CXCR7 (RDC1) promotes breast and lung tumor
growth in vivo and is expressed on
Zhenhua Miao*, Kathryn E. Luker†, Bretton C. Summers*, Rob Berahovich*, Mahaveer S. Bhojani‡,
Alnawaz Rehemtulla‡, Celina G. Kleer§, Jeffrey J. Essner¶, Aidas Nasevicius?, Gary D. Luker†**††,
Maureen C. Howard*, and Thomas J. Schall*‡‡
*ChemoCentryx, Inc., Mountain View, CA 94043; Departments of†Radiology,‡Radiation Oncology,§Pathology, and **Microbiology and Immunology,
University of Michigan, Ann Arbor, MI 48109;¶Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011; and
?Yorktown Technologies, Plant City, FL 33565
Edited by Dan R. Littman, New York University Medical Center, New York, NY, and approved August 3, 2007 (received for review November 24, 2006)
Chemokines and chemokine receptors have been posited to have
lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2),
recently deorphanized as a chemokine receptor that binds chemo-
kines CXCL11 and CXCL12, can regulate these two common malig-
nancies. Using a combination of overexpression and RNA interfer-
ence, we establish that CXCR7 promotes growth of tumors formed
from breast and lung cancer cells and enhances experimental lung
metastases in immunodeficient as well as immunocompetent mouse
models of cancer. These effects did not depend on expression of the
related receptor CXCR4. Furthermore, immunohistochemistry of pri-
mary human tumor tissue demonstrates extensive CXCR7 expression
in human breast and lung cancers, where it is highly expressed on a
majority of tumor-associated blood vessels and malignant cells but
not expressed on normal vasculature. In addition, a critical role for
demonstrated by using morpholino-mediated knockdown of CXCR7
in zebrafish. Taken together, these data suggest that CXCR7 has key
functions in promoting tumor development and progression.
angiogenesis ? cancer ? chemokine
cancer, where these receptors have been implicated in multiple
steps of tumorigenesis and progression to metastatic disease (re-
viewed in ref. 1). In particular, the chemokine CXCL12 (SDF-1)
was thought to act through its “canonical” receptor, CXCR4, to
promote growth of primary tumors and progression to metastatic
disease in breast and lung cancer (reviewed in refs. 2 and 3).
Myofibroblasts associated with breast cancer, but not those in
normal breast tissue, produce CXCL12 and enhance growth of
tumors through mechanisms that include proliferation and survival
of malignant cells and angiogenesis (4, 5). Specific alleles of
CXCL12 are associated with an increased risk of breast cancer (6),
and CXCL12 has been shown to transactivate Her2/neu, an estab-
lished oncogene in breast cancer (7). Furthermore, high levels of
suggesting that gradients of this chemokine account for homing of
breast cancer cells to specific organs (8).
Similar to the effects of CXCL12 and CXCR4 on breast
cancer, chemokine signaling increases proliferation and pro-
metastatic functions of other cancer cells. For example, CXCR4
is expressed in primary small-cell lung cancer cells and, on
small-cell lung cancer cell lines, promotes migration, activation
of integrins, and adhesion of malignant cells to bone marrow
stromal cells (9). CXCR4 is also found in human non-small-cell
lung cancer, and neutralizing antibodies to CXCL12 limit ex-
perimental metastases in mouse models (10). Finally, high levels
of expression of CXCR4 correlate with increased metastases in
patients with non-small-cell lung cancer (11).
in many common malignancies, including breast and lung
Chemokine receptors other than CXCR4 may also regulate
breast and lung cancer. Expression of CCR7 in breast cancer and
non-small-cell lung cancer cells correlates with lymph node metas-
tases and poor prognosis (1, 8, 12). Signaling mediated by the
chemokine CCL5 and its receptor CCR5 in breast cancer cells
activates the tumor suppressor p53, and patients with a nonfunc-
breast tumors and reduced disease-free survival (13). CXCR3 also
significance of this receptor in breast cancer remains to be deter-
mined (14). Furthermore, expression of CXCR2 on cells in the
tumor microenvironment appears important for angiogenesis and
Notwithstanding the collective reports, many questions remain
regarding the direct mechanism of action of chemokines and their
receptors in cancer progression, particularly surrounding the
CXCL12/CXCR4 axis in common malignancies.
Recently, we demonstrated that the orphan receptor RDC1
(CCX-CKR2) functions as a chemokine receptor as demonstrated
by its ability to bind both CXCL11 and CXCL12 and mediate
enhanced growth and adhesion of cells in vitro (16, 17). We have
designated this receptor ‘‘CXCR7.’’ We observed that the intro-
duction of CXCR7 into cell lines correlated with an escape from
apoptosis, that the receptor could be induced to be expressed on
of a small molecule antagonist of CXCR7 correlated with a
decreaseintumorsizeinbothxenograftandsyngenic invivo tumor
growth studies. More recently, ectopic expression of CXCR7 has
been shown to increase cell proliferation of NIH 3T3 in vitro and
enhance tumor formation in nude mice in vivo (18). We wished to
understand whether CXCR7 could function directly to control
tumor development in vivo; to assess whether such control was
manifest in an array of tumor types, particularly breast and lung
tumors; and to determine CXCR7’s potential relevance to human
disease by assessing its presence in a variety of primary human
Author contributions: Z.M. and K.E.L. contributed equally to this work; Z.M., K.E.L., B.C.S.,
R.B., M.S.B., A.R., C.G.K., J.J.E., A.N., and G.D.L. performed research; Z.M., K.E.L., B.C.S.,
M.S.B., A.R., C.G.K., J.J.E., A.N., G.D.L., M.C.H., and T.J.S. analyzed data; and B.C.S., G.D.L.,
and T.J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Abbreviation: LLC, Lewis lung carcinoma.
††To whom correspondence should be addressed regarding experiments involving in vivo
metastasis or photon transfer models. E-mail: email@example.com.
‡‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0610444104 PNAS ?
October 2, 2007 ?
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tumors. Using RNA interference, stable receptor expression stud-
ies, and developmental genetic experiments, we more precisely
interrogated CXCR7’s role in tumor growth. In animal models, a
causal connection between CXCR7 expression and in vivo tumor
progression was determined. Furthermore, to establish a link
normal biopsy tissues from human patients were surveyed to assess
Expression of CXCR7 on Breast and Lung Cancer Cell Lines. We first
analyzed surface expression of CXCR7 in selected lung and breast
mAb (11G8) and radioligand binding assays that exploited the
unique pattern of chemokine binding we defined for this receptor
(16, 17): Specifically, both CXCL11 and CXCL12 interact with
CXCR7, and each chemokine effectively competes with the other
for binding. Detectable levels of CXCR7 were present on the
surface of murine breast tumor 4T1 and Lewis lung carcinoma
(LLC) cell lines, whereas expression of the receptor on the surface
of human breast tumor MDA MB 435s cells was undetectable, in
agreement with our previous studies (16) (Fig. 1 A and B).
Both murine 4T1 and LLC cell lines are known to form primary
and metastatic tumors in mice (19, 20). To quantify the direct
metastatic breast and lung cancer, we generated clonal populations
of 4T1 and LLC cells harboring stable CXCR7 RNA interference
(RNAi) expression vectors. RNAi expression vectors were devel-
oped against two independent sites beginning at nucleotide 35 or
985, respectively, in murine CXCR7 to inhibit expression of en-
reduced expression of this receptor, which are referred to as
4T1-CXCR7-RNAi-35 and -985 cells, respectively, based on the
RNAi target site. 4T1-CXCR7-RNAi-35 cells had essentially un-
detectable levels of CXCR7, whereas levels of the receptor were
substantially reduced in 4T1-CXCR7-RNAi-985 cells by both flow
cytometry and radioligand binding (Fig. 1 A and B Left). As
quantified by branched DNA-based QuantiGene assay, expression
of CXCR7 mRNA in each cell line was reduced to ?10% of that
RNAi of CXCR7 did not induce an IFN response, as measured by
expression of IFN-induced protein with tetratricopeptide repeats-1
was generated with reduced expression of CXCR7 (LLC-CXCR7-
RNAi-985) (Fig. 1 A and B Center). Importantly, none of these cell
lines expressed mRNA or protein for CXCR4, allowing us to
interrogate CXCR7 without potential confounding effects of
CXCL12 interacting with CXCR4 (Fig. 1C and SI Fig. 6). In both
4T1 and the LLC cells, we also attempted to reduce expression of
CXCR7 with an RNAi molecule targeting CXCR7 at nucleotide
200 (4T1-CXCR7-RNAi-control, LLC-CXCR7-RNAi-control).
Neither CXCR7 mRNA nor protein was reduced by this RNA
interference molecule, making these cells ideal controls for further
studies (Fig. 1 A and B).
To test the effects of increased expression of CXCR7, we
generated MDA MB 435s cells that stably overexpressed this
control cell line (MDA MB 435s vector) (Fig. 1 A and B Right).
Similar to parental MDA MB 435s cells, stable MDA MB 435s
CXCR7 cells did not express CXCR4, once again allowing us to
interrogate CXCR7 role in isolation (Fig. 1C).
CXCR7 Promotes Growth of Breast Tumors in Mice. We implanted
MDA MB 435s WT, MDA MB 435s vector, or MDA MB 435s
CXCR7 cells s.c. into SCID mice to investigate the extent to which
CXCR7 affects growth of cell-derived breast tumors (Fig. 2 A and
MDA MB 435s vector control cells, as shown by changes in tumor
volumes over time and tumor weights measured at the end of the
experiment (Fig. 2 A and B). Similar results were obtained by
mammary fat pad implantation of MDA MB 435s clones into nude
differences in expression of CXCR7 in vivo, cells derived from
MDA MB 435s WT, MDA MB 435s vector, or MDA MB 435s
CXCR7 tumors were analyzed by flow cytometry and radioligand
binding assay after resection and tumor dispersion. MDA MB 435s
CXCR7 cells maintained high levels of CXCR7 expression in vivo
binding pattern, whereas cells from MDA MB 435s WT or MDA
MB 435s vector tumors did not (data not shown). All in vivo-grown
12G5, confirming that CXCR4 expression was not induced after
growth in vivo (data not shown).
To establish that CXCR7 promoted growth of cell-derived
and lung cancer cell lines. (A) Surface expres-
sion of CXCR7 on 4T1, LLC, and MDA MB 435s
cell lines was quantified by flow cytometry,
using CXCR7 antibody 11G8. (B) Binding of ra-
diolabeled CXCL12 to 4T1, LLC, and MDA MB
435s cells was quantified in the presence or
absence of nonradiolabeled chemokines and
corresponds to levels of CXCR7 measured by
flow cytometry. (C) Surface expression of
CXCR4 on 4T1, LLC, and MDA MB 435s cell lines
was quantified by flow cytometry using CXCR4
Ab 12G5. wt, wild type.
Surface expression of CXCR7 in breast
www.pnas.org?cgi?doi?10.1073?pnas.0610444104Miao et al.
tumors in immunocompetent mice, we performed similar experi-
ments with 4T1 RNAi cell lines (Fig. 2D). Upon s.c. implantation
of 4T1 cells into BALB/c mice, CXCR7 knockdown lines formed
significantly smaller tumors than WT or 4T1-CXCR7-RNAi-
control cells (Fig. 2D). Growth of 4T1-CXCR7-RNAi-control cells
was identical to WT cells, indicating that the reduced size of the
4T1-CXCR7-RNAi tumors was not due to cell manipulation by
that the differences in levels of CXCR7 in various 4T1 cell lines
were maintained in tumors, further supporting a direct correlation
between breast cancer growth and CXCR7 levels in vivo (data not
shown). Collectively, data from human MDA MB 435s and mouse
4T1 cells demonstrated that CXCR7 expression dramatically en-
hanced growth of cell-derived breast tumors.
CXCR7 Promotes Growth of Lung Cancer Cells in Immunocompetent
cells in vivo, we implanted LLC WT, LLC-CXCR7-RNAi-985, and
LLC-control cells s.c. into C57BL/6 mice. Similar to results with
breast cancer cells, LLC-CXCR7-RNAi-985 cells formed signifi-
cantly smaller tumors than LLC WT or LLC-CXCR7-RNAi-
control cells, with differences in tumor volumes evident as early as
6–8 days after implantation (Fig. 2E). Final weights of LLC-
CXCR7-RNAi-985-derived tumors were significantly less than
those of WT or control cells (Fig. 2E). Collectively, these data
demonstrate that CXCR7 promotes tumor growth in a mouse
model of lung and breast cancers.
CXCR7 Enhances Progression of Experimental Lung Metastases. Hav-
ing established that CXCR7 promotes growth of tumors derived
of the receptor on experimentally induced lung metastases. After
tail vein injection of 4T1 WT or 4T1-CXCR7-RNAi cells, overall
growth of 4T1 WT cells in the lung was greater than that of
4T1-CXCR7-RNAi-35 or -985 cells, as measured by biolumines-
cence imaging of luciferase activity (Fig. 3). By area-under-curve
analysis of luciferase activity, total bioluminescence from 4T1 WT
cells was significantly greater than that of 4T1-CXCR7-RNAi-35
and -985 cells. Necropsy indicated that the lungs from 4T1 WT
cell-injected animals had more tumor mass (load) than 4T1-
CXCR7-RNAi-35 or -985 cells (data not shown). In addition, mice
injected with 4T1 WT cells had to be killed at earlier time points
than RNAi lines because of morbidity (data not shown). We also
experimental lung metastasis model. Overexpression of CXCR7
enhanced initial growth of MDA MB 435s cells relative to vector
control, similar to effects of the receptor in increased proliferation
of 4T1 cells in the lung (data not shown). These data demonstrate
that expression of CXCR7 on breast cancer cells enhances the
ability of these cells to seed and proliferate in the lung, a common
site of metastatic breast cancer.
CXCR7 Expression Marks Various Human Malignancies. To correlate
data from animal models with human malignancy, we analyzed
expression of CXCR7 in breast and lung tissue samples from
patients by immunohistochemistry, using the CXCR7 specific an-
tibody 11G8 (16). In breast tissue obtained from reduction mam-
moplasties, CXCR7 was undetectable or present at very low levels
in normal breast epithelium (Fig. 4A Left). In contrast, expression
of CXCR7 was clearly detected on the transformed cells in ?30%
MB 435s cells were implanted s.c. into the flank (A and B) of scid mice or mammary fat pad of nude mice (C). (D and E) 4T1 (D) and LLC (E) cells were implanted
s.c. into the flank of BALB/c or C57Bl6 mice, respectively. Tumor progression was quantified by tumor volume over time and tumor weight at the end of the
experiment. Studies were repeated twice with n ? 6 mice per group.
Miao et al. PNAS ?
October 2, 2007 ?
vol. 104 ?
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of breast tumor sections from different individuals, including those
from both in situ and invasive ductal and lobular carcinomas
versus invasive malignancies (data not shown). Isotype control
antibody did not react with any samples tested (Fig. 4 B–D Left). In
lung cancer sections, CXCR7-specific reactivity was also readily
apparent in multiple patients, primarily in squamous cell carcino-
mas but also occasionally in adenocarcinomas (Fig. 4B Right).
Immunodetectable CXCR7 was expressed in soft-tissue tumors,
such as rhabdomyosarcoma (Fig. 4C) as well as present in other
malignancies, including cervical (Fig. 4D), renal, and esophageal
tumors such as rhabdomyosarcoma. Overall, our data demonstrate
that CXCR7 is expressed by tumor cells in a substantial number of
patients with breast and lung cancer. Furthermore, our analysis of
other common human cancers suggests that the receptor is ex-
pressed in a broad range of clinical malignancies.
CXCR7 Is Highly Expressed on Tumor Vasculature in Model Systems
and Human Tumors. During the course of CXCR7 protein expres-
sion analysis by immunohistochemistry in mouse tumor models, we
consistently observed staining of tumor vasculature. For example,
observed between CXCR7 and the established endothelial marker
CD31 (Fig. 5A). This result was observed regardless of CXCR7
expression levels on implanted tumor cells themselves (Fig. 5A).
Similar colocalization was also detected in tumor endothelium
associated with 4T1 cell-derived tumors in immunocompetent
BALB/c mice (data not shown). Collectively, these data demon-
strated that CXCR7 was expressed in endothelium of tumor blood
and irrespective of immune status of the animal.
Upon analysis of human sections, CXCR7 protein was expressed
on blood vessels within human tumors (Fig. 5B). For example,
human breast cancer specimens exhibited robust CXCR7 staining
in 97% (106 of 109) of the samples, whereas it was undetectable or
nearly undetectable in blood vessels associated with normal breast
tissues derived from reduction mammoplasties and histologically
normal breast tissues in patients with breast cancer (Fig. 4A Left).
Similar to the data from primary breast cancers, we identified
CXCR7 in vascular endothelium associated with other malignan-
cies. These data suggest that CXCR7 is present on a variety of
breast, lung, and other cancers and on a large percentage of tumor
vasculature from human malignancies.
Early studies examining the role of chemokines in cancer have
focused on CXCL12 and its receptor CXCR4, and how they affect
overall prognosis for patients. Our data establish that a second
receptor for CXCL12, namely CXCR7, heretofore an orphan
receptor that we recently characterized as a chemokine receptor
(16, 17), promotes the growth of both breast and lung cancer.
CXCR7 expression correlated with overall growth of cell-derived
breast and lung cancers and experimentally induced lung metasta-
ses in mouse models. These results are supported by a recent
publication detailing CXCR7’s up-regulation in Kaposi’s sarcoma-
associated herpesvirus-infected endothelial cells and its ability to
promote tumor growth of ectopically expressing cells in mice (18).
CXCR7 is expressed on malignant cells in a substantial percentage
of sections from primary human breast and lung cancers and other
common malignancies. In addition, and perhaps most striking,
all specimens of breast and lung cancer analyzed but not in blood
vessels from nonmalignant tissue.
These studies indicate that CXCR7 plays a critical role in tumor
growth in murine models of disease. Despite this, questions clearly
remain about the mechanism by which CXCR7 mediates these
levels of a number of secreted proteins, most notably matrix
metalloproteinase 3, suggesting a role in regulating extra cellular
matrix modifying proteins (our unpublished data). In addition, the
observation that CXCR7 expressed is on both the tumor vascula-
ture and malignant cells suggests a possible role in chemokine
presentation or adhesion in the tumor microenvironment. This
hypothesis is supported by adhesion studies (16) demonstrating, in
vitro, that CXCR7 expression is regulated by inflammatory cyto-
kines on endothelial cells and promotes maximal cell–cell interac-
metastases. WT and CXCR7 RNAi 4T1 lines were injected intravenously into
BALB/c mice via tail vein. Tumor growth in lung was quantified by biolumines-
cence imaging. The study was repeated twice with n ? 4 mice per group.
RNA interference of CXCR7 reduces growth of experimental lung
Undetectable expression of CXCR7 in normal breast tissue from a reduction
mammoplasty. (Right) Primary invasive ductal carcinoma of the breast with
increased amounts of CXCR7. Isotype control (Left) versus CXCR7 (Right)
staining of sections from lung adenocarcinoma (B), Rhabdomyosarcoma (C),
or Cervix squamous cell carcinoma (D). Nuclei were counterstained with
hematoxylin (blue). (Magnification: ?400.)
www.pnas.org?cgi?doi?10.1073?pnas.0610444104 Miao et al.
tions when expressed concomitantly on both tumor cells and
activated endothelium. In addition, preliminary studies using mor-
pholino oligo-mediated knockdown of CXCR7 in zebrafish have
organization during development (SI Fig. 7 and SI Movies 1–4).
Indeed, CXCR7 morphant embryos strongly resemble VEGF-A
morphants in the development of enlarged pericardium and major
blood vessel deficiencies (21). Further studies are ongoing to
determine the specific pathway(s) and mechanisms by which
CXCR7 mediates its effect.
The hypothesis that CXCR7 plays a role in human disease is
supported not only by animal models but also by the observation
that CXCR7 is expressed on malignant but not normal human
tissue biopsies. We observed CXCR7 expression on a wide variety
of human malignancies, suggesting a wide role for this receptor in
and lung cancer growth through expression in malignant cells and
may regulate tumorigenesis in a variety of other common malig-
nancies. The significance of CXCR7 in cancer is also emphasized
by the recent observation that a specific small molecule antagonist
of this molecule limits growth of tumor in both syngenic and
xenograft models (16). Collectively, these data show that therapeu-
tic strategies to inhibit CXCR7 represent a unique opportunity to
improve treatment of breast, lung, and possibly other cancers
because of the potential to specifically target both malignant cells
and tumor blood vessels.
Materials and Methods
Reagents. Chemokines and anti-CXCR4 antibody 12G5 were pur-
chased from R&D Systems and PeproTech. Anti-mCXCR4 anti-
body 2B11 was purchased from BD Biosciences. Anti-huCXCR7
antibody 11G8 was generated as reported in ref. 16. mRNA
quantification QuantiGene assay kits were purchased from Geno-
PerkinElmer. All other reagents were from Sigma.
125I-ITAC were purchased from
Cell Lines. Mouse4T1andhumanMDAMB435sbreastcancercell
lines were purchased from American Type Culture Collection.
Mouse LLC lung carcinoma cells were generously provided by C.
Kuo (Stanford University, Stanford, CA). 4T1 cells were cultured
cells were maintained in DMEM with 10% FBS. Generation of
MDA MB 435s stable transfectants is detailed in ref. 16.
Cell Lines with Stable RNA Interference Against CXCR7. We designed
short hairpin RNA molecules targeted against sites beginning at
nucleotides 35 and 985 in mouse CXCR7 (NM?007722). The
following oligonucleotides were synthesized (Invitrogen): (i) posi-
tion 35, 5?-caccGCAACTACTCTGACATCAACTcgaaAGTTG-
ATGTCAGAGTAGTTGC-3? and 5?-aaaaGCAACTACTCTGA-
CATCAACTttcgAGTTGATGTCAGAGTAGTTGC-3? and (ii)
gaaCGAGTACTTGAAGATGAAGGC-3? and 5?-aaaaGCCT-
AGGC-3? (lowercase letters represent linkers).
U6 RNAi entry vector and inserted into pBLOCK-iT 3-DEST
shRNA pBLOCK-iT U6 RNAi entry vectors in 293 cells stably
transfected with mouse CXCR7. 4T1 and LLC cells were trans-
fected with shRNA constructs, using Lipofectamine 2000 (Invitro-
gen) according to the manufacturer’s protocol. Stable transfectants
were selected and cultured in medium containing 1 mg/ml G418.
Lentiviruses. To construct a lentiviral vector that expresses firefly
luciferase and a monomeric orange fluorescent protein (mKO)
(Stratech, Cary, NC), we removed the gene for firefly luciferase
(FL) from pGL3 basic (Promega, Madison, WI) with NheI and
XbaI and blunt end-ligated it into the BamHI site in the FUW
lentiviral vector (22) (gift of D. Baltimore, California Institute of
Technology, Pasadena, CA). mKO was removed with BamHI and
HindIII and blunt end-ligated to the NotI site in pBUDCE4.1
(Invitrogen). The EF-1? promoter from pBUDCE4.1 and mKO
were excised with NheI and BglII and blunt end-ligated into the
PacI site of FUW. The resulting lentiviral transfer vector (FUW-
promoter to constitutively express FL, respectively.
Lentiviral stocks were prepared as described (20, 22) and used to
transduce various cell lines. Stably transduced cell lines were
identified by orange fluorescence and sorted by flow cytometry for
Flow Cytometry. Cells were stained with mouse monoclonal anti-
bodies against human CXCR7 (11G8, Chem; Centryxo), human
CXCR4 (clone 12G5; R&D Systems), or mouse CXCR4 (clone
conjugated to PE (Beckman Coulter). Samples were analyzed on a
(Genospectra) was used to quantify mRNAs in various samples
instructions. Briefly, cells were harvested at the day for FACS and
binding analysis. Cell lysates and specific probe sets were incubated
in the capture plate and hybridized overnight at 53°C. Amplifier
reagents and label probe sets were incubated in the capture plate
for 60 min at 53°C after wash. Substrate reagents were added in the
end for 30 min, and plates were read on a chemiluminescent plate
Radioligand Binding Assays. Assays to assess radioligand binding to
CXCR7 expressed on various cells were performed as described in
ref. 23. Cells were incubated for 3 h at 4°C with125I-SDF1? (final
MB 435s cells were stained with antibodies to the vascular endothelial marker
CD31 (green) or CXCR7 (red). Merged image shows colocalization of CXCR7 and
CD31 in tumor blood vessels (yellow). (B) Intense CXCR7 staining is observed on
the tumor vascular of sections from breast carcinoma (a), lung adenocarcinoma
(b), ovary mucinous adenocarcinoma (c), breast adenocarcinoma (d), lung squa-
mous cell carcinoma (e), liver hepatocellular carcinoma (f), bladder transitional
cell carcinoma (g), kidney renal cell carcinoma (h), and liver cholangiocarcinoma
(i). No staining was observed with isotype control antibody.
Miao et al. PNAS ?
October 2, 2007 ?
vol. 104 ?
no. 40 ?
concentration ?0.05 nM) or125I-CXCL11 (final concentration
?0.01 nM) in buffer (25 mM Hepes/140 mM NaCl/1 mM CaCl2/5
mM MgCl2/0.2% BSA, adjusted to pH 7.1) in the presence of an
excess (100 nM) unlabeled chemokine. Reactions were aspirated
onto PEI-treated GF/B glass filters, using a cell harvester (Pack-
35 ?l) was added to the filters and counted in a Packard Topcount
scintillation counter. Data were analyzed and plotted by using
GraphPad software (GraphPad Software).
Animal Experiments. All animal procedures were approved by
ChemoCentryx Institutional Animal Care and Use Committee or
the University of Michigan Committee on Use and Care of Ani-
or 4T1-CXCR7-RNAi cells were implanted into 6- to 8-week-old
or MDA MB 435s CXCR7 cells were injected into 6- to 8-week-old
female Ncr nude mice (Taconic) or SCID mice (Charles River
Laboratories). For some experiments, volumes of cell-derived
tumors were quantified as the product of caliper measurements in
two dimensions and calculated by the equation of width (mm) ?
width (mm) ? length (mm) ? 0.52. Animals were killed when
tumor volumes reached 1,000 mm3or animals lost ?20% of initial
body weight. Tumor weights were taken at termination of each
study. In other experiments, growth of viable breast cancer cells in
viable tumors was quantified by bioluminescence imaging.
To produce experimental lung metastases, 1 ? 1064T1 breast
cancer cells were injected intravenously via a tail vein in 100 ?l of
sterile 0.9% NaCl. Bioluminescence imaging was used to quantify
overall proliferation of metastatic cells.
amidate oligonucleotides were designed against the 5?UTR region
of zebrafish CXCR7: CXCR7mo1, 5?-TCACGTTCACACT-
CATCTTGGTCCG-3?; CXCR7mo2, 5?-TGTTATCGTCAA-
CACTTCAGTGACC-3?. For the experiments shown here, a mix-
ture of CXCR7mo1 (1 ng/embryo) and CXCR7mo2 (12 ng/
embryo) was injected between the one- and eight-cell stages.
each morpholino phosphodiamidate oligonucleotide alone. For
microangiography experiments, embryos were anesthetized in tric-
aine solution and injected with FITC-Dextran (20 mg/ml) into the
sinus venosa. Data are representative of multiple experiments:
microangiography (n ? 67).
Bioluminescence Imaging. Bioluminescence imaging and data anal-
ysis for photon flux produced by primary and metastatic tumors
were performed as described in ref. 20. For experimental lung
metastases, data for photon flux in the lung were normalized to
values obtained 3 h after injection to normalize for variations in
actual numbers of cells successfully injected (20).
Immunofluorescence and Immunohistochemistry. For immunofluo-
rescence microscopy, tumors were frozen in OCT compound and
sectioned at 10-?m intervals. We processed specimens for immu-
nofluorescence microscopy as described in ref. 24, using 1 ?g/ml
final concentrations of the mouse monoclonal antibody against
CXCR7 or a rat polyclonal antibody against CD31 present on
endothelium of blood vessels (eBioscience). Primary antibodies
were detected with Cy3-conjugated donkey anti-mouse-IgG and
Cy2-conjugated donkey anti-rat IgG secondary antibodies, respec-
tively (Jackson ImmunoResearch). Images of each fluorophore
were merged electronically using commercially available Spotfire
Immunohistochemistry was performed on paraffin-embedded
breast tissue sections arrayed in a high-density tissue microarray as
performed in ref. 25. Tissues were obtained from the surgical
contained largely consecutive invasive carcinoma tissue samples
characterized in ref. 25. In addition, normal breast tissues derived
from five different reduction mammoplasties were used. Tumor
microarrays were purchased from Imgenex, Zymed/Invitrogen,
Cybrdi, US Biomax, Biochain, and Petagen/Telechem. Specimens
methods; detection was performed with biotinylated rabbit anti-
mouse IgG (Jackson ImmunoResearch) coupled with ABC-AP
and fuchsin? kits (Dako). Mayer’s hematoxylin (Sigma) was used
as a counterstain. An irrelevant mouse IgG1 antibody was used as
an isotype control in all cases to demonstrate that staining was
specific for CXCR7.
Statistical Analysis. Area-under-the-curve analysis was done with
Prism software (GraphPad). Data are reported as mean values ?
SEM and compared with Student’s t test. Values ? 0.05 were
We thank Linda Ertl, Trageen Baumgart, Kevin Moore, Dan Dairaghi, Nu
Lai, Niky Zhao, Ton Dang, Anita Melikian, and J. J. Kim Wright for
contributions to this manuscript. This work was supported by National
Institutes of Health National Institute of Allergy and Infectious Diseases
Grant 1 U19 AI056690 (to ChemoCentryx); the Susan B. Komen Foun-
of Health Grants P50 CA93990 (to G.D.L. and A.R.), R01CA107469 (to
C.G.K.), and R24CA083099 (for the University of Michigan Small Animal
1. Balkwill F (2004) Nat Rev Cancer 4:540–550.
2. Hartmann T, Burger M, Burger J (2004) J Biol Regul Homeost Agents 18:126–130.
3. Luker K, Luker G (2006) Cancer Lett 23830–41.
4. Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, Huang H, Porter D, Hu M,
Chin L, Richardson A, et al. (2004) Cancer Cell 6:17–32.
5. Orimo A, Gupta P, Sgroi D, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey V,
Richardson A, Weinberg R (2005) Cell 121:335–348.
6. Razmkhah M, Talei A, Doroudchi M, Khalili-Azad T, Gharderi A (2005) Cancer Lett
7. Cabioglu N, Summy J, Miller C, Parikh N, Sahin A, Tuzlali S, Pumiglia K, Gallick G, Price
J (2005) Cancer Res 65:6493–6497.
8. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanon M, McClanahan T, Murphy E,
Yuan W, Wagner S, et al. (2001) Nature 410:50–56.
9. Burger M, Glodek A, Hartmann T, Schmitt-Graff A, Silberstein L, Fujii N, Kipps T, Burger
J (2003) Oncogene 22:8093–8101.
10. Phillips R, Burdick M, Lutz M, Belperio J, Keane M, Strieter R (2003) Am J Respir Crit Care
11. Su L, Zhang J, Xu H, Wang Y, Chu Y, Liu R, Xiong S (2005) Clin Cancer Res
12. Takanami I (2003) Int J Cancer 105:186–189.
13. Man ˜es S, Mira E, Colomer R, Montero S, Real LM, Go ´mez-Mouto ´n C, Jime ´nez-Baranda
S, Garzo ´n A, Lacalle RA, Harshman K, et al. (2003) J Exp Med 198:1381–1389.
A (2004) Immunol Lett 92:171–178.
15. Keane M, Belperio J, Xue Y, Burdick M, Strieter R (2004) J Immunol 172:2853–2860.
16. Burns J, Summers B, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold M, Sunshine M,
Littman D, Kuo C, et al. (2006) J Exp Med 203:2201–2213.
WO04058705 (7/15/2004) and USA patent publication US 20040170634 (9/2/2004).
18. Raggo C, Ruhl R, McAllister S, Koon H, Dezube B, Fruh K, Moses A (2005) Cancer Res
19. Young M, Duffie G, Lozano Y, Young M, Wright M (1990) Cancer Res 50:2973–2978.
20. Smith M, Luker K, Garbow J, Prior J, Jackson E, Piwnica-Worms D, Luker G (2004) Cancer
21. Nasevicius A, Larson J, Ekker S (2000) Yeast 17:294–301.
22. Lois C, Hong E, Pease S, Brown E, Baltimore D (2002) Science 295:868–872.
23. Dairaghi D, Fan R, McMaster B, Hanley M, Schall T (1999) J Biol Chem 274:21569–21574.
24. Luker G, Pica C, Kumar A, Covey D, Piwnica-Worms D (2000) Biochemistry 39:7651–7661.
25. Kleer C, Cao Q, Varambally S, Shen R, Ota I, Tomlins S, Ghosh D, Sewalt R, Otte A, Hayes
D, et al. (2003) Proc Natl Acad Sci USA 100:11606–11611.
www.pnas.org?cgi?doi?10.1073?pnas.0610444104Miao et al.