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Hypoxia-inducible factor-dependent signaling
betweentriple-negative breast cancer cells and mesenchymalstem
cells promotes macrophage recruitmentPallavi Chaturvedia,b, Daniele
M. Gilkesa,b, Naoharu Takanoa,b, and Gregg L. Semenzaa,b,c,1
aVascular Program, Institute for Cell Engineering,
bMcKusick-Nathans Institute of Genetic Medicine, and cDepartments
of Pediatrics, Medicine, Oncology,Radiation Oncology, and
Biological Chemistry, Johns Hopkins University School of Medicine,
Baltimore, MD 21205
Contributed by Gregg L. Semenza, April 14, 2014 (sent for review
March 20, 2014)
Intratumoral hypoxia induces the recruitment of stromal cells,
suchas macrophages and mesenchymal stem cells (MSCs), which
stim-ulate invasion and metastasis by breast cancer cells (BCCs).
Pro-duction of macrophage colony-stimulating factor 1 (CSF1) by
BCCsis required for macrophage recruitment, but the
mechanismsunderlying CSF1 expression have not been delineated.
Triple-negative breast cancers have increased expression of
genesregulated by hypoxia-inducible factors (HIFs). In this study,
wedelineate two feed-forward signaling loops between
humanMDA-MB-231 triple-negative BCCs and human MSCs that
drivestromal cell recruitment to primary breast tumors. The first
loop,in which BCCs secrete chemokine (C-X-C motif) ligand 16
(CXCL16)that binds to C-X-C chemokine receptor type 6 (CXCR6) on
MSCsand MSCs secrete chemokine CXCL10 that binds to receptor
CXCR3on BCCs, drives recruitment of MSCs. The second loop, in
whichMSCs secrete chemokine (C-C motif) ligand 5 that binds to
C-Cchemokine receptor type 5 on BCCs and BCCs secrete cytokineCSF1
that binds to the CSF1 receptor on MSCs, drives recruitmentof
tumor-associated macrophages and myeloid-derived suppressorcells.
These two signaling loops operate independent of eachother, but
both are dependent on the transcriptional activity ofHIFs, with
hypoxia serving as a pathophysiological signal that syn-ergizes
with chemokine signals from MSCs to trigger CSF1 genetranscription
in triple-negative BCCs.
HIF-1 | mammary fat pad | orthotopic implantation |lymph node
metastasis | lung metastasis
Breast cancer metastasis transforms a local disease that iscured
by surgical excision into a systemic disease that re-sponds poorly
to available therapies and is the major cause ofpatient mortality
(1). Although somatic mutations have beencataloged in hundreds of
human breast cancers and many genesthat promote or suppress
metastasis have been identified, theanalysis of genetic alterations
cannot reliably distinguish meta-static from nonmetastatic cancers
(1–3). Multiple stromal celltypes, including mesenchymal stem cells
(MSCs) and tumor-associated macrophages (TAMs), are recruited to
the tumor mi-croenvironment and promote metastasis (4, 5). In mouse
models,MSCs produce chemokines, including chemokine (C-C motif)
li-gand 5 (CCL5) and chemokine (C-X-C motif) ligand 10
(CXCL10),which bind to their cognate receptors, chemokine receptor
type5 (CCR5) and C-X-C chemokine receptor type 3
(CXCR3),respectively, on breast cancer cells (BCCs) to stimulate
invasionand metastasis (6–9). TAMs are abundant in breast cancerand
outnumber the BCCs in some cases (10). The density ofTAMs in
primary breast cancer biopsies is correlated with me-tastasis and
patient mortality (11–13). In mouse models, mac-rophage
colony-stimulating factor 1 (CSF1) and the chemokineCCL2 are
secreted by BCCs and bind to their cognate receptors,CSF1 receptor
(CSF1R) and CCR2, on TAMs, leading to theirrecruitment to the tumor
microenvironment, where they produceEGF and other secreted proteins
that promote invasion andmetastasis (14–19).
Intratumoral hypoxia is another major microenvironmentalfactor
that is associated with invasion, metastasis, and patientmortality
(20–22). Cancer cells respond to the hypoxic micro-environment
through the activity of hypoxia-inducible factors(HIFs), which are
heterodimeric transcription factors composedof an O2-regulated
HIF-1α or HIF-2α subunit and a constitu-tively expressed HIF-1β
subunit (23). In primary tumor biopsies,elevated HIF-1α or HIF-2α
protein levels are associated with anincreased risk of metastasis
and mortality that is independent ofbreast cancer grade or stage
(24–28). HIF-1α, HIF-2α, or bothare required for the
transcriptional activation of a battery ofhypoxia-inducible genes
whose protein products are required fordiscrete steps in the
process of breast cancer invasion and me-tastasis (29–34). High
expression of HIF target genes is com-monly observed in
triple-negative breast cancers (TNBCs), whichlack estrogen
receptor, progesterone receptor, and human epi-dermal growth factor
receptor 2 (HER2) expression and respondpoorly to chemotherapy
(2).Both MSCs and TAMs are recruited to the hypoxic breast
tumor microenvironment (9, 35), although the underlying
mech-anisms are not fully understood. In the present study, we
hy-pothesized that the presence of MSCs in the primary breasttumor
may facilitate TAM recruitment. Our studies of humanMDA-MB-231 TNBC
cells in immunodeficient mice revealedthat HIFs regulate the
hypoxia-induced expression of CXCL16in BCCs, which was required for
MSC recruitment. CCL5→CCR5signaling between MSCs and BCCs was
required for CSF1expression by BCCs, which was also induced by
hypoxia. Ex-pression of CSF1 and CCR5 by BCCs was required for
TAMrecruitment and BCC metastasis. HIF-dependent recruitment of
Significance
The recruitment of host stromal cells, such as macrophages
andmesenchymal stem cells (MSCs), to the primary tumor is a
crit-ical step toward cancer malignancy. We have identified
signalsthat are exchanged between breast cancer cells (BCCs)
andMSCs. This signaling increases the recruitment of both MSCsand
macrophages to primary tumors and increases metastasisof BCCs to
lymph nodes and lungs. Reduced oxygen levels(hypoxia) in breast
cancers are associated with increased risk ofmetastasis and
decreased patient survival. We show thathypoxia stimulates
signaling between BCCs and MSCs due tothe activity of
hypoxia-inducible factors (HIFs). Drugs that blockHIF activity
prevent signaling and macrophage recruitment,which suggests that
they may be useful additions to breastcancer therapy.
Author contributions: G.L.S. designed research; P.C., D.M.G.,
and N.T. performed research;P.C. and G.L.S. analyzed data; and P.C.
and G.L.S. wrote the paper.
The authors declare no conflict of interest.1To whom
correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406655111/-/DCSupplemental.
E2120–E2129 | PNAS | Published online May 5, 2014
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TAMs was also demonstrated after implantation of mouse 4T1TNBC
cells into the mammary fat pad (MFP) of immunocom-petent mice.
Taken together, these results delineate molecularmechanisms by
which intratumoral hypoxia regulates the re-cruitment of MSCs and
TAMs, and their interaction with TNBCs,to stimulate invasion and
metastasis.
ResultsCXCL16 Expression by BCCs Stimulates MSC Recruitment. We
previ-ously demonstrated that hypoxia-induced expression of
placentalgrowth factor (PGF) by MDA-MB-231 BCCs provides a
signalfor the recruitment of MSCs to primary breast tumors
andstimulates MSCs to express CXCL10, which binds to CXCR3on BCCs
(CXCL10MSC→CXCR3BCC) to stimulate invasion andmetastasis (9). We
also showed that CXCR3 expression by BCCswas required for CXCL10
expression by MSCs, but the re-sponsible signal from BCCs to MSCs
was not identified (9).CXCL16 expression by prostate cancer cells
was shown to pro-mote MSC recruitment (36), and CXCL16 expression
by BCCshas been reported (37). We cocultured GFP-expressing
humanMDA-MB-231 BCCs with human MSCs at a 1:1 ratio for 48 hunder
20% or 1% O2, and flow cytometry was performed usingGFP and CD105
immunofluorescence to sort for BCCs andMSCs, respectively. RNA was
isolated from the sorted cells andRT-quantitative real-time PCR
(qPCR) revealed that CXCL16expression by BCCs was induced following
coculture and thathypoxia further enhanced expression (Fig. 1A).
The expressionof CXCR6, a receptor for CXCL16, was induced by
hypoxia inBCCs, as previously reported (38), as well as in MSCs,
but co-culture did not enhance its expression (Fig. 1B).We
hypothesized that CXCL10MSC→CXCR3BCC signaling might
stimulate CXCL16BCC→CXCR6MSC reciprocal signaling. To testthis
hypothesis, we analyzed BCC subclones stably transfected withshRNAs
that inhibit CXCR3 expression (9). CXCL16 expression
inCXCR3-deficient BCCs was not induced by coculture with MSCsor by
hypoxia (Fig. 1C). We next added neutralizing antibody(NAb) against
CXCL10 or control IgG to cocultures of MSCs andBCCs. CXCL16 mRNA
expression was significantly decreased inthe presence of CXCL10 NAb
(Fig. 1D).We also hypothesized that CXCL16BCC→CXCR6MSC signal-
ing might stimulate CXCL10MSC→CXCR3BCC reciprocal sig-naling. To
test this hypothesis, we generated BCC subclones thatwere stably
transfected with shRNAs that inhibit CXCL16 ex-pression (Fig. S1).
CXCL10 expression in MSCs cocultured withCXCL16-deficient BCCs was
significantly decreased and was notinduced by hypoxia (Fig. 1E).To
investigate whether CXCL16 secretion from BCCs stimu-
lated the motility of MSCs, we isolated conditioned medium(CM)
from CXCL16-deficient BCCs or BCCs expressing a non-targeting
control (NTC) shRNA. CM from CXCL16-deficientBCCs induced
significantly lessMSCmigration in aBoyden chamberassay, and the
augmented effect of CM from hypoxic control BCCswas not observed
with CXCL16-deficient BCCs (Fig. 1F). To de-termine whether CXCL16
secretion from BCCs promotes re-cruitment ofMSCs to the primary
tumor, BCCs were orthotopicallyimplanted in the MFP of female SCID
mice. When the tumorsreached a volume of 250 mm3, MSCs that were
originally derivedfrom a male donor were injected via the tail
vein. Primary tumorswere harvested 16 h later, and MSC recruitment
was determinedby qPCR assay of genomic DNA using Y
chromosome-specificprimers (9). CXCL16 deficiency in BCCs
significantly decreasedthe recruitment of MSCs to the primary tumor
(Fig. 1G). Takentogether, these results delineate
CXCL16BCC→CXCR6MSC andCXCL10MSC→CXCR3BCC reciprocal signaling,
which creates afeed-forward loop between BCCs and MSCs that drives
MSC re-cruitment to the primary tumor (Fig. 1H).
CXCL16 Promotes Metastasis of BCCs to Lymph Nodes and
Lungs.Because CXCL16BCC→CXCR6MSC signaling was requiredfor
CXCL10MSC→CXCR3BCC signaling, which stimulates in-vasion and
metastasis (9), we hypothesized that CXCL16 ex-pression was
required for efficient metastasis. To test thishypothesis, control
or CXCL16-deficient BCCs were ortho-topically implanted in the MFP
of female SCID mice. CXCL16deficiency had no effect on primary
tumor growth (Fig. 2A).However, mice bearing CXCL16-deficient
tumors had signifi-cantly decreased numbers of circulating tumor
cells (Fig. 2B),
0
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)
Fig. 1. CXCL16 expression by MDA-MB-231 BCCs is induced by
hypoxia andis required for MSC recruitment. (A) GFP-expressing BCCs
were coculturedwith MSCs at 20% or 1% O2 for 48 h and then
flow-sorted based on GFPfluorescence for BCCs and CD105
immunofluorescence for MSCs. CXCL16mRNA levels were determined in
flow-sorted BCCs and MSCs from coculturesand in BCCs or MSCs
cultured alone. RNA levels were normalized to resultsfor BCCs
cultured alone at 20% O2. *P < 0.05 vs. 20%;
##P < 0.001 vs. BCCsalone. (B) BCCs, MSCs, and BCCs +MSCs
were incubated at 20% or 1% O2 for48 h, and CXCR6 mRNA levels were
determined and normalized to BCCsat 20%. *P < 0.05 vs. 20%. (C)
MDA-MB-231 subclones expressing shRNAagainst CXCR3 (shCR3-1 and
shCR3-3) or an NTC shRNA were cultured aloneor with MSCs and
exposed to 20% or 1% O2 for 48 h. CXCL16 mRNA levelswere normalized
to NTC − MSCs at 20%. *P < 0.05; **P < 0.001 vs. 20%;#P <
0.05 vs. NTC; ##P < 0.001 vs. NTC. (D) BCCs + MSCs were treated
withCXCL10 NAb or IgG and exposed to 20% or 1% O2. CXCL16 mRNA
levelswere normalized to IgG at 20%. **P < 0.001 vs. 20%; ##P
< 0.001 vs. IgG. (E)MDA-MB-231 subclones expressing shRNA
against CXCL16 (shCX16-1 andshCX16-3) or NTC shRNA were cultured
alone or with MSCs and exposedto 20% or 1% O2. CXCL10 mRNA levels
were normalized to NTC at 20%.*P < 0.05; **P < 0.001 vs. NTC
− MSCs; ##P < 0.001 vs. NTC + MSCs. (F) Mi-gration of MSCs in
response to CM from BCC subclones cultured at 20% or1% O2 was
determined and normalized to NTC at 20%. *P < 0.01 vs. 20%NTC;
#P < 0.01 vs. 1% NTC. (G) BCC subclones were implanted in the
MFP,and MSC recruitment to the primary tumor was analyzed by qPCR
for Ychromosome sequences and normalized to NTC. *P < 0.01 vs.
NTC. (H) Bi-directional signaling between BCCs and MSCs generates a
feed-forwardloop that stimulates MSC recruitment.
Chaturvedi et al. PNAS | Published online May 5, 2014 |
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metastatic cancer cells in the lungs by qPCR (Fig. 2C)
andmetastatic foci in the lungs by histology (Fig. 2 D and E),
andmetastatic cancer cells in the ipsilateral axillary lymph node
(Fig.2 F and G). Taken together, these results demonstrate
thatCXCL16 expression by BCCs plays a significant role in
pro-moting breast cancer metastasis.
HIF-Dependent BCC–MSC Interactions Promote TAM and
Myeloid-Derived Suppressor Cell Recruitment. To investigate whether
in-teraction of MSCs with BCCs facilitates the recruitment ofTAMs
to primary breast tumors, we cocultured MSCs with BCCsfor 48 h,
followed by MFP implantation of 1 × 106 coculturedcells. Controls
included mice implanted with 1 × 106 BCCs aloneor BCCs mixed with
MSCs immediately before implantation(Fig. 3A). When primary tumors
reached 450 mm3, they wereexcised and single-cell suspensions were
analyzed by flow cy-tometry for the presence of TAMs, which express
CSF1R andF4/80 on their cell surface, and myeloid-derived
suppressor cells(MDSCs; TAM progenitors), which express CD11b and
Ly6C.CSF1R+F4/80+ and CD11b+Ly6C+ cells were increased in tu-mors
derived from BCC +MSC coculture compared with tumorsderived from
BCCs alone or BCC + MSC coinjection (Fig. 3 Band C and Fig. S2 A
and B).Recent studies have shown that exposure of macrophages
to
levels of hypoxia that are comparable to what is observed
intumors leads to induction of HIF-1α and HIF-2α, which, in
turn,activate a broad array of genes with proangiogenic,
proinvasive,and prometastatic functions (39, 40). TAM numbers are
gener-ally higher in tumors containing high overall levels of
hypoxia,as seen in primary human breast carcinomas (41) and
various
animal tumors (42). To investigate whether HIF activity in
BCCsplays a role in TAM recruitment, we used the
MDA-MB-231double-knockdown (DKD) subclone, which is stably
trans-fected with vectors encoding shRNAs that inhibit HIF-1α
andHIF-2α, and the empty vector (EV) subclone (9). The BCCsubclones
were injected into the MFP of female SCID mice,and tumors were
harvested for flow cytometric analysis whenthey reached a volume of
450 mm3 (Fig. 3D). The recruitmentof TAMs and MDSCs to tumors
derived from DKD cells wassignificantly decreased compared with EV
tumors (Fig. 3 E andF and Fig. S2 C and D).We next examined the
effect of HIFs on macrophage re-
cruitment by treating tumor-bearing mice with digoxin to
inhibitHIF activity (30, 31). When tumors reached 200 mm3, mice
re-ceived daily i.p. injections of either saline or digoxin (2
mg/kg)for 7 d, followed by tumor excision and flow cytometric
analysis(Fig. 3G). Recruitment of TAMs and MDSCs was
significantlydecreased in primary tumors of digoxin-treated mice
(Fig. 3 Hand I and Fig. S2 E and F). Taken together, these results
dem-onstrate that HIFs play an important role in recruiting TAMsand
MDSCs to primary breast tumors.
Hypoxia and Coculture Induce HIF-Dependent CSF1 and
CSF1RExpression. Previous studies demonstrated reciprocal
paracrineinteractions in which BCCs secrete CSF1 and sense
EGF,whereas TAMs sense CSF1 and secrete EGF (16–18), but thetrigger
for CSF1 expression was not determined. CSF1 binds toits cognate
receptor, CSF1R, which is expressed by TAMs;however, CSF1R is also
expressed in >50% of breast tumors,
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s
Fig. 2. CXCL16 promotes lung and lymph node metastasis of
MDA-MB-231BCCs. BCC subclones were implanted in the MFP of SCID
mice. (A) Primarytumor volumes were determined serially. (B)
Circulating tumor cells in pe-ripheral blood were determined by
qPCR using primers specific for human18S rRNA and normalized to
NTC. *P < 0.01 vs. NTC. (C) Lung DNA wasanalyzed by qPCR with
primers specific for human HK2 sequences and nor-malized to NTC. *P
< 0.01 vs. NTC. (D) Photomicrographs of H&E-stained
lungsections. (Scale bars: 2 mm.) (E) Lung sections were scored for
metastatic fociper field. **P < 0.001 vs. NTC. (F) Lymph node
sections were stained with anantibody that specifically recognizes
human vimentin. (Scale bars: 0.5 mm.) (G)Stained area was
quantified by image analysis. **P < 0.001 vs. NTC.
0 0.5
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Tumor FACS
F4/80CSF1R
CD11bLy6C
MonocyticMDSCs
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MonocyticMDSCs
M
F4/80CSF1R
CD11bLy6C
FACSTumor
Saline Digoxinor
HIF
7 days(200 mm )3
(450 mm )3
(450 mm )3
MFP
Tumor FACS
F4/80CSF1R
CD11bLy6C
MonocyticMDSCs
Fig. 3. Macrophage recruitment is dependent on HIF activity. (A)
1 × 106
MDA-MB-231 BCCs alone, 1 × 106 BCCs mixed with 1 × 106 MSCs
immediatelybefore injection, or 1 × 106 BCCs cocultured with 1 ×
106 MSCs for 48 h wereimplanted in the MFP of SCID mice. The
percentage of CSF1R+F4/80+ (B) andCD11b+Ly6C+ (C) cells in 450-mm3
primary tumors was determined by fluo-rescence-activated cell
sorting (FACS). *P < 0.05; **P < 0.01 vs. BCCs. (D)MDA-MB-231
EV or DKD cells were implanted in the MFP of SCID mice.
Thepercentage of CSF1R+F4/80+ (E) and CD11b+Ly6C+ (F) cells in
450-mm3 pri-mary tumors was determined by FACS. **P < 0.01 vs.
EV. (G) Mice bearing200-mm3 MDA-MB-231 tumors were treated with
saline or digoxin for 7 d.The percentage of CSF1R+F4/80+ (H) and
CD11b+Ly6C+ (I) cells in primarytumors was determined. **P <
0.01 vs. saline.
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et al.
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which suggests that CSF1 mediates both autocrine and
paracrinesignaling (14–18). Coculture of GFP-expressing human
MDA-MB-231 BCCs with human MSCs and flow sorting of GFP+
BCCs and CD105+ MSCs revealed that CSF1 mRNA expressionwas
induced in BCCs (Fig. 4A), whereas CSF1R mRNA ex-pression was
induced in both MSCs and BCCs (Fig. 4C). Ex-pression of both CSF1
and CSF1R was augmented in BCCssubjected to hypoxia (Fig. 4 A and
C).To investigate the role of HIFs in these phenomena, MSCs
were cocultured with the DKD or EV subclone of BCCs. Ex-pression
of CSF1 and CSF1R mRNA was significantly decreasedin DKD + MSC,
compared with EV + MSC, cocultures at both20% and 1% O2 (Fig. 4 B
and D). Levels of secreted CSF1protein were also significantly
decreased in CM isolated fromDKD + MSC, compared with EV + MSC,
cocultures (Fig. S3A).Pharmacological inhibition of HIF activity
using acriflavine, whichblocks the dimerization of HIF-1α or HIF-2α
with HIF-1β (43),inhibited coculture- and hypoxia-induced CSF1 and
CSF1RmRNA expression (Fig. 4 E and F). Taken together, these
resultsindicate that cross-talk between BCCs and MSCs is
mediatedthrough HIF-dependent CSF1→CSF1R signaling.
CSF1 Promotes Macrophage Recruitment and Metastasis. We
gen-erated MDA-MB-231 subclones that were stably transfected
withvector encoding either of two different shRNAs targeting
CSF1.Efficient knockdown of CSF1 mRNA expression and
proteinsecretion was confirmed by RT-qPCR and ELISA,
respectively(Fig. S3 B and C). Coculture-induced CSF1 secretion was
alsoabrogated (Fig. S3C). CSF1 deficiency had no effect on
primarytumor growth after MFP implantation (Fig. 5A). Mice
bearingtumors derived from CSF1-deficient BCCs had significantly
de-creased numbers of circulating tumor cells (Fig. 5B),
metastaticcancer cells (Fig. 5C) and metastatic foci (Fig. 5 D and
E) in thelungs, metastatic cancer cells in the ipsilateral axillary
lymphnode (Fig. 5 F and G), and CSF1R+F4/80+ TAMs (Fig. 5H)
andCD11b+Ly6C+ MDSCs (Fig. 5I) recruited to the primary tumor.These
data demonstrate that CSF1 expression in BCCs playsa significant
role in promoting macrophage recruitment and me-tastasis to both
lymph nodes and lungs.
CCL5→CCR5 Signaling Between MSCs and BCCs Is Required for
CSF1Expression. Previous studies showed that CCL5→CCR5
signalingstimulates breast cancer metastasis (6) and that coculture
andhypoxia induce the expression of CCL5 in MSCs and its
cognate
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1R m
RN
A
20% O2
1% O2
MSCsMSCs
Veh ACF Veh ACF
*
#
##
*#
**
##
* *** **
A
D
E
B
0
50
100
150
200
**
##
0
10
20
30
40
50
BCCs MSCs BCCs MSCsCoculture
CSF
1R m
RN
A
20% O2
1% O2
EV DKD EV
MSCs
DKD
MSCs
MSCs+ +
**
**
20% O2
1% O2
20% O2
1% O2
CSF
1 m
RN
A
C
F
BCCs MSCs BCCs MSCsCoculture
**
BCCs
BCCsBCCs
+ MSCsBCCs + MSCs
Fig. 4. HIFs mediate coculture- and hypoxia-induced expression
of CSF1 andCSF1R. (A and C) GFP-expressing MDA-MB-231 BCCs were
cultured in-dividually or cocultured with MSCs at 20% or 1% O2 for
48 h, and GFP
+ BCCsand CD105+ MSCs were then sorted by FACS. CSF1 (A) and
CSF1R (C) mRNAlevels were determined and normalized to BCCs at 20%
O2. **P < 0.001 vs.20% BCCs; ##P < 0.001 vs. 1% BCCs. (B and
D) EV BCCs, DKD BCCs, MSCs, EV +MSCs or DKD + MSCs were cultured at
20% or 1% O2 for 48 h. CSF1 (B) andCSF1R (D) mRNA levels were
determined and normalized to EV at 20%. *P <0.05 vs. 20% EV; **P
< 0.001 vs. 1% EV; #P < 0.01 vs. 1% EV; ##P < 0.001 vs.1%
EV +MSCs. (E and F) BCCs, MSCs, or BCCs +MSCs were treated with 1
μMacriflavine (ACF) or DMSO vehicle (Veh) and exposed to 20% or 1%
O2 for48 h. CSF1 (E) and CSF1R (F) mRNA levels were normalized to
BCC Veh at20%. *P < 0.05 vs. Veh; **P < 0.001 vs. Veh.
0
200
400
600
800
1000
1200
1400
21 28 35 42 48 56
Days
NTC shCSF1-1 shCSF1-5
Tum
or v
olum
e (m
m3 )
0 10 20 30 40 50 60 70
BCCs
(% o
f LN
area
)
NTC shCSF1-1 shCSF1-5
NTC shCSF1-1 shCSF1-5
No.
of l
ung
foci
0 1 2 3 4 5 6 7 8 9
0 0.5
1 1.5
2 2.5
3 3.5
4 4.5
NTC shCSF1-1 shCSF1-5 NTC shCSF1-1 shCSF1-5
NTC shCSF1-1 shCSF1-5
0
0.2
0.4
0.6
0.8
1
1.2
NTC shCSF1-1 shCSF1-5
Circ
ulat
ing
tum
or c
ells
NTC shCSF1-1 shCSF1-5 0 2 4 6 8
10 12
A B
D E
F G
H I
** **
0
0.2
0.4
0.6
0.8
1
1.2 C
NTC shCSF1-1 shCSF1-5
* *
** **
****
***
***
% C
SF1R
+ F4/
80+ c
ells
% C
D11b
Ly6
C c
ells
++
Met
asta
tic B
CC
s in
lung
s
Fig. 5. CSF1 promotes metastasis of MDA-MB-231 BCCs.
MDA-MB-231subclones expressing shRNA against CSF1 (shCSF1-1 and
shCSF1-5) or NTCshRNA were implanted in the MFP of female SCID
mice. (A) Primary tu-mor volumes were determined serially. (B)
Circulating tumor cells weredetermined by qPCR and normalized to
NTC. *P < 0.01 vs. NTC. (C )Metastatic BCCs in the lungs were
determined by qPCR and normalizedto NTC. **P < 0.001 vs. NTC.
(D) Photomicrographs of H&E-stained lungsections. (Scale bars:
0.5 mm.) (E ) Lung sections were scored for metastaticfoci per
field. **P < 0.001 vs. NTC. (F) Lymph node sections were stained
withhuman-specific vimentin. (Scale bars: 0.5 mm.) (G) Stained
lymph node (LN)area was quantified by image analysis. **P <
0.001 vs. NTC. (H and I) Thepercentage of CSF1R+F4/80+ (H) and
CD11b+Ly6C+ (I) cells in primary tumorswas determined by FACS. *P
< 0.01 vs. NTC; **P < 0.001 vs. NTC.
Chaturvedi et al. PNAS | Published online May 5, 2014 |
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-
receptor CCR5 in BCCs (9, 44). However, neither the
upstreamtrigger nor the downstream effector of CCL5→CCR5
signalingwas delineated. To investigate whether CCL5→CCR5
signalingregulates CSF1 expression, CCL5 NAb was added to
coculturesof MSCs and BCCs. Expression of both CCL5 (Fig. 6A)
andCCR5 (Fig. 6B) mRNA was significantly decreased in the pres-ence
of CCL5 NAb. Blocking CCL5→CCR5 signaling also sig-nificantly
decreased CSF1 mRNA levels (Fig. 6C). Next, wegenerated MDA-MB-231
subclones stably transfected with vec-tor encoding either of two
different shRNAs, which inhibitedexpression of CCR5 mRNA (Fig. S4A)
and cell surface protein(Fig. S4B). CCR5 deficiency in BCCs blocked
the induction ofCSF1 mRNA expression by coculture or hypoxia (Fig.
6D). Re-markably, CSF1 deficiency in BCCs abrogated hypoxia- and
co-culture-induced CCL5 mRNA expression by MSCs (Fig. 6E).Taken
together, these results demonstrate that CSF1 expressionby BCCs is
regulated by CCL5MSC→CCR5BCC signaling and thatCCL5 expression by
MSCs is regulated by CSF1BCC→CSF1RMSCsignaling, indicating the
existence of a second feed-forward loop,which, in this case,
promotes the recruitment of TAMs andMDSCs to primary breast tumors
(Fig. 6F).
CCR5 Promotes Macrophage Recruitment and Metastasis. To
in-vestigate the role of CCR5 in breast cancer pathogenesis
further,CCR5-deficient BCCs were implanted in the MFP of femaleSCID
mice. CCR5 deficiency had no effect on primary tumorgrowth (Fig.
7A). However, mice bearing CCR5-deficient tumorsshowed
significantly decreased numbers of circulating tumor cells(Fig.
7B), metastatic cancer cells (Fig. 7C) and metastatic foci(Fig. 7 D
and E) in the lungs, metastatic cancer cells in lymphnodes (Fig. 7
F and G), and TAMs (Fig. 7H) and MDSCs (Fig.7I) recruited to the
primary tumor. These results indicate thatCCR5 expression by BCCs
promotes macrophage recruitmentand metastasis.
CSF1 and CCR5 Are HIF Target Genes. Analysis of the human
CSF1gene sequence revealed two candidate HIF binding sites
located0.6 kb (site 1) and 2.5 kb (site 2) 5′ to the transcription
start site(Fig. 8A). To determine whether HIFs bind to these sites,
ChIPassays were performed with MDA-MB-231 BCCs, which dem-onstrated
hypoxia-induced binding of HIF-1α (Fig. 8B), HIF-1β(Fig. 8C), and
HIF-2α (Fig. 8D) to site 1 and hypoxia-inducedbinding of HIF-1α
(Fig. 8E) and HIF-1β (Fig. 8F), but not HIF-2α (Fig. 8G), to site
2.
- MSCs +MSCs
0 2 4 6 8
10 12 14 16 18 20
0
5
10
15
20
25
-MSCs +MSCs
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
CCL5 NAb CCL5 NAb
20% O2 1% O2
IgG IgG
CC
R5
mR
NA
CS
F1 m
RN
A
20% O2 1% O2
NTC shCCR5-4 shCCR5-5 NTC shCCR5-4 shCCR5-5 NTC shCSF1-1
shCSF1-5 NTC shCSF1-1 shCSF1-5
CS
F1 m
RN
A
CC
L5 m
RN
A20% O2 1% O2
20% O2 1% O2
B
0
1
2
3
4
5
CC
L5 m
RN
A
CCL5 NAbIgG
20% O2 1% O2
**
##
*
*
# #
**
## ##
**
## ##*
# #
E
A C
D
BCC MSC
CCL5
TAM
CSF1
CSF1R
CCR5
homing to BCC
CSF1R
F
TAM
Fig. 6. CCL5-CCR5 signaling between MSCs and BCCs is required
for CSF1expression. (A–C) MDA-MB-231 BCCs +MSCs were treated with
CCL5 NAb orIgG and exposed to 20% or 1% O2 for 48 h. CCL5 (A), CCR5
(B), and CSF1 (C)mRNA levels were normalized to 20% IgG. *P <
0.01 vs. 20% IgG; **P < 0.001vs. 20% IgG; #P < 0.01 vs. 1%
IgG; ##P < 0.001 vs. 1% IgG. (D) MDA-MB-231subclones expressing
NTC shRNA or shRNA against CCR5 (shCCR5-4 andshCCR5-5) were
cultured alone or with MSCs and exposed to 20% or 1% O2for 48 h.
CSF1 mRNA levels were normalized to 20% NTC. *P < 0.05 vs.
20%NTC; **P < 0.001 vs. 20% NTC; #P < 0.05 vs. 1% NTC; ##P
< 0.001 vs. 1% NTC +MSCs. (E) MDA-MB-231 subclones expressing
NTC shRNA or shRNA againstCSF1 (shCSF1-1 and shCSF1-5) were
cultured alone or with MSCs and exposedto 20% or 1% O2 for 48 h.
CCL5 mRNA levels were normalized to 20% NTC.**P < 0.001 vs. 20%
NTC + MSCs; ##P < 0.001 vs. 1% NTC + MSCs. (F) Bi-directional
signaling between BCCs and MSCs generates a feed-forward loopthat
stimulates tumor-associated macrophage (TAM) recruitment.
0 0.5
1 1.5
2 2.5
3 3.5
4
0
200
400
600
800
1000
1200
21 28 35 42 49
Tum
or V
olum
e (m
m3
)
Days
*
NTC shCCR5-4 shCCR5-5
A B C
0
0.2
0.4
0.6
0.8
1
1.2
**
NTC shCCR5-4 C
ircul
atin
g tu
mor
cel
ls
0
0.2
0.4
0.6
0.8
1
1.2
shCCR5-5 NTC shCCR5-4 shCCR5-5
** **
D
0
1
2
3
4
5
6
7
NTC shCCR5-4 shCCR5-5
H
NTC shCCR5-4 shCCR5-5
* * **
NTC shCCR5-4 shCCR5-5 E
FG
0
2
4
6
8
10
12
NTC shCCR5-4 shCCR5-5
** **
No.
of l
ung
foci
I
NTC shCCR5-4 shCCR5-5
0
10
20
30
40
50
60
70
NTC shCCR5-4 shCCR5-5
****
BC
Cs
(% o
f LN
are
a)
% C
SF1R
+ F4/
80+ c
ells
% C
D11b
Ly6
C c
ells
++
Met
asta
tic B
CC
s in
lung
s
Fig. 7. CCR5 promotes macrophage recruitment and breast cancer
metas-tasis. MDA-MB-231 subclones were implanted in the MFP of SCID
mice. (A)Primary tumor volumes were determined serially. (B)
Circulating tumor cellswere determined by qPCR and normalized to
NTC. *P < 0.05 vs. NTC. (C)Metastatic BCCs in the lungs were
determined by qPCR. **P < 0.001 vs. NTC.(D) Photomicrographs of
H&E-stained lung sections. (Scale bars: 0.5 mm.) (E)Lung
sections were scored for metastatic foci per field. **P < 0.001
vs. NTC.(F and G) Lymph node sections were stained with
human-specific vimentinantibody (F), and staining was quantified by
image analysis (G). **P < 0.001vs. NTC. (Scale bars: F, 0.5 mm.)
The percentage of CSF1R+F4/80+ (H) andCD11b+Ly6C+ (I) cells in
primary tumors was determined by FACS. *P < 0.01vs. NTC.
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Analysis of the human CCR5 gene sequence revealed candi-date HIF
binding sites in the 5′-flanking and 3′-untranslated regions,1,370
bp 5′ (site 1) and 8,065 bp 3′ (site 2) of the transcription
startsite, respectively (Fig. 8H). ChIP assays revealed
hypoxia-inducedbinding of HIF-2α and HIF-1β, but not HIF-1α, to
site 1 (Fig. 8 I–K)and hypoxia-induced binding of HIF-1α and
HIF-1β, but not HIF-2α, to site 2 (Fig. 8 L–N). Taken together, the
ChIP data demon-strate that the human CSF1 and CCR5 genes are
directly regulatedby both HIF-1 and HIF-2.
CCL5 and CSF1 Stimulate Macrophage Migration. To study
macro-phage migration directly, we isolated bone marrow cells
fromBALB/c mice and incubated them in the presence of CSF1
tostimulate macrophage (BM-Mϕ) differentiation, with CSF1R+F4/80+
cells representing 80% of the final population (Fig. 9A).CM from
BCCs +MSCs cocultured at 20% O2 stimulated BM-Mϕmigration, and the
effect was augmented when CM from hypoxiccocultures was used (Fig.
9B). The stimulatory effect of CM fromcocultures was abrogated when
CCL5 NAb was added to the CM(Fig. 9C) or when CSF1-deficient BCCs
were used (Fig. 9D). These
results indicate that coculture- and hypoxia-induced CCL5
andCSF1 expression stimulate BM-Mϕ migration.
HIFs Are Required for Macrophage Recruitment and Metastasis ina
Syngeneic Mouse Mammary Carcinoma Model. To investigate therole of
HIFs in macrophage recruitment in an immunocompe-tent model of
TNBC, we used 4T1 mouse mammary carcinomacells, which form primary
tumors and metastases similar to hu-man TNBC after implantation
into the MFP of syngeneic BALB/cmice (45). We generated 4T1
subclones that were stably trans-fected with vectors encoding NTC
shRNA or shRNA(s) thatinhibited the expression of HIF-1α (sh1α),
HIF-2α (sh2α), or both
0
1
2
3
4
5
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6
IgG HIF2
0 0.5 1
1.5 2
2.5 3
3.5 4
IgG HIF1
E1 E2 E3 E4 E5 E6 E9 E7 E8
CSF1 ~600 nt~2500 nt
*
*
Site 1Site 2
E1 E2 E3
CCR5 -1370 nt
Site 1
+8065 nt
Site 2
**
0 0.2 0.4 0.6 0.8
1 1.2 1.4
0
1
2
3
4
5
6 *
0
1
2
3
4
5 20% O2 1% O2
*
*
0
1
2
3
4
5
Bind
ing
at s
ite 2
20% O2 1% O2
IgG HIF1B
indi
ng a
t site
2*
Bind
ing
at s
ite 2
Bin
ding
at s
ite 1
Bind
ing
at s
ite 1
Bin
ding
at s
ite 1
HIF10
0.5 1
1.5 2
2.5 3
3.5
0
1
2
3
4
5
IgG HIF1
Bin
ding
at s
ite 1
Bind
ing
at s
ite 1
20% O2 1% O2
IgG
20% O2 1% O2
HIF2IgG
Bind
ing
at s
ite 1
A
B C D
E G
H
F
I J K
L M N
0 1 2 3 4 5 6 7 8 9
0 1 2 3 4 5 6 7 20% O2
1% O2 **
Bin
ding
at s
ite 2
Bin
ding
at s
ite 2
Bin
ding
at s
ite 2
20% O2 1% O2
*
20% O2 1% O2
20% O2 1% O2
20% O2 1% O2
IgG HIF1 IgG HIF1 IgG HIF2
IgG HIF1 IgG HIF1 IgG HIF2
20% O2 1% O2
20% O2 1% O2
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6 20% O2
1% O2
Fig. 8. CSF1 and CCR5 are HIF target genes. (A) Candidate HIF
binding siteswere identified in the 5′-flanking region of the human
CSF1 gene. (B–G)MDA-MB-231 cells were incubated at 20% or 1% O2 for
16 h, and ChIP assayswere performed using IgG or antibody against
HIF-1α, HIF1β, or HIF-2α.Primers flanking site 1 and site 2 were
used for qPCR, and values werenormalized to IgG 20%. *P < 0.05
vs. all other conditions, Student’s t test onlog-converted values.
(H) Candidate HIF binding sites were identified in the5′-flanking
region (site 1) and 3′-untranslated region (site 2) of the
humanCCR5 gene. (I–N) MDA-231 cells were incubated at 20% or 1% O2
for 16 h,and ChIP assays were performed using IgG or antibodies
against HIF-1α, HIF-1β, or HIF-2α. Primers flanking site 1 or site
2 were used for qPCR, and valueswere normalized to IgG 20%. *P <
0.05 vs. all other conditions; **P < 0.001.
BCCs
MSCs
BCCs + MSCs
20% O2 1% O2
CM: 20% O2 1% O2
0
5
10
15
20
BCCs MSCs
CCL5 NAb
IgG Ab
CCL5 NAb
BCCs
0
5
10
15
BCCs IgG
BCCs+MSCsCCL5 NAb
20% O2 1% O2
A
B C
*
**
#
*
**
#
##
IgG Ab
CM:
NTC shCSF1-1 NTC shCSF1-1
-MSCs +MSCs
BCCs + MSCs
BCCs + MSCs
CM: 20% O2 1% O2
BCCs + MSCs
CM:
20% O2
1% O2
BM
79.5%
BM-MΦ
F4/80
0 2 4 6 8
10 12 14 16 18
NTC shCSF1-1 NTC shCSF1-1
**
#
##
*
Mig
ratio
n of
BM
-Mϕ
Mig
ratio
n of
BM
-Mϕ
Mig
ratio
n of
BM
-Mϕ 20
-MSCs +MSCs
CS
F1R
+
+
CM:
0.03%
D
Fig. 9. Coculture- and hypoxia-induced CCL5 and CSF1 expression
is re-quired for migration of bone marrow-derived macrophages
(BM-Mϕ). (A)Bone marrow cells were isolated from the BALB/c mice
and differentiatedinto macrophages (BM-Mϕ) by supplementing the
culture media with CSF1.The efficiency of differentiation was
determined by FACS analysis of CSF1Rand F4/80 surface antigen
expression, using antibodies conjugated to phy-coerythrin (PE-A) or
allophycocyanin (APC-A). (B) BM-Mϕ were seeded in theupper
compartment of a Boyden chamber, and the number of cells
thatmigrated through the filter in response to CM from nonhypoxic
(20% O2) orhypoxic (1% O2) MDA-MB-231 cells (BCCs), MSCs, or BCCs +
MSCs in thelower compartment were counted under light microscopy
after stainingwith crystal violet. (Scale bars: 0.2 mm.) Data were
normalized to CM fromBCCs at 20% O2. *P < 0.05 vs. 20% BCCs; **P
< 0.01 vs. 20% BCCs;
#P < 0.01vs. 20% BCCs + MSCs. (C) BM-Mϕ migration in response
to CM isolated fromBCCs or BCCs + MSCs (supplemented with IgG or
CCL5 NAb) was determinedand normalized to CM from BCCs at 20% O2.
(Scale bars: 0.2 mm.) *P < 0.05vs. 20% BCCs; **P < 0.01 vs.
20% BCCs; #P < 0.05 vs. 20% IgG; ##P < 0.001 vs.1% IgG. (D)
BM-Mϕ migration in response to CM isolated from NTC orshCSF1-1 BCCs
(alone or cocultured with MSCs) was determined and nor-malized to
CM from NTC cells at 20% O2. (Scale bars: 0.2 mm.) *P < 0.05
vs.20% NTC − MSCs; **P < 0.01 vs. 20% NTC − MSCs; #P < 0.05
vs. 1% NTC -MSCs; ##P < 0.001 vs. 1% NTC + MSCs.
Chaturvedi et al. PNAS | Published online May 5, 2014 |
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-
0
200
400
600
800
1000
1200
5 10 15 20
* *
*
*
Tum
or V
olum
e (m
m )3
0 2 4 6 8
10 12 14 16 18 20
0
5
10
15
20
25
* *
*
*
** 0 2 4 6 8
10 12 14 16
****
*
No.
of L
N fo
ci
A
C
0
5
10
15
20
***
F
Days
0
0.5
1
1.5
2
2.5
Tum
or w
eigh
t (g)
****
B
D
No.
of l
ung
met
asta
ses
E
% C
SF1R
+ F4/
80+ c
ells
% C
D11b
Ly6
C c
ells
++
sh1sh2
sh2sh1
NTC
DKD
NTC DKD
DKD
DKDNTC
NTC
sh1
sh1
sh2
sh2
sh2sh2
DKDDKD
NTCNTC sh1
sh1
sh2 DKDNTC sh1 sh2 DKDNTC sh1
Fig. 10. HIFs are required for macrophage recruitment and
metastasis in the immunocompetent 4T1 mouse mammary carcinoma model
of TNBC. (A) 4T1subclones expressing NTC shRNA or shRNA against
HIF-1α (sh1α), HIF-2α (sh2α), or both HIF-1α and HIF-2α (DKD) were
implanted in the MFP of femalesyngeneic BALB/c mice. Primary tumor
volumes were determined serially. *P < 0.05 vs. NTC or sh2α. (B)
Tumor weights were measured at the end of theexperiment. **P <
0.01 vs. NTC. (C) Whole-mount inflated lungs were stained with
India blue dye. The number of metastatic nodules (arrows) was
counted.*P < 0.05 vs. NTC; **P < 0.01 vs. NTC. (D) Lymph node
sections were stained with H&E. (Scale bars: 2 mm.) The number
of metastatic foci (arrows) was counted.*P < 0.05 vs. NTC; **P
< 0.01 vs. NTC. (E and F) The percentage of CSF1R+F4/80+ (E) and
CD11b+Ly6C+ (F) cells in primary tumors was determined by FACS. *P
<0.05 vs. NTC; **P < 0.01 vs. NTC.
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(DKD) (Fig. S5A). After orthotopic implantation in the MFP
offemale BALB/c mice, the growth of primary tumors derived fromsh1α
or DKD cells was significantly decreased compared withtumors
derived from sh2α or NTC cells (Fig. 10 A and B). Thenumbers of
metastatic nodules in lungs (Fig. 10C) and metastaticfoci in
axillary lymph nodes (Fig. 10D) harvested from miceimplanted with
sh1α, sh2α, or DKD cells were significantlydecreased compared those
from mice implanted with NTCcells. Recruitment of TAMs (Fig. 10E
and Fig. S5B) and MDSCs(Fig. 10F and Fig. S5C) was significantly
decreased in primarytumors derived from sh1α and DKD cells compared
with tu-mors derived from sh2α or NTC cells. Taken together, these
dataindicate that HIFs are required for primary tumor
growth,macrophage recruitment, and metastasis of TNBCs in
immuno-competent mice.
Breast Cancer Gene Expression Data Suggest Signaling Pathways
AreClinically Relevant. To determine whether the two
bidirectionalsignaling loops that were delineated in BCC-MSC
cocultures areclinically relevant, we analyzed microarray gene
expression dataobtained from 530 primary human breast cancers, 63
samples ofadjacent normal breast tissue, and 6 metastases (2). We
dem-onstrated that CXCR3 expression (and activation) in BCCs
wasrequired for the induction of CXCL16 expression in BCCs
inresponse to coculture with MSCs and exposure to hypoxia (Fig.
1),and Pearson’s test revealed that CXCR3 and CXCL16 mRNAlevels
were highly correlated (P = 10−13) in the clinical
specimens.Similarly, we demonstrated that CCR5 expression (and
activation)in BCCs was required for the induction of CSF1
expression inBCCs in response to coculture and hypoxia (Fig. 6),
and Pearson’stest revealed that CCR5 and CSF1 mRNA levels were
highlycorrelated (P = 10−16) in the clinical specimens.
DiscussionIn this study, we have delineated two HIF-regulated
feed-forwardsignaling loops between BCCs and MSCs that drive
stromal cellrecruitment to primary breast tumors (Fig. 11). The
first loop, in-volving CXCL16BCC→CXCR6MSC and
CXCL10MSC→CXCR3BCCsignaling arms, with each arm stimulating the
activity of theother, drives recruitment of MSCs. The second loop,
involvingCCL5MSC→CCR5BCC and CSF1BCC→CSF1RMSC signalingarms, with
each arm stimulating the activity of the other,drives recruitment
of TAMs and MDSCs. These two pathwaysoperate independent of each
other, but both are dependent onHIF activity, with hypoxia serving
as a physiological signal thatsynergizes with chemokine signals
from MSCs to trigger CSF1and CCR5 gene transcription in BCCs.The
recruitment of MSCs promotes cancer progression and
metastasis (6–9). In a previous study, we showed that PGF
se-cretion by BCCs facilitates the recruitment of MSCs to
breasttumors and that PGF binds to VEGF receptor 1 (VEGFR1) onMSCs
to stimulate CXCL10 expression (9). In the present study,we have
demonstrated that in addition to PGF, CXCL16 secre-tion by hypoxic
BCCs recruits MSCs to primary breast tumors.Furthermore, whereas
PGFBCC→VEGFR1MSC signaling is unidi-rectional, we show that
CXCL16BCC→CXCR6MSC signaling ispart of a bidirectional feed-forward
loop that activates CXCR3signaling in BCCs (Fig. 11), which is
proinvasive and prometastatic(9). As a result, CXCL16 deficiency
markedly impaired lymphnode and lung metastasis of BCCs.Elegant
studies have demonstrated that CSF1 expression by
BCCs promotes macrophage recruitment and metastasis inmouse
models of breast cancer (15–18). Elevated serum levels ofCSF1
predict lymph node involvement in women with early- stagebreast
cancer and decreased overall survival in postmenopausalpatients
with breast cancer (46). However, the mechanisms bywhich CSF1
expression is induced in BCCs had not been de-lineated. Similarly,
CCL5MSC→CCR5BCC signaling was linked toinvasion and metastasis (6,
47), but the upstream trigger anddownstream effector had not been
delineated. We have nowunified these observations by showing that
CCL5MSC→CCR5BCCsignaling induces CSF1 expression in BCCs, which
serves torecruit CSF1R+ TAMs, as well as feeding back to
stimulateCCL5 expression by MSCs (Fig. 6H).HIF activity in cancer
cells plays critical roles in the pro-
duction of angiogenic growth factors and the mobilization ofbone
marrow-derived angiogenic cells, which are blocked bytreating
tumor-bearing mice with HIF inhibitors (43). Treatmentof mice
bearing primary breast tumors with acriflavine or digoxinpotently
inhibits metastatic niche formation and blocks lungand lymph node
metastasis (30, 31, 48). Hypoxia induces HIF-dependent expression
of genes in BCCs that activate cell motility(34), ECM remodeling
(32, 33), lymphangiogenesis, and lymphnode metastasis (31), as well
as extravasation and lung metastasis(30). The present study now
identifies recruitment of MSCs,TAMs, and MDSCs as additional
components of the metastaticprocess that are stimulated by
intratumoral hypoxia in an HIF-dependent manner. Finally, both
human MDA-MB-231 andmouse 4T1 cells are preclinical models for the
15% of humanbreast cancers, designated as TNBC, that do not express
pro-gesterone, estrogen, or HER2 receptors and manifest the
basal/claudin-low gene expression pattern. Further studies are
re-quired to determine whether these same HIF-driven
intercellularsignaling mechanisms are also activated by hypoxia in
estrogen/progesterone receptor-positive and HER2+ breast
cancers.Targeted therapies are not available for TNBCs, which
are
treated with cytotoxic chemotherapy to which
-
digoxin, blocked signaling and recruitment of TAMs and
MDSCs,suggesting that addition of HIF inhibitors to existing
thera-peutic regimens may improve the clinical outcome in
patientswith TNBC.
Materials and MethodsCell Lines and Culture. Mycoplasma-free and
molecularly authenticated hu-man MDA-MB-231 BCCs were maintained in
high-glucose (4.5 mg/mL)DMEMwith 10% FBS and 1%
penicillin/streptomycin. The 4T1mouse mammarycarcinoma cells
(American Type Culture Collection) were maintained in RPMI-1640
with 10% FBS and 1% penicillin/streptomycin. Human bone
marrow-derived MSCs (49) were obtained from the Tulane Center for
Gene Therapy.MSCs were maintained in α-MEM supplemented with 20%
FBS and 1%penicillin/streptomycin. Cells were maintained at 37 °C
in a 5% CO2 and 95%air incubator (20% O2). Hypoxic cells were
maintained at 37 °C in a modularincubator chamber
(Billups–Rothenberg) flushed with a gas mixture containing1%O2, 5%
CO2, and 94% N2. For coculture experiments, equal numbers of
MSCsand BCCs were seeded in a 1:1 ratio of DMEM/10% FBS and
α-MEM/20% FBS.
Transduction with shRNA Vectors. The pLKO.1-puro lentiviral
vectors encodingshRNA targeting human CSF1 (clone ID: NM_000757),
human CCR5 (clone ID:NM_000579), mouse HIF-1α (clone ID:
NM_010431), and mouse HIF-2α (cloneID: NM_010137) were purchased
from Sigma–Aldrich. The recombinant vectorswere cotransfected with
plasmid pCMV-dR8.91 and plasmid encoding vesicularstomatitis virus
G protein into 293T cells using FuGENE 6 (Roche Applied Sci-ence).
Viral supernatant was collected 48 h posttransfection, filtered
(0.45-μmpore size), and added to MDA-MB-231 cells in the presence
of 8 μg/mL poly-brene (Sigma–Aldrich). Puromycin (0.5 μg/mL) was
added to the medium ofcells transduced with pLKO.1-puro vectors for
selection.
RT-qPCR. Total RNA was extracted from cells using TRIzol
(Invitrogen) andtreated with DNase I (Ambion). One microgram of
total RNA was used forfirst-strand DNA synthesis with the iScript
cDNA Synthesis system (BioRad).qPCR was performed using
human-specific primers and SYBR Green qPCRMasterMix (Fermentas).
For each primer pair, the annealing temperaturewasoptimized by
gradient PCR. The expression of each target mRNA relative to18S
rRNA was calculated based on the threshold cycle (Ct) as 2−Δ(ΔCt),
whereΔCt = Cttarget − Ct18S and Δ(ΔCt) = ΔCttest − ΔCtcontrol (22).
Primer sequencesare provided in Table S1.
Animal Studies. Female 5- to 7-wk-old SCID (National Cancer
Institute) orBALB/c (Charles River Laboratories) mice were studied
according to protocolsapproved by the Johns Hopkins University
Animal Care and Use Committeethat were in accordance with the
National Institutes of Health Guide for theCare and Use of
Laboratory Animals (50). Digoxin and saline for injectionwere
obtained from the research pharmacy of the Johns Hopkins
Hospital.BCCs were harvested by trypsinization, washed twice in
PBS, counted, andsuspended at 107 cells/mL in a 1:1 solution of PBS
and Matrigel (Corning).Mice were anesthetized, and 2 × 106 cells
were injected into the MFP. Pri-mary tumors were measured in three
dimensions (a, b, and c), and volume (V)was calculated as V = abc ×
0.52. Primary tumors and the ipsilateral axillarylymph node were
harvested (31). Lungs were perfused with PBS, and onelung was
inflated for formalin fixation, paraffin embedding, and
stainingwith H&E; the other lung was used to isolate genomic
DNA for qPCR toquantify human HK2 and mouse 18S rRNA gene
sequences, as previouslydescribed (30).
Circulating Tumor Cell Assay. Total RNA isolated from 0.5 mL of
whole bloodwas subjected to qPCR using primers specific for human
18S rRNA (31).
MSC Recruitment Assay. 2 × 106 MDA-MB-231 cells were implanted
in theMFP of female SCID mice. When the tumor reached a volume of
450 mm3,5 × 105 MSCs (derived from a male donor) were injected i.v.
and the primary
tumor was harvested after 16 h. MSCs recruited to the primary
tumor weredetermined by qPCR analysis of SRY copy number (9).
Flow Cytometry. Tumor tissue was minced and digested with 1
mg/mL type 1collagenase (Sigma) at 37 °C for 30 min. Digested
tissues were filtered through70-μm cell strainers. Cells were
incubated with Fc Block (BD Pharmingen). Toidentify TAMs, cells
were stained with peridinin chlorophyll protein-conju-gated CSF1R
antibody (BD Biosciences) and allophycocyanin-conjugated
F4/80antibody (Novus Biologicals), and subjected to flow cytometry.
To identifyMDSCs, cells were stained with
allophycocyanin-conjugated CD11b antibody(BD Biosciences) and
peridinin chlorophyll protein-conjugated Ly6C antibody(BD
Biosciences), and subjected to flow cytometry. Unstained control
andsingle-stained cells were prepared in every experiment for
gating. Dead cellswere gated out by side-scatter and
forward-scatter analysis.
Isolation of Mouse Bone Marrow-Derived Macrophages. Bone marrow
cellswere isolated and cultured for 10 d in RPMI-1640 supplemented
with 10%FBS, 1% penicillin/streptomycin, and 100 ng/mL CSF1
(R&D Systems) accordingto standard protocols (51, 52).
Migration Assays. Cells were seeded onto an uncoated filter in
the uppercompartment of a 24-well Boyden chamber (8-mm pore size;
Costar) andallowed to migrate for 8 h in response to CM in the
lower compartment. Thecells that migrated to the underside of the
filter were stained with crystalviolet and counted under
bright-field microscopy.
India Ink Staining of Lungs. Mice were euthanized, and India ink
(15%) wasinjected into the lungs through the trachea. The lungs
were fixed in Feketet’ssolution (100 mL of 70% alcohol, 10 mL of
formalin, and 5 mL of glacialacetic acid) at room temperature.
After de-staining, metastatic nodules appearwhite on a black
background (53).
Analysis of Lymph Node Metastasis. Immunohistochemical analyses
wereperformed on formalin-fixed, paraffin-embedded lymph node
sections aspreviously described (9). Staining was performed using
an antibody thatspecifically recognizes human vimentin (Santa Cruz
Biotechnology) and an-alyzed by ImageJ software (National
Institutes of Health). The acquiredimages in red-green-blue (RGB)
color were separated into different colorchannels by a color
deconvolution method (54).
ChIP Assays. MDA-MB-231 cells were cross-linked with
formaldehyde andlysed with SDS lysis buffer. Chromatin was sheared
by sonication, and lysateswere precleared with salmon sperm
DNA/protein A-agarose slurry (Millipore)and incubated with antibody
against HIF-1α (Santa Cruz Biotechnology),HIF-1β (Novus
Biologicals), or HIF-2α (Novus Biologicals), or with IgG (SantaCruz
Biotechnology or Novus Biologicals) as previously described
(9).
Statistical Analysis. All data are presented as mean ± SEM (n =
3). Differencesbetween groups were analyzed by one-way ANOVA,
unless otherwise stated,before normalization of data. Level 3 gene
expression data from 596 patientsin the Breast Invasive Carcinoma
dataset (2) were obtained from The CancerGenome Atlas Data Portal
(http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp).GraphPad Prism
software was used to calculate Pearson’s correlation coef-ficients
and determine P values for coexpression.
ACKNOWLEDGMENTS. We thank Karen Padgett of Novus Biologicals,
Inc.for providing IgG and antibodies against F4/80, HIF-1β, and
HIF-2α. We thankDarwin Prockop for providing human MSCs from the
Tulane Center for GeneTherapy, which was supported by Grant
P40-RR017447 from the National Insti-tutes of Health. G.L.S. is an
American Cancer Society Research Professor and theC. Michael
Armstrong Professor at Johns Hopkins University School of
Medicine.This work was supported by funds from the American Cancer
Society and Na-tional Cancer Institute. D.M.G. was supported by a
postdoctoral fellowship fromthe Susan G. Komen Foundation, and N.T.
was supported by the Japan Scienceand Technology Agency.
1. Marino N, et al. (2013) Breast cancer metastasis: Issues for
the personalization of itsprevention and treatment. Am J Pathol
183(4):1084–1095.
2. Cancer Genome Atlas Network (2012) Comprehensive molecular
portraits of humanbreast tumours. Nature 490(7418):61–70.
3. Bohl CR, Harihar S, Denning WL, Sharma R, Welch DR (2014)
Metastasis suppressors inbreast cancers: Mechanistic insights and
clinical potential. J Mol Med (Berl) 92(1):13–30.
4. Joyce JA, Pollard JW (2009) Microenvironmental regulation of
metastasis. Nat RevCancer 9(4):239–252.
5. Hanahan D, Coussens LM (2012) Accessories to the crime:
Functions of cells recruitedto the tumor microenvironment. Cancer
Cell 21(3):309–322.
6. Karnoub AE, et al. (2007) Mesenchymal stem cells within
tumour stroma promotebreast cancer metastasis. Nature
449(7162):557–563.
7. Mi Z, et al. (2011) Osteopontin promotes CCL5-mesenchymal
stromal cell-mediatedbreast cancer metastasis. Carcinogenesis
32(4):477–487.
8. El-Haibi CP, et al. (2012) Critical role for lysyl oxidase in
mesenchymal stem cell-drivenbreast cancer malignancy. Proc Natl
Acad Sci USA 109(43):17460–17465.
9. Chaturvedi P, et al. (2013) Hypoxia-inducible
factor-dependent breast cancer-mesenchy-mal stem cell bidirectional
signaling promotes metastasis. J Clin Invest 123(1):189–205.
10. Kelly PMA, Davison RS, Bliss E, McGee JO (1988) Macrophages
in human breast dis-ease: A quantitative immunohistochemical study.
Br J Cancer 57(2):174–177.
E2128 | www.pnas.org/cgi/doi/10.1073/pnas.1406655111 Chaturvedi
et al.
Dow
nloa
ded
by g
uest
on
June
22,
202
1
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406655111/-/DCSupplemental/pnas.201406655SI.pdf?targetid=nameddest=ST1http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jspwww.pnas.org/cgi/doi/10.1073/pnas.1406655111
-
11. Leek RD, Landers RJ, Harris AL, Lewis CE (1999) Necrosis
correlates with high vasculardensity and focal macrophage
infiltration in invasive carcinoma of the breast. Br JCancer
79(5-6):991–995.
12. Tsutsui S, et al. (2005) Macrophage infiltration and its
prognostic implications inbreast cancer: The relationship with VEGF
expression and microvessel density. OncolRep 14(2):425–431.
13. Medrek C, Pontén F, Jirström K, Leandersson K (2012) The
presence of tumor asso-ciated macrophages in tumor stroma as a
prognostic marker for breast cancer pa-tients. BMC Cancer
12:306.
14. O’Sullivan C, Lewis CE, Harris AL, McGee JO (1993) Secretion
of epidermal growthfactor by macrophages associated with breast
carcinoma. Lancet 342(8864):148–149.
15. Lin EY, Nguyen AV, Russell RG, Pollard JW (2001)
Colony-stimulating factor 1 pro-motes progression of mammary tumors
to malignancy. J Exp Med 193(6):727–740.
16. Wyckoff J, et al. (2004) A paracrine loop between tumor
cells and macrophages isrequired for tumor cell migration in
mammary tumors. Cancer Res 64(19):7022–7029.
17. Pollard JW (2004) Tumour-educated macrophages promote tumour
progression andmetastasis. Nat Rev Cancer 4(1):71–78.
18. Goswami S, et al. (2005) Macrophages promote the invasion of
breast carcinoma cellsvia a colony-stimulating factor-1/epidermal
growth factor paracrine loop. Cancer Res65(12):5278–5283.
19. Qian BZ, et al. (2011) CCL2 recruits inflammatory monocytes
to facilitate breast-tumourmetastasis. Nature
475(7355):222–225.
20. Harris AL (2002) Hypoxia—A key regulatory factor in tumour
growth. Nat Rev Cancer2(1):38–47.
21. Vaupel P, Mayer A, Höckel M (2004) Tumor hypoxia and
malignant progression.Methods Enzymol 381:335–354.
22. Sullivan R, Graham CH (2007) Hypoxia-driven selection of the
metastatic phenotype.Cancer Metastasis Rev 26(2):319–331.
23. Semenza GL (2012) Hypoxia-inducible factors: Mediators of
cancer progression andtargets for cancer therapy. Trends Pharmacol
Sci 33(4):207–214.
24. Schindl M, et al.; Austrian Breast and Colorectal Cancer
Study Group (2002) Over-expression of hypoxia-inducible factor 1α
is associated with an unfavorable prognosisin lymph node-positive
breast cancer. Clin Cancer Res 8(6):1831–1837.
25. Bos R, et al. (2003) Levels of hypoxia-inducible factor-1α
independently predict prognosisin patients with lymph node negative
breast carcinoma. Cancer 97(6):1573–1581.
26. Dales JP, et al. (2005) Overexpression of hypoxia-inducible
factor HIF-1α predicts earlyrelapse in breast cancer: Retrospective
study in a series of 745 patients. Int J Cancer116(5):734–739.
27. Generali D, et al. (2006) Hypoxia-inducible factor-1α
expression predicts a poor re-sponse to primary chemoendocrine
therapy and disease-free survival in primary hu-man breast cancer.
Clin Cancer Res 12(15):4562–4568.
28. Helczynska K, et al. (2008) Hypoxia-inducible factor-2α
correlates to distant re-currence and poor outcome in invasive
breast cancer. Cancer Res 68(22):9212–9220.
29. Wong CC, et al. (2011) Hypoxia-inducible factor 1 is a
master regulator of breastcancer metastatic niche formation. Proc
Natl Acad Sci USA 108(39):16369–16374.
30. Zhang H, et al. (2012) HIF-1-dependent expression of
angiopoietin-like 4 and L1CAMmediates vascular metastasis of
hypoxic breast cancer cells to the lungs.
Oncogene31(14):1757–1770.
31. Schito L, et al. (2012) Hypoxia-inducible factor 1-dependent
expression of platelet-derived growth factor B promotes lymphatic
metastasis of hypoxic breast cancer cells.Proc Natl Acad Sci USA
109(40):E2707–E2716.
32. Gilkes DM, et al. (2013) Collagen prolyl hydroxylases are
essential for breast cancer
metastasis. Cancer Res 73(11):3285–3296.33. Gilkes DM, et al.
(2013) Procollagen lysyl hydroxylase 2 is essential for
hypoxia-
induced breast cancer metastasis. Mol Cancer Res
11(5):456–466.34. Gilkes DM, et al. (2014) Hypoxia-inducible
factors mediate coordinated RhoA-ROCK1
expression and signaling in breast cancer cells. Proc Natl Acad
Sci USA 111(3):E384–E393.35. Murdoch C, Giannoudis A, Lewis CE
(2004) Mechanisms regulating the recruitment of
macrophages into hypoxic areas of tumors and other ischemic
tissues. Blood 104(8):
2224–2234.36. Jung Y, et al. (2013) Recruitment of mesenchymal
stem cells into prostate tumours
promotes metastasis. Nat Commun 4:1795.37. Matsumura S, et al.
(2008) Radiation-induced CXCL16 release by breast cancer cells
attracts effector T cells. J Immunol 181(5):3099–3107.38. Lin S,
et al. (2009) Chemokine C-X-C motif receptor 6 contributes to cell
migration
during hypoxia. Cancer Lett 279(1):108–117.39. Lewis C, Murdoch
C (2005) Macrophage responses to hypoxia: Implications for
tumor
progression and anti-cancer therapies. Am J Pathol
167(3):627–635.40. Murdoch C, Lewis CE (2005) Macrophage migration
and gene expression in response
to tumor hypoxia. Int J Cancer 117(5):701–708.41. Leek RD,
Harris AL (2002) Tumor-associated macrophages in breast cancer. J
Mammary
Gland Biol Neoplasia 7(2):177–189.42. De Palma M, Lewis CE
(2013) Macrophage regulation of tumor responses to anti-
cancer therapies. Cancer Cell 23(3):277–286.43. Lee K, et al.
(2009) Acriflavine inhibits HIF-1 dimerization, tumor growth, and
vas-
cularization. Proc Natl Acad Sci USA 106(42):17910–17915.44. Lin
S, et al. (2012) Chemokine C-C motif receptor 5 and C-C motif
ligand 5 promote
cancer cell migration under hypoxia. Cancer Sci
103(5):904–912.45. Aslakson CJ, Miller FR (1992) Selective events
in the metastatic process defined by
analysis of the sequential dissemination of subpopulations of a
mouse mammary
tumor. Cancer Res 52(6):1399–1405.46. Aharinejad S, et al.
(2013) Elevated CSF1 serum concentration predicts poor overall
survival in women with early breast cancer. Endocr Relat Cancer
20(6):777–783.47. Velasco-Velázquez M, et al. (2012) CCR5
antagonist blocks metastasis of basal breast
cancer cells. Cancer Res 72(15):3839–3850.48. Wong CC, et al.
(2012) Inhibitors of hypoxia-inducible factor 1 block breast
cancer
metastatic niche formation and lung metastasis. J Mol Med (Berl)
90(7):803–815.49. Colter DC, Class R, DiGirolamo CM, Prockop DJ
(2000) Rapid expansion of recycling
stem cells in cultures of plastic-adherent cells from human bone
marrow. Proc Natl
Acad Sci USA 97(7):3213–3218.50. Committee on Care and Use of
Laboratory Animals (1985) Guide for the Care and Use
of Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ.
No. (NIH) 85-23.51. Zhang X, Goncalves R, Mosser DM (2008) The
isolation and characterization of murine
macrophages. Curr Protoc Immunol Chapter 14:Unit 14. 1.52.
Weischenfeldt J, Porse B (2008) Bone Marrow-Derived Macrophages
(BMM): Isolation
and Applications. CSH Protoc 2008:pdb prot5080.53. Wang Y, et
al. (2011) Integrin subunits α5 and α6 regulate cell cycle by
modulating the
chk1 and Rb/E2F pathways to affect breast cancer metastasis. Mol
Cancer 10:84.54. Ruifrok AC, Johnston DA (2001) Quantification of
histochemical staining by color
deconvolution. Anal Quant Cytol Histol 23(4):291–299.
Chaturvedi et al. PNAS | Published online May 5, 2014 |
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