into a firm adhesion, mediated by interac-
tions between ICAM-1 and SDF-1.(33) CXCL12/SDF-1
may also regulate the development of pseudopodia in
breast cancers that may lead to invasive metastasis.(7) These
findings are important in that blockade of CXCR4 signaling
may result in decreased metastases. Part of the mechanism
whereby CXCR4 inhibition may function is to limit extrav-
asation of circulating tumors by limiting binding or by yet
another unidentified mechanism. For example, Bertolini
326
FIG. 7. Reduction in bone resorption markers in serum after
blockade of CXCR4. Intracardiac injection of PC3Luc cells was
performed in the presence or absence of neutralizing antibody to
CXCR4 or IgG control. Antibody treatments were repeated 2 and
24 h after PC3 injection. At 1 month, the animals were killed, and
serum was collected. Calcium and the collagen specific pyridino-
line cross-links levels were used as markers of bone turnover. (A)
Overall, there was a decrease in the total serum calcium in the
anti-CXCR4–treated group relative to controls. The PYD levels
were lower in the CXCR4 antibody group compared with those of
the control group. (B) In animals that did have osseous tumors,
there were no differences in the number of osteoclasts (OCs)/mm
bone, the percentage of the growth plate occupied by tumor, or
percent tumor/bone area between controls and anti-CXCR4 an-
tibody-treated animals. (C) Tibial tumor representative of anti-
CXCR4–treated group showing multinucleated osteoclasts lining
bone surfaces (arrows).
et al.(27) enumerated the number of circulating tumor neu-
tralization in non-Hodgkin’s lymphoma (NHL) cells in the
peripheral blood of mice injected with an NHL cell line
intravenously after CXCR4 neutralization. One day after
tumor injection, circulating tumor cells were undetectable.
For mice injected with CXCR4-neutralized NHL cells, the
frequency of circulating tumor cells was >0.5%. These ob-
servations, along with data that show that antibody to
SUN ET AL.
CXCR4 impairs transendothelial/stromal cell migration,
suggest a pivotal role of the CXCR4-CXCL12/SDF-1 in
tumor extra- and intravasation.(27) Whether SDF-1 serves a
similar role in CaPs is not known, but seems likely.
The autocrine production of SDF-1 resulting in prolifera-
tion is an exciting aspect of the biology of the SDF-1/
CXCR4 chemokine axis in CaP cells,(5) more so in that
CaPs use SDF-1 to localize to end-organs that supply high
levels of ligand (e.g., bone, lung, liver, lymph node(7)). Un-
fortunately, we have not been able to consistently show
SDF-1 at the protein level using tissue microarrays, al-
though weak nuclear staining was observed in localized
cancers (data not shown). Recently, Zhou et al.(34) showed
that overexpression of CXCR4 in glioblastoma cell lines
enhanced their growth in soft agar, and expression of anti-
sense CXCR-4 in glioblastoma cell lines caused neurite out-
growth and cellular differentiation. Moreover, treatment of
the glioblastoma cell lines with antibody to CXCR4 or
SDF-1 inhibited proliferation, suggesting that the CXCR-4
gene is required to prevent apoptosis after serum with-
drawal.(34,35) Similarly systemic administration of CXCR4
antagonist AMD 3100 inhibits growth of intracranial glio-
blastoma and medulloblastoma xenografts by increasing
apoptosis and decreasing the proliferation of tumor
cells.(35) Moreover, in some instances, CXCR4 may not be
responsible for invasion but rather be critical for the out-
growth of micrometastases.(36) However, not all tumors ex-
pressing CXCR4 proliferate in response to SDF-1, includ-
ing rhabdomyosarcoma cells.(37) Likewise, chemokines may
stimulate proliferation of tumor cells directly, as was shown
with melanoma growth stimulatory activity (MGSA), caus-
ing the growth of melanoma and pancreatic cell lines; in-
terleukin (IL)-8 stimulates growth of non–small-cell lung
and prostate carcinomas.(28,38–40) Several tumor-derived
chemokines may protect neoplasms from immune attack of
tumor-infiltrating lymphocytes including CCR5 ligands
(CCL5 [RANTES], CCL3 [MIP-1␣], CCL4 [MIP-1␤]) and
SDF-1 (CXCL12).(41) Conversely, the release of chemotac-
tic chemokines has been shown to recruit leukocyte infil-
trates that may limit tumor growth.(38,40,41–44)
A further possibility is that SDF-1 secretion facilitates
retention of the CaP cells in the marrow. This mechanism
may be envisioned through the continued activation of in-
tegrin molecules, or autocrine produced SDF-1 may desen-
sitize CaP cells to further CXCR4 signaling. Recently, it
was reported from several groups that the uncoupling of
G-coupled receptors including CXCR4 after receptor inter-
nalization by endocytosis may persist even after the recep-
tor is recycled to the cell surface.(45,46) This would result in
receptor ligand interactions that fail to induce downstream
signals such as calcium fluxes and/or MAP kinase signaling
or cell migration. Additionally, the production of SDF-1 by
CaP cells may help to establish a “repellant migratory” sys-
tem to localize the tumor cells between sources of SDF-1 in
the marrow (endothelium, osteoblasts). A similar system
was shown in T-cells, when different concentrations of
SDF-1 were immobilized in vitro, and time-lapse video mi-
croscopy was used to show a bidirectional effect.(46) T-cells
were found to move away from the high-concentration sites
of SDF-1 and toward those of low SDF-1 concentra-
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER 327

FIG. 8. Blockade of CXCR4 reduces intratibial tumor growth. PC3Luc cells were inoculated intratibially (105 cells) to measure the
effect of blocking CXCR4 on tumor growth.(25) One day later, anti-CXCR4 antibody and anti-CXCR4 peptide (TN14003) or control
(IgG and scrambled peptide) were administered daily for 4 weeks intraperitoneally. (A) Skeletal lesions identified by bioluminescence
imaging show a reduction in tumor size after administration of anti-CXCR4 antibody or TN14003 peptide compared with control. (B)
Representative histology of skeletal lesions depicting severe bone remodeling and the presence of tumor in control-treated animals. (C)
PYD and serum calcium levels showing that the PYD levels were lower in the anti-CXCR4 antibody and anti-CXCR4 peptide groups
compared with those of the control group. (D) Histomorphometry of intraosseous tumors evaluating the number of osteoclasts/mm
bone, the percentage of the growth plate occupied by tumor, or percent tumor/bone area between controls, anti-CXCR4 antibody, and
anti-CXCR4 peptide-treated animals.

tions.(46) Moreover, antigen-induced T-cell recruitment into
the peritoneal cavity was reversed by high but not low con-
centrations of SDF-1. Whether such findings represent
mechanisms to explain why high SDF-1–producing organs
such as the thymus and bone marrow are not chronically
infiltrated with T-cells is not known.
Our hypothesis was among the very first to suggest a role
for SDF-1/CXCR4 in prostate tumor metastasis. Recently
Muller et al.(7) and Kang et al.(6) reported that CXCR4 and
SDF-1 are central players in regulating metastasis by show-
ing that normal breast tissues express little CXCR4,
whereas breast neoplasms express high levels of CXCR4,
and antibody to CXCR4 blocked the metastatic spread of
the tumors to the lung and lymph nodes. Similarly, CXCR4
mRNA levels are elevated in glioblastoma multiforme in
regions of angiogenesis and degeneration but decreased in
areas of rapid cell proliferation and may be a general char-
acteristic of neuroblastoma cells that supports their prefer-
ential metastasis into the bone marrow.(8) Results consis-
tent with these have also been reported for human
melanoma cell lines, melanoma cells that had macroscopi-
cally infiltrated draining lymph nodes,(9) and pancreatic as
well as renal carcinomas.(11,47) These results are also con-
sistent with those of Zeelenberg et al.,(48) who transfected
SDF-1 fused to a KDEL sequence into mouse T-cell hy-
bridoma TAM2D2. The SDF-KDEL fusion protein was re-
tained in the endoplasmic reticulum by the KDEL receptor
and bound to CXCR4, which was therefore also retained,
preventing the metastasis of the cell line to many different
tissues.(48) However, little is known about the role of SDF-
1/CXCR4 in CaP. These in vivo metastasis data provide
critical support for a role of SDF-1/CXCR4 in skeletal me-
tastasis. Most importantly, these data show that SDF-1/
CXCR4 participate in localizing tumors to the bone mar-
row for CaP.
ACKNOWLEDGMENTS
Expertise on the Xenogen IVIS imaging system was pro-
vided by Dan Hall of the University of Michigan’s Small
328
Animal Imaging Resource Core (http://www.med.
umich.edu/msair/). These investigations were supported in
part by the Tissue Procurement Core of the University of
Michigan Comprehensive Cancer Center and National In-
stitutes of Health Grants DK067688 (RST), R01 DE13701
(RST), R01 AR46024 (RST), P01 CA46952 (KJP, ETK,
LKM, RST), P50 CA69568 (KJP), CA93900 (ETK, LKM,
RST), and the Department of Defense DAMD17-02-1-
0100 (RST).
REFERENCES
1. Rubens RD 1998 Bone metastases—the clinical problem. Eur
J Cancer 34:210–213.
2. Cooper CR, Chay CH, Gendernalik JD, Lee HL, Bhatia J,
Taichman RS, McCauley LK, Keller ET, Pienta KJ 2003 Stro-
mal factors involved in prostate carcinoma metastasis to bone.
Cancer Res 97:739–747.
3. Horuk R 2001 Chemokine receptors. Cytokine Growth Factor
Rev 12:313–335.
4. Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman N
McCauley LK 2002 Use of the stromal cell-derived factor-1/
CXCR4 pathway in prostate cancer metastasis to bone. Cancer
Res 62:1832–1837.
5. Sun Y-X, Wang J, Shelburne CE, Lopatin DE, Chinnaiyan
AM, Pienta KJ, Rubin MA, Taichman RS 2003 The expression
of CXCR4 and CXCL12 (SDF-1) in human prostate cancers
(PCa) in vivo. J Cell Biochem 89:462–473.
6. Kang YB, Siegel PM, Shu WP, Drobnjak M, Kakonen SM,
Cordon-Cardo C, Guise TA, Massague J 2003 A multigenic
program mediating breast cancer metastasis to bone. Cancer
Cell 3:537–549.
7. Muller CA, Homey B, Sato H, Ge N, Catron D, Buchanan M,
McClanahan T, Murphy E, Yuan W, Wagners S, Barrera J,
Mohar A, Verastegui E, Zlotnik A 2001 Involvement of che-
mokine receptors in breast cancer metastasis. Nature 410:50–
56.
8. Geminder H, Sagi-Assif O, Goldberg L, Meshel T, Rechavi G,
Witz IP, Ben-Baruch A 2001 A possible role for CXCR4 and
its ligand, the CXC chemokine stromal cell-derived factor-1, in
the development of bone marrow metastases in neuroblas-
toma. J Immunol 167:4747–4757.
9. Robledo MM, Bartolome RA, Longo N, Rodriguez-Frade JM,
Mellado M, Longo I 2001 Expression of functional chemokine
receptors CXCR3 and CXCR4 on human melanoma cells. J
Biol Chem 276:45098–45105.
10. Gerritsen ME, Peale FV Jr, Wu T 2002 Gene expression pro-
filing in silico: Relative expression of candidate angiogenesis
associated genes in renal cell carcinomas. Exp Nephrol 10:114–
119.
11. Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima
S, Kawaguchi M, Kobayashi H, Doi R, Hori T, Fujii N,
Imamura M 2000 Expression of stromal cell-derived factor 1
and CXCR4 ligand receptor system in pancreatic cancer: A
possible role for tumor progression. Clin Cancer Res 6:3530–
3535.
12. Aiuti A, Tavian M, Cipponi A, Ficara F, Zappone E, Hoxie J,
Peault B, Bordignon C 1999 Expression of CXCR4, the recep-
tor for stromal cell-derived factor-1 on fetal and adult human
lympho-hematopoietic progenitors. Eur J Immunol 29:1823–
1831.
13. Nagasawa T, Hirota S, Tachibana K, Takakura N 1996 Defects
of B-cell lymphopoiesis and bone-marrow myelopoiesis in
mice lacking the CXC chemokine PBSF/SDF-1. Nature
382:635–638.
14. Peled A, Petit I, Kollet O, Magid M, Ponomaryov T 1999 De-
pendence of human stem cell engraftment and repopulation of
NOD/SCID mice on CXCR4. Science 283:845–848.
15. Nagasawa T, Nakajima T, Tachibana K, Iizasa H, Bleul CC,
Yoshie O, Matsushima K, Yoshida N, Springer TA, Kishimoto
SUN ET AL.
T 1996 Molecular cloning and characterization of a murine
pre-B-cell growth-stimulating factor/stromal cell-derived factor
1 receptor, a murine homolog of the human immunodeficiency
virus 1 entry coreceptor fusin. Proc Natl Acad Sci USA
93:14726–14729.
16. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K,
Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishi-
kawa S, Kishimoto T, Nagasawa T 1998 The chemokine recep-
tor CXCR4 is essential for vascularization of the gastrointes-
tinal tract. Nature 393:591–594.
17. Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman RL,
Moore MA, Rafii S 1998 Transendothelial migration of mega-
karyocytes in response to stromal cell-derived factor 1 (SDF-1)
enhances platelet formation. J Exp Med 188:539–548.
18. Wang JF, Liu ZY, Groopman JE 1998 The alpha-chemokine
receptor CXCR4 is expressed on the megakaryocytic lineage
from progenitor to platelets and modulates migration and ad-
hesion. Blood 92:756–764.
19. Nyati MK, Symon Z, Kievit E, Dornfeld KJ, Rynkiewicz SD,
Ross BD, Rehemtulla A, Lawrence TS 2002 The potential of
5-fluorocytosine/cytosine deaminase enzyme prodrug gene
therapy in an intrahepatic colon cancer model. Gene Ther
9:844–849.
20. Kalikin LM, Schneider A, Griffin LB, Dunn RL, Rosol TJ,
Shah RB, Rehemtulla A, McCauley LK, Pienta KJ 2003
In vivo visualization of metastatic prostate cancer and quanti-
tation of disease progression in immunocompromised mice.
Cancer Biol Ther 2:656–660.
21. Tamamura H, Omagari A, Hiramatsu K, Gotoh K, Kanamoto
T, Xu YN, Kodama E, Matsuoka M, Hattori T, Yamamoto N,
Nakashima H, Otaka A, Fujii N 2001 Development of specific
CXCR4 inhibitors possessing high selectivity indexes as well as
complete stability in serum based on an anti-HIV peptide
T140. Bioorg Med Chem Lett 11:1897–1902.
22. Tamamura H, Sugioka M, Odagaki Y, Omagari A, Kan Y,
Oishi S, Nakashima H, Yamamoto N, Peiper SC, Hamanaka
N, Otaka A, Fujii N 2001 Conformational study of a highly
specific CXCR4 inhibitor, T140, disclosing the close proximity
of its intrinsic pharmacophores associated with strong anti-
HIV activity. Bioorg Med Chem Lett 11:359–362.
23. Meador-Woodruff JH, Damask SP, Wang J, Haroutunian V,
Davis KL, Watson SJ 1996 Dopamine receptor mRNA expres-
sion in human striatum and neocortex. Neuropsychopharma-
cology 15:17–29.
24. Rehemtulla A, Stegman LD, Cardozo SJ, Gupta S, Hall DE,
Contag CH, Ross BD 2000 Rapid and quantitative assessment
of cancer treatment response using in vivo bioluminescence
imaging. Neoplasia (New York) 2:491–495.
25. Zhang J, Dai J, Qi Y, Lin DL, Smith P, Strayhorn C, Mizokami
A, Fu Z, Westman J, Keller ET 2001 Osteoprotegerin inhibits
prostate cancer-induced osteoclastogenesis and prevents pros-
tate tumor growth in the bone. J Clin Invest 107:1235–1244.
26. Zhou H, Wu T, Wu X, Lou H, Yu X, Taichman R, Lau S,
Howard E, Nie S, Umbreit J, Shim H 2004 Inhibition of breast
cancer metastasis by selective synthetic polypeptide against
CXCR4 chemokine receptor. Cancer Res 64:4302–4308.
27. Bertolini F, Dell’Agnola C, Mancuso P, Rabascio C, Burlini A,
Monestiroli S, Gobbi A, Pruneri G, Martinelli G 2002 CXCR4
neutralization, a novel therapeutic approach for non-
Hodgkin’s lymphoma. Cancer Res 62:3106–3112.
28. Addison CL, Arenberg DA, Morris SB, Xue YY, Burdick MD,
Mulligan MS, Iannettoni MD, Strieter RM 2000 The CXC che-
mokine, monokine induced by interferon-gamma, inhibits non-
small cell lung carcinoma tumor growth and metastasis. Hum
Gene Ther 11:247–261.
29. Schneider A, Kalikia LM, Mattos AC, Keller ET, Allen MJ,
Pienta KJ, McCauley LK 2004 Bone turnover mediates pref-
erential localization of prostate cancer in the skeleton. (in
press).
30. Netelenbos T, Zuijderduijn S, van den Born J, Kessler FL,
Zweegman S, Huijgens PC, Drager AM 2002 Proteoglycans
guide SDF-1-induced migration of hematopoietic progenitor
cells. J Leukoc Biol 72:353–362.
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER
31. Amara A, Lorthioir O, Valenzuela A, Magerus A, Thelen M,
Montes M, Virelizier JL, Delepierre M, Baleux F, Lortat-Jacob
H, Arenzana-Seisdedos F 1999 Stromal cell-derived factor-
1alpha associates with heparan sulfates through the first beta-
strand of the chemokine. J Biol Chem 274:23916–23925.
32. Mbemba E, Gluckman JC, Gattegno L 2000 Glycan and gly-
cosaminoglycan binding properties of stromal cell-derived fac-
tor (SDF)-1{alpha}. Glycobiology 10:21–29.
33. Peled A, Grabovsky V, Habler L, Sandbank J, Arenzana-
Seisdedos F, Petit I, Ben-Hur H, Lapidot T, Alon R 1999 The
chemokine SDF-1 stimulates integrin-mediated arrest of
CD34(+) cells on vascular endothelium under shear flow. J
Clin Invest 104:1199–1211.
34. Zhou Y, Larsen PH, Hao C, Yong VW 2002 CXCR4 is a major
chemokine receptor on glioma cells and mediates their sur-
vival. J Biol Chem 9:9–99.
35. Rubin JB, Kung AL, Klein RS, Chan JA, Sun Y, Schmidt K,
Kieran MW, Luster AD, Segal RA 2003 A small-molecule
antagonist of CXCR4 inhibits intracranial growth of primary
brain tumors. Proc Natl Acad Sci USA 100:13513–13518.
36. Zeelenberg IS, Ruuls-Van Stalle L, Roos E 2003 The chemo-
kine receptor CXCR4 is required for outgrowth of colon car-
cinoma micrometastases. Cancer Res 63:3833–3839.
37. Libura JD 2002 CXCR4-SDF-1 signaling is active in rhabdo-
myosarcoma cells and regulates locomotion, chemotaxis, and
adhesion. Blood 100:2597–2606.
38. Owen JD, Strieter R, Burdick M, Haghnegahdar H, Nanney L,
Shattuck-Brandt R, Richmond A 1997 Enhanced tumor-
forming capacity for immortalized melanocytes expressing
melanoma growth stimulatory activity/growth-regulated cyto-
kine beta and gamma proteins. Int J Cancer 73:94–103.
39. Luan J, Shattuck-Brandt R, Haghnegahdar H, Owen JD, Stri-
eter R 1997 Mechanism and biological significance of consti-
tutive expression of MGSA/GRO chemokines in malignant
melanoma tumor progression. J Leukoc Biol 62:588–597.
40. Moore BB, Arenberg DA, Stoy K, Morgan T, Addison CL,
Morris SB 1999 Distinct CXC chemokines mediate tumorige-
nicity of prostate cancer cells. Am J Pathol 154:1503–1512.
41. Luan J, Shattuck-Brandt R, Haghnegahdar H, Owen JD, Stri-
eter R 1997 Mechanism and biological significance of consti-
tutive expression of MGSA/GRO chemokines in malignant
melanoma tumor progression. J Leukoc Biol 62:588–597.
42. Maghazachi AA, Al-Aoukaty A 1998 Chemokines activate
329
natural killer cells through heterotrimeric G-proteins: Implica-
tions for the treatment of AIDS and cancer. FASEB J 12:913–
924.
43. Mellado M, de Ana AM, Moreno MC, Martinez C, Rodriguez-
Frade JM 2001 A potential immune escape mechanism by
melanoma cells through the activation of chemokine-induced
T cell death. Curr Biol 11:691–696.
44. Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE,
Wang XY, Algate PA, Franklin RA, Oberhaus SM, Steelman
LS, McCubrey JA 1999 Signal transduction, cell cycle regula-
tory, and anti-apoptotic pathways regulated by IL-3 in hema-
topoietic cells: Possible sites for intervention with anti-
neoplastic drugs. Leukemia 13:1109–1166.
45. Zhang J, Ferguson SS, Barak LS, Aber MJ, Giros B, Lefkowitz
RJ, Caron MG 1997 Molecular mechanisms of G protein-
coupled receptor signaling: Role of G protein-coupled receptor
kinases and arrestins in receptor desensitization and resensiti-
zation. Receptors Channels 5:193–199.
46. Shen H, Cheng T, Olszak I, Garcia-Zepeda E, Lu Z, Herrmann
S, Fallon R, Luster AD, Scadden DT 2001 CXCR-4 desensi-
tization is associated with tissue localization of hemopoietic
progenitor cells. J Immunol 166:5027–5033.
47. Eitner F, Cui Y, Hudkins KL, Alpers CE 1998 Chemokine
receptor (CXCR4) mRNA-expressing leukocytes are in-
creased in human renal allograft rejection. Transplantation
66:1551–1557.
48. Zeelenberg IS, Ruuls-Van SL, Roos E 2001 Retention of
CXCR4 in the endoplasmic reticulum blocks dissemination of
a T cell hybridoma. J Clin Invest 108:269–277.
Address reprint requests to:
Russell S Taichman, DMD, DMSc
Department of Periodontics, Prevention, Geriatrics
University of Michigan School of Dentistry
1011 North University Avenue
Ann Arbor, MI 48109-1078, USA
E-mail: rtaich@umich.edu
Received in original form May 24, 2004; revised form August 2,
2004; accepted September 8, 2004.