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JBMR 041109
JBMR 041109
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
INTRODUCTION cancer are at high risk for pathologic fractures, spinal cord
compression, and pain due in part to dysregulated cycles of
P ROSTATE CANCER (CaP) frequently metastasizes to the
bone marrow, resulting in significant morbidity and
mortality. Nearly 90% of patients with advanced prostate
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 authors have no conflict of interest. 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 319
chemotactic responses to bone-derived factors, preferential In this report, the goal was to delineate the role of SDF-
adhesion to bone marrow endothelium, and the interaction 1/CXCR4 receptor in CaP disease. Two independent stud-
of CaP cells with the bone microenvironment leading to ies were performed. First, to establish a positive correlation
tumor in the bone marrow, play considerable roles.(2) between the SDF-1 gene and protein expression with tumor
Chemokines are a group of molecules known to play metastasis, SDF-1 levels were characterized for a variety of
significant roles as activators and chemoattractants for murine tissues by ELISA and in situ hybridization. In an in
many blood cell types as well as an increasing array of vivo metastasis model, the role of CXCR4 in metastasis of
non-hematopoietic cell types (e.g., epithelial cells, fibro- prostate cancer to the bone was examined. In this model,
blasts, and endothelial cells). Chemokines are 8- to 10-kDa neutralizing antibody to CXCR4 limited the extent of bone
proteins subdivided into four structural groups on the basis metastases. Finally, antibody and blocking peptide to
of the relative position of the first two cysteine residues in CXCR4 limited the growth of intraosseous CaPs after CaP
the mature protein (CXC, ML, C3XC, and C). At present, cell intratibial injections. Together, these in vivo metastasis
>50 chemokines have been identified, most of which are data provide critical support that SDF-1/CXCR4 plays a
thought to activate cell migration by binding to G-protein– role in skeletal metastasis. Most importantly, these novel
coupled cell surface receptors. Currently, there are at least data show that SDF-1/CXCR4 participates in localizing tu-
18 known chemokine receptors.(3) Activation of chemokine mors to the bone marrow in CaP and may help in design of
receptors triggers activation of many downstream pathways therapeutic treatments to prevent the spread and growth of
including nonreceptor tyrosine kinases, inositol trisphos- metastasis.
phate, MAPK, protein kinase C, and calcium mobilization.
Previously, we determined that the stromal-derived MATERIALS AND METHODS
factor-1 (SDF-1)/CXCR4 chemokine axis is activated in
CaP metastasis to bone.(4,5) Specifically, CXCR4 expres- CaP cell line
sion is related to increasing tumor grade.(5) Moreover, we PC3 prostate cancer cells originally isolated from a ver-
have shown that SDF-1 signaling through CXCR4 triggers tebral metastasis of a human prostate cancer patient were
the adhesion of CaPs to bone marrow endothelial cells. obtained from American Type Culture Collection (Rock-
Similar demonstrations have also been made suggesting ville, MD, USA) and were maintained in RPMI containing
that the SDF-1/CXCR4 axis may play parallel roles in other 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.
tumors that also metastasize to the marrow.(6,7) For ex- PC3 cells infected with the pLazarus retroviral construct
ample, Muller et al.(7) reported that CXCR4 and SDF-1 are expressing luciferase were selected for stable transfectants
central players in regulating metastasis by showing that nor- in G418.(19,20)
mal breast tissues express little CXCR4, whereas breast
neoplasms express high levels of CXCR4. Furthermore, an- SDF-1 ELISA
tibody to CXCR4 blocks the metastatic spread of the tu- Bone marrow and tissue extracts were derived from
mors to the lung and lymph nodes. Similarly, mRNA levels C57BL6 mice in PBS with protease inhibitors (Sigma). In
for CXCR4 are elevated in regions of angiogenesis and some cases, the femur and tibia were dissected free of soft
degeneration but decreased in areas of rapid cell prolifera- tissues, and 2-mm sections of the bones were generated on
tion in glioblastoma multiforme that may prime the cells for ice using a 0.5-mm diamond mandrill set at low speed in a
metastasis.(8) Results consistent with these have also been dental drill. Harvested tissues were subjected to sonication
reported for human melanoma cell lines, melanoma cells and spun, and the resulting supernatants were stored at
that had macroscopically infiltrated draining lymph −80°C until assayed for cytokine SDF-1 levels by double-
nodes,(9) and for pancreatic, neuroblastoma, and renal antibody sandwich method assembled with commercially
carcinomas.(10,11) available components according to the directions of the
Common to most of these reports is the analogy with manufacturer (sensitivity, 31.25 pg/ml; range, 62.5–5000 pg/
hematopoietic stem cell homing where gene knockout ap- ml; R&D Systems). SDF-1 levels are presented as mean ±
proaches in mice have shown that SDF-1 and CXCR4 play SE picograms per milliliter for duplicate determinations.
an important role in embryologic development.(12–14) Spe- Conditioned media from human osteoblasts (HOBs) and
cifically, SDF-1–deficient mice die in utero with severe car- the human osteosarcoma cell line MG-63 (ATCC
diac septum defects and poorly developed marrow. CXCR4 CRL1424) were used as positive controls for SDF-1.
receptor deletion is similarly lethal, resulting from circula-
tory, CNS, immune, and hematopoietic defects.(13,15) The SDF-1 bioactivity in tissues
phenotype of the SDF-1−/− mouse so closely followed the Bone and liver tissue extracts were generated from 20-
CXCR4−/− that these investigations showed that the recep- day-old C57BL6 mice in RPMI. In vitro invasion assays
tor-ligand pair play important roles in cardiac, CNS, and were performed using a reconstituted extracellular matrix
hematopoietic stem cell homing.(16) In addition, CXCR4 membrane (BD BioCoat Matrigel Invasion Chamber; BD
expression levels correlate with the ability of human pro- Biosciences, San Jose, CA, USA) using PC3 cells after de-
genitors to engraft into the marrow in nude mice, and an- termining the levels of SDF-1 in the extract by ELISA. For
tibody to CXCR4 prevented the engraftment of progenitors these investigations, test cells were placed in the upper
in the bone marrow.(14) Finally, osteoblasts and marrow chamber (1 × 105 cells/well) in RPMI medium with 1%
endothelial cells express SDF-1, which may thereby localize serum and 0–200 ng/ml SDF-1 or 50% tissue extract: RPMI
hematopoietic progenitor cells in the marrow.(14,17,18) (vol/vol) was added to the lower chamber. Spontaneous
320 SUN ET AL.
invasion was compared with invasion supported by a SDF-1 O-phenylenediamine dihydrochloride (OPD; Sigma) in ci-
gradient. Invasion into the matrix was assayed after 24 h by trate buffer was added for development.
quantification with MTT. The effect of 30 g/ml CXCR4
blocking antibody (MAB173; R&D Systems), IgG control, In vivo metastasis assay
or peptides designed to block SDF-1 binding to the CXCR4 For these studies, 2 × 105 PC3luc cells that were stably
receptor (TC14012 or TN14003(21,22)) synthesized by the transfected with luciferase before the experiment were
Emory Microchemical Facility served as positive controls. used. Two experimental groups were established; sodium
They were added to the top chamber of the Transwell to azide-free monoclonal antibodies, anti-CXCR4 (clones
provide additional proof that observed responses are de- MAB171 from R&D and 12G5 from BD PharMingen, San
pendent on CXCR4 receptor binding. Diego, CA, USA) and polyclonal anti-SDF-1 (R&D), were
used to neutralize the CXCR4-CXCL12/SDF-1 circuitry.
Histological evaluation Appropriate irrelevant antibodies (sodium azide-free
Femurs and tibia were trimmed of musculature, fixed in 44708.111 clone [MBA171] monoclonal antibody [IgG2a];
10% formalin at 4°C, decalcified in 10% EDTA (pH R&D Systems) or IgG matched controls were used. Before
7.4) for 10 days, and embedded in paraffin. Longitudinal inoculation, the tumor cells were preincubated with anti-
sections of tibias were cut and stained with H&E for histo- body to CXCR4 or control at 5 g per 1 × 104 injected cells.
logical evaluation. Where indicated, histomorphometric Our choice of reagents and dosing was based on the re-
analyses of the femurs/tibias were performed using a ported use in blocking human hematopoietic CD34+ cell
computer-assisted bone histomorphometric analyzing sys- engraftment into the bone marrows of NOD/SCID ani-
tem (Image-Pro Plus version 4.0; Media Cybernetics, Silver mals,(14) the ability to block invasion of LNCaP, C4-2B cells
Spring, MD, USA). and PC3 invasion in vitro,(4) and the ability to block SDF-1
binding to PC3 cells. Moreover, these reagents were used
Digoxigenin in situ hybridization by Muller et al.(7) to block metastasis of breast cancer cell
and immunohistochemistry lines mediated by SDF-1 in vivo. Inbred HSD:athymic male
Normal murine bones were harvested and fixed in 10% mice were purchased from Harlan Bioscience (Indianapo-
buffered formalin and decalcified in EDTA, and 2- to 3-M lis, IN, USA) and were housed under constant humidity
paraffin embedded slides were prepared. Human bone was and temperature, with 12-h light and 12-h dark cycles. The
obtained from patients undergoing orthopedic surgery in animals were anesthetized with a mixture of xylazine (100–
accordance with the University of Michigan’s Investiga- 200 mg/kg) and ketamine HCL (5–16 mg/kg; Ketaset; Fort
tional Review Board. Each slide was dewaxed and rehy- Dodge Laboratories, Fort Dodge, IA, USA), and the left
drated and subsequently treated with proteinase K, 10% cardiac ventricle was punctured percutaneously and in-
formalin, and washed in SSC. Hybridization was performed jected using a 25-gauge needle attached to a 1-ml syringe as
with a digoxin-labeled SDF-1 sense or antisense SDF-1 previously described.(20) The animals were subsequently in-
riboprobe at 55°C overnight. After incubation, the slides jected 2 and 24 h afterward with equal doses of the same
were washed in 4× SSC, RNase A, 2× SSC-10 mM DTT, 1× antibody.
SSC-10 mM DTT, and 0.5× SSC-10 mM DTT.(23) The slides After 4 weeks, bioluminescence imaging was used to fol-
were washed, blocked in 2% blocking buffer for 1 h, 1:500 low prostate-derived bone metastases as a primary out-
antibody (anti-digoxigenin), and color developed using come. The mice were injected intraperitoneally with lu-
nitro-blue tetrazolium chloride (NBT) and 5-bromo-4- ciferin (100 l at 40 mg/ml in PBS) before imaging. This
chloro-3⬘-indolyphosphate p-toluidine salt (BCIP). In some dose and route of administration have been shown to be
cases, antibody to SDF-1 (MAb Clone 79018; R&D Sys- optimal for rodent studies with a 10- to 20-minute after
tems) or IgG control (Sigma) were used in conjunction with luciferin injection.(24) Mice were anesthetized with 1.5%
a DAB Chromogen kit (LSAB + Peroxidase Kit; Dako, isoflurane/air, and the Xenogen IVIS cryogenically cooled
Carpinterina, CA, USA) to identify the protein in tissues. imaging system was used as described.(20) Selected mice
were imaged weekly after tumor injection to monitor tumor
SDF-1 binding assay development. Bioluminescence generated by the luciferin/
To show the validity of the antibody and peptide re- luciferase reaction served as a locator for cancer growth and
agents, PC3 cells were plated in 96-well plates at an initial was used for quantification using the LivingImage software
density of 2 × 104 cells/well. After 24 h, antibody to SDF-1 on a red (high intensity/cell number) to blue (low intensity/
(polyclonal and monoclonal), unlabeled SDF-1 (0–100 g), cell number) visual scale. A digital grayscale animal image
anti-CXCR4 (clone 12G4 or MAB 171 [clone 44708.111]), was acquired followed by acquisition and overlay of a pseu-
and anti-CXCR4 peptide or IgG control (0–100 g/ml) docolor image representing the spatial distribution of de-
were added to select wells (all reagents from R&D Sys- tected photon counts emerging from active luciferase within
tems). Similarly, peptides designed to block SDF-1 binding the animal. Signal intensity was quantified as the sum of all
to the CXCR4 receptor (TC14012 or TN14003(21,22)) were detected photons within the region of interest during a
synthesized by the Emory Microchemical Facility and 1-minute luminescent integration time.
added to several wells. Biotinylated SDF-1 was added to
the wells, and binding was allowed to proceed for 1 h at 4°C. Intratibial injections
Thereafter, the cells were washed, streptavidin horseradish PC3Luc cells were inoculated intratibially to measure the
peroxidase (HRP) was added to wells for 0.5 h at 4°C, and effect of blocking CXCR4 on tumor growth.(25) Intratibial
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER 321
Inhibition of SDF-1 binding by antibodies ated SDF-1, antibody to CXCR4, which binds to the ligand-
to CXCR4 binding domain of CXCR4, neutralizing polyclonal or
monoclonal antibody to SDF-1, or the peptide TC14012
Previous reports suggest that metastasis is frequently reduced binding of labeled SDF-1 to PC3 cells (Fig. 5).
found at sites of high bone turnover, including the region These data confirmed that these reagents were appropriate
near the growth plate. Our findings that SDF-1 levels were for further investigation.
elevated in the growth plate region and that it is bioactive
suggest that sites of elevated SDF-1 production may be In vivo metastasis investigations
particularly prone to CaP metastasis. To test this hypoth-
esis, agents that blocked the binding of SDF-1 to CXCR4 To test our hypothesis that the SDF-1/CXCR4 pathway is
on CaP cells are desired. To validate the employment of critical for the development of bone metastases, in vivo we
several commercial reagents for use in our investigations, established two experimental groups (Fig. 6A). In each
we first evaluated whether anti-CXCR4 or anti-SDF-1 an- case, PC3 cells tagged with luciferase were preincubated
tibodies would block the binding of biotinylated SDF-1 to with antibody to CXCR4 or an isotype-matched nonspecific
PC3 prostate cancer cells. For these investigations PC3 cul- antibody control group on ice immediately before intracar-
tures were established in 96-well plates, where unlabeled diac injection. The antibody that was chosen to block
SDF-1 (0–100 g), antibody to SDF-1 (polyclonal and CXCR4 (MBA171 monoclonal antibody; R&D Systems)
monoclonal), anti-CXCR4 antibodies (clone 12G4 or clone was used at 5 g/1 × 104 injected cells. The animals were
44708.111), antibody IgG controls (0–100 g/ml), or an subsequently injected intraperitoneally twice at 2 and 24 h.
anti-CXCR4 peptide designed to block SDF-1 (TC14012) After 4 weeks, bioluminescence imaging to follow the CaP
binding to the CXCR4 receptor were added to the wells. cell-derived metastases was used as our primary out-
Subsequently biotinylated SDF-1 was added to the wells, come.(24) As shown in Fig. 6A, we observed that all of the
and binding was allowed to proceed for 1 h at 4°C. There- animals that received the IgG control antibody established
after, the cells were washed, and streptavidin HRP and osseous metastases. Antibody to CXCR4 significantly re-
OPD were added for detection. duced the total metastatic load of the animals compared
The data showed that either unlabeled SDF-1, which with IgG control, regardless of whether the animal was im-
competes for binding of the CXCR4 receptor with biotinyl- aged from the dorsal or ventral surface (Fig. 6C). Exami-
SDF-1/CXCR4 AND OSSEOUS METASTASIS IN PROSTATE CANCER 323
nation of individual sites of bone metastasis also showed Figs. 7B and 7C). We interpret these data to suggest that,
that administration of antibody to CXCR4 significantly re- whereas the tibia/femur markers were not significant on a
duced the total luminescent signal (i.e., total tumor bur- per animal basis with many metastatic lesions in the IgG
den), further showing that the SDF-1/CXCR4 chemokine control group, the systemic PYD levels were elevated, re-
axis in part regulates osseous metastasis, including femur/ flecting the total metastatic burden. These data suggest that
tibia regions, spine, and maxilla/mandible areas (Fig. 6D). if CaP cells localize to the skeleton, the early treatment to
Radiographic analysis confirmed these findings, suggesting block CXCR4 had no influence on tumor growth under
that the antibody to CXCR4 had a significant effect on these conditions.
reducing osseous metastases.
Because antibody to CXCR4 decreased the metastatic Growth of intraosseous tumors in vivo
load, we tested if this also affected tumor-induced bone
resorption. For these evaluations, both serum calcium and The observation that antibody to CXCR4 blocked me-
serum pyridinoline cross-links levels were evaluated as tastasis induced by PC3 and our prior observation that an-
markers of bone turnover. There was no difference in total tibody to SDF-1 decreased PC3 proliferation in vitro(5)
serum calcium concentration in the experimental group in- prompted us to determine what effect blocking CXCR4
jected with the IgG antibody compared with those animals would have on the growth of prostate cancers in bone. Ac-
receiving the CXCR4 antibody (Fig. 7A). The PYD levels cordingly, PC3 cells were injected intratibially into nude
were lower in the CXCR4 antibody group compared with mice. One day later, anti-CXCR4 antibody, anti-CXCR4
those of the control group, indicating that there was an peptide (TC14012), or control antibody was administered
overall decrease in osteoclastic activity in the CXCR4 ex- daily for 4 weeks intraperitoneally. Skeletal lesions identi-
perimental groups (Fig. 7A). In animals with bone lesions, fied by bioluminescence imaging were used as our primary
however, there were no statistical differences in the tumor outcome. Tumors were identified in all of the control
size/bone area in anti-CXCR4 antibody treated versus IgG groups. The majority of the animals treated with either the
control-treated animals (41.8 ± 9.3 versus 45.5 ± 5.6 mm), in anti-CXCR4 antibody or TC14012 also had tumors; how-
number of osteoclasts/mm of tumor adjacent to the trabec- ever, the tumors were significantly smaller (Fig. 8A). His-
ular or cortical bone surfaces (1.8 ± 1.0 versus 1.0 ± 0.8 tological analyses revealed that tumor infiltration in all the
mm), or for the percentage of the growth plate that was control-treated animals was greater than the anti-CXCR4
associated with tumor (38.7 ± 13.1% versus 57.69 ± 19.3%; antibody or TC14012-treated mice (Fig. 8B). The levels of
324 SUN ET AL.
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 CaP cancer cell lines (Y-X Sun and RS Taichman, unpub-
addition to the SDF-1/CXCR4 axis in humans. lished data, 2004). Clearly, binding to endothelium is a ma-
To evaluate what role SDF-1 has on intraosseous growth jor event regulating the establishment of metastasis of
of CaPs, intratibial injection of PC3 cells was performed. blood-borne tumors. Recently, it has been reported that
Antibody or specific peptide against CXCR4 decreased the SDF-1 is bound to bone marrow endothelial cells by endo-
growth of pre-established CaP cell lesions in bone. These thelial heparan sulfate and chondroitin/dermatan proteo-
data correlated with our previous finding that showed that glycans, where it would activate adhesion receptors on the
human CaPs express elevated levels of SDF-1 mRNA rela- tumor cells to facilitate transendothelial migration.(30–32)
tive to benign hyperplasia in situ and that CaP cell lines that For hematopoietic cells, it is well established that efficient
secrete biologically active SDF-1 protein regulate their ex- rolling on endothelial cells requires P-selectin, E-selectin,
pression of SDF-1 mRNA in response to exogenous SDF- and the CD44 under physiologic flow conditions. On the
1.(5) Correlating with the in vivo data, neutralizing antibody other hand, intercellular adhesion molecule-1 (ICAM-1)
to SDF-1 decreased the proliferation metastatic tumor cells alone does not seem to facilitate binding of hematopoietic
that were isolated from bone, but not those isolated from cells under flow conditions. In the presence of surface-
other tissues.(5) These findings, together with those from bound SDF-1, however, rolling on endothelial cells is rap-
our metastasis study, suggest that neutralization of the idly developed into a firm adhesion, mediated by interac-
SDF-1/CXCR4 axis may prevent osseous metastasis by tions between ICAM-1 and SDF-1.(33) CXCL12/SDF-1
both inhibiting metastatic spread within the vasculature, may also regulate the development of pseudopodia in
and once in bone, preventing tumor growth. breast cancers that may lead to invasive metastasis.(7) These
We recently reported that SDF-1 activates binding of findings are important in that blockade of CXCR4 signaling
prostate cancer cells to marrow endothelial cells.(4) We de- may result in decreased metastases. Part of the mechanism
termined that part of the mechanism that facilitates CaP whereby CXCR4 inhibition may function is to limit extrav-
cell binding to endothelium in response to SDF-1 may be asation of circulating tumors by limiting binding or by yet
through activation of ␣v3 integrins already expressed on another unidentified mechanism. For example, Bertolini
326 SUN ET AL.
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 melanoma cell lines, melanoma cells that had macroscopi-
the peritoneal cavity was reversed by high but not low con- cally infiltrated draining lymph nodes,(9) and pancreatic as
centrations of SDF-1. Whether such findings represent well as renal carcinomas.(11,47) These results are also con-
mechanisms to explain why high SDF-1–producing organs sistent with those of Zeelenberg et al.,(48) who transfected
such as the thymus and bone marrow are not chronically SDF-1 fused to a KDEL sequence into mouse T-cell hy-
infiltrated with T-cells is not known. bridoma TAM2D2. The SDF-KDEL fusion protein was re-
Our hypothesis was among the very first to suggest a role tained in the endoplasmic reticulum by the KDEL receptor
for SDF-1/CXCR4 in prostate tumor metastasis. Recently and bound to CXCR4, which was therefore also retained,
Muller et al.(7) and Kang et al.(6) reported that CXCR4 and preventing the metastasis of the cell line to many different
SDF-1 are central players in regulating metastasis by show- tissues.(48) However, little is known about the role of SDF-
ing that normal breast tissues express little CXCR4, 1/CXCR4 in CaP. These in vivo metastasis data provide
whereas breast neoplasms express high levels of CXCR4, critical support for a role of SDF-1/CXCR4 in skeletal me-
and antibody to CXCR4 blocked the metastatic spread of tastasis. Most importantly, these data show that SDF-1/
the tumors to the lung and lymph nodes. Similarly, CXCR4 CXCR4 participate in localizing tumors to the bone mar-
mRNA levels are elevated in glioblastoma multiforme in row for CaP.
regions of angiogenesis and degeneration but decreased in
areas of rapid cell proliferation and may be a general char- ACKNOWLEDGMENTS
acteristic of neuroblastoma cells that supports their prefer-
ential metastasis into the bone marrow.(8) Results consis- Expertise on the Xenogen IVIS imaging system was pro-
tent with these have also been reported for human vided by Dan Hall of the University of Michigan’s Small
328 SUN ET AL.
Animal Imaging Resource Core (http://www.med. T 1996 Molecular cloning and characterization of a murine
umich.edu/msair/). These investigations were supported in pre-B-cell growth-stimulating factor/stromal cell-derived factor
1 receptor, a murine homolog of the human immunodeficiency
part by the Tissue Procurement Core of the University of virus 1 entry coreceptor fusin. Proc Natl Acad Sci USA
Michigan Comprehensive Cancer Center and National In- 93:14726–14729.
stitutes of Health Grants DK067688 (RST), R01 DE13701 16. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K,
(RST), R01 AR46024 (RST), P01 CA46952 (KJP, ETK, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishi-
LKM, RST), P50 CA69568 (KJP), CA93900 (ETK, LKM, kawa S, Kishimoto T, Nagasawa T 1998 The chemokine recep-
tor CXCR4 is essential for vascularization of the gastrointes-
RST), and the Department of Defense DAMD17-02-1- tinal tract. Nature 393:591–594.
0100 (RST). 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)
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