Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RAMesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557-563

Article (PDF Available)inNature 449(7162):557-63 · November 2007with177 Reads
Impact Factor: 41.46 · DOI: 10.1038/nature06188 · Source: PubMed
Abstract
Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.

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ARTICLES
Mesenchymal stem cells within tumour
stroma promote breast cancer metastasis
Antoine E. Karnoub
1
, Ajeeta B. Dash
2
, Annie P. Vo
1
, Andrew Sullivan
2
, Mary W. Brooks
1
, George W. Bell
1
,
Andrea L. Richardson
3
, Kornelia Polyak
4
, Ross Tubo
2
& Robert A. Weinberg
1
Mesenchymal stem cells have been recently des cribed to localize to breast carcinomas, where they integrate into the
tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour
pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells,
when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their
metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour
xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from
mesenchymal stem cells, which then acts in a paracrine fashio n on the cancer cells to enhan ce their motility, invasion and
metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine
receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by
eliciting reversible changes in the phenotype of cancer cells.
The origins of the invasive and metastatic phenotypes of carcinoma
cells have been the subjects of intense investigation. Whereas some
current models depict these phenotypes as cell-autonomous altera-
tions specified by the genomes of cancer cells, alternative views pro-
pose that metastatic traits are acquired through exposure of epithelial
cancer cells to paracrine signals that they receive from mesenchymal
cell types within the tumour-associated stroma. Although several
lines of evidence demonstrate the contributions of stromal cells to
primary tumour growth
1
, direct experimental demonstration of the
influence of these various cells on the metastatic abilities of cancer
cells has been difficult to obtain. This is due, in part, to the complexity
of the mesenchymal cell types that are recruited into the stroma, and
to the elusive nature of the putative paracrine signals that are
exchanged between the mesenchymal and epithelial compartments
of a tumour. Recent reports proposed that the bone-marrow-derived
mesenchymal stem cell (MSC) is a cell type that is recruited in large
numbers to the stroma of developing tumours
2
. To characterize bet-
ter the role of this stromal cell in tumorigenesis, we set out to deter-
mine whether MSCs could supply contextual signals that serve to
promote cancer metastasis.
Mesenchymal stem cells are pluripotent progenitor cells that con-
tribute to the maintenance and regeneration of a variety of connect-
ive tissues, including bone, adipose, cartilage and muscle
3
. Although
MSCs reside predominantly in the bone marrow, they are also dis-
tributed throughout many other tissues, where they are thought to
serve as local sources of dormant stem cells
4,5
. The contributions of
MSCs to tissue formation become apparent only in cases of tissue
remodelling after injury or chronic inflammation. These conditions
are typically accompanied by the release of specific endocrinal signals
from the injured or inflamed tissue that are then transmitted to the
bone marrow, leading to the mobilization of multi-potent MSCs and
their subsequent recruitment to the damage site
6
. For example, MSCs
have been shown to contribute to the formation of fibrous scars after
injury
7
.
The formation of breast carcinomas is often accompanied by a
well-orchestrated desmoplastic reaction, which involves the recruit-
ment of a variety of stromal cells with both pro- and anti-tumorigenic
activities
1
. Such response closely resembles wound healing and scar
formation, and entails the constant deposition of growth factors,
cytokines and matrix-remodelling proteins that render the tumour
site a ‘wound that never heals’
8
. This suggests that, similar to sites of
injury, actively growing tumours recruit MSCs through the release of
various endocrine and paracrine signals. Indeed, as we have found,
mouse stroma prepared from developing human MCF7/Ras or
MDA-MB-231 breast cancer xenografts is rich in cells with an ability
to generate fibroblastoid colony-forming units (CFU-F) in vitro
(Supplementary Fig. 1a), a hallmark of MSCs
3
. The absence of such
colonies from control Matrigel plugs or from neighbouring tissues
(negative control; Supplementary Fig. 1a) suggested that endogenous
murine MSCs localize specifically to sites of neoplasia.
To investigate whether human breast cancer cells also have the
ability to attract human MSCs, we established a transwell assay
in which bone-marrow-derived human MSCs were allowed to
migrate towards media derived from MCF7/Ras or MDA-MB-231
cultures. We found that human MSCs migrated much more avidly
(,11-fold more) towards media derived from these cancer cells
than towards control media (Supplementary Fig. 1b). More im-
portantly, green fluorescent protein (GFP)-labelled human MSCs
infused into the venous circulation of mice bearing MCF7/Ras
or MDA-MB-231 human breast cancer xenografts localized speci-
fically to the developing tumours, with no observable accumulation
in other tissues, such as the kidneys (Supplementary Fig. 1c), liver
and spleen (data not shown). Such findings indicated that MSCs
are specifically recruited by subcutaneous breast xenografts, and cor-
roborated recent studies that described the localization of system-
ically infused MSCs to other types of malignancy, such as gliomas
9,10
,
colon carcinomas
11,12
, ovarian carcinomas
13
, Kaposi’s sarcomas
14
and
melanomas
15
.
1
Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
2
Genzyme Corporation, Framingham,
Massachusetts 01701, USA.
3
Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA.
4
Department of Medical Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, Bo ston, Massachusetts 02115, USA.
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MSCs enhance breast cancer metastasis
To investigate the functional consequences of the heterotypic inter-
actions between MSCs and mammary carcinoma cells, we established
a xenograft model in which GFP-labelled MCF7/Ras, MDA-MB-231,
MDA-MB-435 and HMLER (see Methods) human breast cancer
cells (BCCs) were mixed with bone-marrow-derived human MSCs
(hereafter referred to as MSCs) and injected subcutaneously into
immunocompromised mice. The growth kinetics of the MSC-
containing tumours (BCCs plus MSCs) were compared to those of
BCCs injected alone (BCCs) over the subsequent 8–12 weeks, after
which the histopathology of the resulting tumours was studied.
We found that MSCs accelerated the growth of MCF7/Ras
tumours without affecting the kinetics of MDA-MB-231-, MDA-
MB-435- or HMLER-containing tumours (Fig. 1a). More impor-
tantly, whereas mice carrying tumours composed only of BCCs
exhibited few microscopic metastases in the lungs (Fig. 1b, d), mice
bearing the mixed MCF7/Ras1MSC, MDA-MB-2311MSC, MDA-
MB-4351MSC and HMLER1MSC tumours displayed a marked
increase in the numbers of micro- and macroscopic lung metastases
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs
of BCC1MSC-bearing mice compared to their BCC-control litter-
mates revealed two-, three-, four- and sevenfold enhancements in
the overall numbers of detectable HMLER, MDA-MB-435, MCF7/
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c).
Furthermore, in contrast to the MDA-MB-231-bearing mice, the
MDA-MB-2311MSC-bearing mice showed metastases to various
other tissues, including the mammary glands (Supplementary
Table 1). Although all four of the tested cell lines exhibited enhanced
metastatic potential after admixture of MSCs, we chose to focus
further analysis on the MDA-MB-231 tumour model, because it
displayed the greatest relative increase in MSC-induced metastasis
without any concomitant effect on either tumour cell proliferation
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall prim-
ary tumour growth kinetics.
We note that admixture of other types of mesenchymal cells—
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 3
and data not shown)—to MDA-MB-231 cancer cells before injection
into host mice did not result in either enhanced growth kinetics
(Supplementary Fig. 3a, b) or increased numbers of lung metastases
(Supplementary Fig. 3c, d). Taken together, these observations indi-
cated that the metastasis-enhancing powers were a specific property
of admixed MSCs or derivatives thereof.
Reversible metastasis
Implantation of MSCs either contralaterally to MDA-MB-231 cells or
in nearby separate sites of injection did not affect the metastatic
potential of the resulting primary tumours (data not shown), indi-
cating that MSCs could enhance cancer metastasis only when they
were in close proximity to the engrafted BCCs. This influence might
be ascribed to various effects that MSCs exert on the commingled
carcinoma cells. Thus, the MSCs might favour the outgrowth of rare
variants within the MDA-MB-231 cell populations that exhibit
unusually high metastatic powers. Alternatively, the MSCs might
cause otherwise weakly metastatic MDA-MB-231 cells to acquire
enhanced metastatic abilities. This latter mechanism suggests the
possibility that the acquisition of the metastatic phenotype might
be reversible, in that carcinoma cells might revert to a lower meta-
static state once they were no longer in close contact with MSCs.
To resolve between these two mechanisms, explants of MDA-MB-
231 cells were prepared from BCC plus MSC primary tumours (T-
explants) as well as from their derived lung metastases (L-explants),
expanded in vitro, cleared from contaminating stromal components,
and then re-injected into subcutaneous sites in host mice in order
to evaluate their respective metastatic powers (Fig. 2a). Although
the growth rate of the resulting L-explant primary tumours was
marginally enhanced compared to their T-explant counterparts
(Fig. 2b, c), these L-explant cells were no more metastatic than the
parental T-explant cancer cells (Fig. 2d). This suggested that the
a
dc
Days after injection
Tumour volume (mm
3
)
10 19 24 31 38 4610 17 24 31 38 46 72 80 89 13 19 26 33 40 47 54 61 68 75 81
b
1 mm
1 mm
1 mm1 mm
300 µm
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC
MCF7/Ras MCF7/Ras+MSC
100 µm
300 µm
100 µm
MDA-MB-231
MDA-MB-435
MCF7/Ras
**
Metastasis index (fold)
0
1
2
3
4
5
6
7
8
9
*
**
**
MSC + +–+–+
*
HMLER
700
1,200
1,000
800
600
400
200
0
800
700
600
500
400
300
200
100
0
3,500
0
500
1,000
1,500
2,000
2,500
3,000
600
500
400
300
200
100
0
0 2131384956637078
MCF7/Ras alone
MCF7/Ras+MSC
MDA-MB-231 alone
MDA-MB-231+MSC
MDA-MB-435 alone
MDA-MB-435+MSC
HMLER alone
HMLER+MSC
HMLER HMLER+MSC
MCF7/Ras MCF7/Ras+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC
Figure 1
|
MSCs promote breast cancer metastasis. a, Tumour volume
measurements (mean 6 s.e.m.) of 500,000 GFP-labelled BCCs injected
subcutaneously into nude mice with or without 1.5 3 10
6
MSCs.
Representative data from multiple experiments are shown. Diamonds, BCCs
alone, n 5 5–7 mice per group; squares, BCCs plus MSCs, n 5 5–8 mice per
group.
b, Representative bright-field/fluorescence images of lungs of mice
bearing the indicated tumours. Cancer colonies are in green. MCF7/Ras-
bearing mice were killed at approximately day 150 to allow these tumours to
grow to comparable sizes to their MCF7/Ras1MSC counterparts.
c, The
lung metastasis indices pooled within each cohort of mice in
a are expressed
as fold increase (6s.e.m.) over controls. Data shown are representative of
multiple repeats. Asterisk, P , 0.01, double asterisk, P , 0.05 using one-
tailed Student’s t-test.
d, Representative haematoxylin-and-eosin-stained
sections of lungs of mice bearing the indicated tumours. Metastases are
delineated by a dashed line.
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MSC-induced metastatic powers reflected a reversibly induced trait
of the MDA-MB-231 cells, and that the ability of these cells to metas-
tasize to the lungs was a consequence of their ‘education’ by MSCs in
the primary tumour rather than the selection of rare variants of
MDA-MB-231 cells that display elevated metastatic potency in a
stable fashion.
The effects that the MSCs exerted on the BCCs might have
occurred within the site of primary tumour formation. Alterna-
tively, the MSCs might have accompanied the metastasizing BCCs
to sites of metastasis formation. To distinguish between these two
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled
MDA-MB-231 cells and implanted the mixture subcutaneously in
host mice. We found that the tumour-derived lung metastases con-
tained green-labelled MDA-MB-231 cells but no detectable red-
labelled MSCs (or their derivatives; Supplementary Fig. 4a) when
scored 4, 5 or 6 weeks after primary tumour implantation. The
absence of red-labelled MSCs from the lung metastatic sites cannot
be ascribed to an inhospitable lung parenchyma, as MSCs that lodge
in the lungs of recipient animals after tail-vein infusion survive in that
environment for ,6 weeks after injection (Supplementary Fig. 4b).
Hence, it appeared that the admixed MSCs do not migrate in large
numbers to the sites of metastasis, and that they exerted their pro-
metastatic effects on BCCs in the context of primary tumours.
CCL5 in MSC-induced metastasis
The aforementioned observations indicate that MSCs supply locally
acting paracrine cues that induce BCCs within primary tumours to
metastasize. To understand this crosstalk better, in vitro co-cultures
of MDA-MB-231 breast cancer cells and MSCs were established and
their conditioned media were screened for the levels of various cyto-
kines, chemokines and growth factors using the Luminex-based Bio-
Plex suspension array system (Fig. 3a). In some cases, the resulting
a
bdc
Days after injection
Tumour volume
(× 100 mm
3
)
0
5
10
15
20
25
14 17 21 24 28 31 34 38 42 45 48 66 71 77
Primary tumour explants
Lung explants
MDA-MB-231
MSC
+
Lung explants
Primary tumour
explants
Antibiotic
BCC selection
T-e
xplant
L-explant
T-e
xplant
L-e
xplant
Tumour mass (g)
3.0
2.0
1.0
00
0.4
0.8
1.2
1.6
2.0
#
Metastasis index (fold)
##
Figure 2
|
MSC-induced increase in the metastasis of MDA-MB-231 cells
involves reversible mechanisms. a
, BCCs were recovered from lung or
primary tumour tissues, cleared of stromal contaminants by culture in
blasticidin-containing media (5 mgml
21
), and re-injected as primary
subcutaneous tumours in recipient animals.
b, Tumour growth
(means 6 s.e.m.)of 500,000 GFP-labelled lung-derived (L-explant) or primary
tumour-derived (T-explant) MDA-MB-231 cells inoculated subcutaneously.
Data shown are representative of multiple independent experiments in which
four different paired batches of L-explant and T-explant cultures were assayed
in parallel. MDA-MB-231-T-explant (n 5 8 mice); MDA-MB-231-L-explant
(n 5 10 mice).
c, Masses (means 6 s.e.m.)of tumours in b. Hash, P . 0.4 using
one-tailed Student’s t-test and indicates no statistical significance.
d,Lung
metastasis index of mice in
c.Doublehash,P . 0.3 using one-tailed Student’s
t-test and indicates no statistical significance.
a
CCL4
bFGF
VEGF
IFN-γ
TNF-α
G-CSF
GM-CSF
CCL3
CCL5
MMP1
MMP3
MMP9
MMP13
IL-1α
IL-1β
IL-4
IL-5
IL-6
IL-7
IL-8
IL-10
IL-12
IL-13
IL-17
IL-2
TGF-β
Fold induction
MSC alone
MDA alone
MDA+MSC (2:1 ratio)
*** *** **** *** ***
1
3
5
7
9
11
13
60
bc
0.4 µm
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
00
5
10
15
20
25
30
35
40
45
50
d1 d2 d3 d4
MDA alone
MSC alone
MDA+MSC
CCL5 levels (pg ml
–1
)
Co-culture
Fold CCL5 induction
MDA alone
MSC alone
MDA+MSC
d
CCL5 (A.U. × 100)
30
25
20
15
10
5
0
+
MSC.c
+
MSC.1
+
MSC.5
MDA
MSC
MDA.1
MDA.c
MDA.5
TC-MSC
MSC (from MDA tumour)
BCC (from MDA tumour)
Control (MDA/CCL5)
CCL5
GAPDH
e
Figure 3
|
The interaction of BCCs with MSCs causes a rise in the levels of
CCL5. a
, MDA-MB-231, MSCs, or MDA-MB-2311MSCs were cultured in
complete media for 3 days. The levels of various factors in the cell-free culture
supernatants were measured by xMAP Bio-Plex cytokine arrays at day 3, and
were normalized to the levels observed in the media of BCCs cultured alone.
Data are expressed as fold induction 6 s.d. of triplicates. Asterisk indicates
undetectable levels.
b, CCL5 ELISA on the media of MDA-MB-231, MSCs, or
MDA-MB-2311MSC cultures (1:3 MDA:MSCs) at the indicated time points.
Data points represent means 6 s.d. of quadruplicates.
c, BCCs were separated
from co-cultured MSCs by a 0.4-mm membrane. CCL5 levels were probed by
ELISA on the culture supernatants. Data are expressed as fold induction over
levels seen in MDA-MB-231 culture supernatants (mean 6 s.d. of triplicates).
d, CCL5 ELISA on the supernatants of MSC-siluc (MSC.c), MSC-siCCL5.1
(MSC.1) and MSC-siCCL5.5 (MSC.5) co-cultured with MDA-MB-231-siluc
(MDA.c), MDA-MB-231-siCCL5.1 (MDA.1), or MDA-MB-231-siCCL5.5
(MDA.5). Data are expressed as means 6 s.d. of triplicates in arbitrary units
(A.U.).
e, RT–PCR analyses of CCL5 in MSCs and BCCs sorted from
GFP–MSC1MDA-MB-231 tumours (3:1 ratio) 4 weeks after tumour
implantation. Tissue-cultured MSCs (TC-MSC) and MDA-MB-231/CCL5
cells were used as controls. GAPDH was used for equal loading.
NATURE
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levels of certain released factors (for example, interferon-c or
tumour-necrosis factor-a) reflected the additive contributions of
the two cell types when cultured on their own. Notably, the levels
of only one cytokine, CCL5, reflected a synergistic interaction
between the MSCs and BCCs, as it accumulated to levels ,60-fold
higher than those produced by pure BCC cultures (Fig. 3a). This
cooperative induction of CCL5 was proportional to the numbers of
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was appar-
ent as early as the third day of co-culture (Fig. 3b). Moreover, this
induction required close physical contact between MSCs and cancer
cells, because it failed to occur when the two cell populations were
separated by a permeable membrane (Fig. 3c).
We undertook to determine the source of the CCL5 produced
under conditions of co-culture. To do so, we stably reduced the
expression of CCL5 in MDA-MB-231 cells by .80% using short
hairpin (sh)RNA (variant siCCL5.1; Supplementary Fig. 6). Impor-
tantly, however, subsequent co-culture of these MDA-MB-231.1 cells
with MSCs continued to allow accumulation of CCL5 in the culture
supernatants to levels that were comparable to those observed in the
co-cultures of MSCs and control cancer cells (Fig. 3d). This suggested
that the source of CCL5 was the admixed neighbouring MSCs.
Indeed, inhibition of CCL5 protein expression in MSCs using the
same shRNA hairpin vector (MSC.1; Fig. 3d) resulted in more than
75% reduction of CCL5 protein levels in the co-cultures, indicating
that the MSCs were the major source of the CCL5 observed on co-
culture of the two cell types. In support of this conclusion, analysis of
CCL5 levels in the media of MSCs or MDA-MB-231 cells separated
from one another after 3 days of co-culture indicated a strong induc-
tion of CCL5 in the culture of MSCs, but not that of BCCs (Sup-
plementary Fig. 5b). Finally, polymerase chain reaction with reverse
transcription (RT–PCR) analysis of the RNA prepared from these co-
culture-derived MSCs (Supplementary Fig. 5c), as well as from the
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after
tumour implantation (Fig. 3e), indicated a strong accumulation of
CCL5 messenger RNA, suggesting that an active signal transduction
pathway is triggered in MSCs by the nearby BCCs.
A series of observations has linked CCL5 signalling and cancer. For
example, CCL5 levels in the plasma of breast cancer patients have
been correlated with the severity of the disease, and localized CCL5
protein expression was found to be elevated in invasive tumours
when compared to in situ ductal tumours or benign lesions
16,17
.
However, the precise contributions of CCL5 to cancer development
and progression are poorly understood. To investigate further the
possible causal role of CCL5 in cancer cell metastasis, we over-
expressed this chemokine in the MDA-MB-231 BCCs (Supple-
mentary Fig. 7a) and analysed its effects on cancer cell growth and
tumorigenesis. The overexpressed CCL5 did not confer any pro-
liferative advantage on cultured cancer cells when compared with
those lacking such overexpression (Supplementary Fig. 7b), and had
no effect on the ability of BCCs either to grow in an anchorage-
independent fashion in vitro (Supplementary Fig. 7c), or to form
primary subcutaneous tumours in immunocompromised mice (Fig.
4a). However, these tumours exhibited a ,5-fold enhancement in
their metastatic potential when compared with control tumours
lacking ectopic CCL5 (Fig. 4a). Similarly, overexpression of CCL5
in WI-38 fibroblasts sufficed to enable these cells to promote the
metastasis of admixed MDA-MB-231 BCCs (Fig. 4b), indicating that
the actions of CCL5 are responsible for much, if not all, of the observed
MSC-induced metastasis by the BCCs.
CCL5 promotes lung colonization
Previous reports have described an important role for CCL5 as a
chemoattractant for stromal cells, such as macrophages, that express
one of the receptors for CCL5, CCR5 (refs 18, 19). Furthermore,
CCL5 expression has been associated with increased tumour neovas-
cularization, suggesting that endothelial cells, which express a variety
of chemokine receptors, may also be attracted by CCL5 to sites of
tumour formation, thereby enhancing tumour angiogenesis
20
. Such
observations suggest that CCL5 may contribute to breast cancer
metastasis through the recruitment of a number of stromal cell types
to sites of primary tumour growth.
However, immunohistochemical analyses indicated that the
MDA-MB-231 control and CCL5-overexpressing MDA-MB-231
(MDA-MB-231/CCL5) tumours exhibited comparable numbers of
tumour-infiltrating macrophages and had similar vessel densities (as
evident by F4/80 and MECA-32 staining for macrophages and
endothelial cells, respectively; Supplementary Fig. 8). In addition,
we found that ectopic CCL5 expression did not cause an accumulation
of other stromal cells, such as SMA-positive cells, in the examined
tumours (Supplementary Fig. 8a). Together, these data indicated that
the observed CCL5-induced metastasis could not be attributed to
significant effects on the numbers of the major constituents of the
stroma or to the vascularity of these tumour xenografts.
Invasion and metastatic dissemination of carcinoma cells are often
facilitated by their transdifferentiation through the process termed
df
Bcl-XL
Bcl-2
β-Actin
S473-Akt
Motility
0.5
%
1
0
%
LY + +
g
Extravasated clusters (mean)
Migration (fold)
*
e
0
MDA/vector
MDA/CCL5
***
I
nvasion
10%
10%
I
nvas
ion
0.5%
10%
Migration (fold)
**
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*
MDA/vector
MDA/CCL5
a
b
*
Nodules per lung (mean)
c
30
25
20
15
10
5
0
Tumour mass (mg)
30
25
20
15
10
5
00
50
100
150
200
250
0
50
100
150
200
250
300
350
Tumour mass (mg)
Ctrl
CCL5
Ctrl
CCL5
Ctrl
CCL5
Metastasis index (fold)
Metastasis index (fold)
0
1
2
3
4
5
6
*
0
1
2
3
4
5
6
7
8
*
MDA/vector
MDA/CCL5
MDA+WI-38/vector
MDA+WI-38/CCL5
1
2
3
4
*
Figure 4
|
CCL5 enhances breast cancer cell migration, invasion and
metastasis. a
, A total of 500,000 MDA-MB-231/vector (ctrl) or MDA-MB-
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour
masses (mean 6 s.e.m., n 5 6 each group) were taken at 10 weeks. Lung
metastasis indices are expressed as fold increase (6s.e.m.) over controls. Data
shown are representative of multiple repeats. Asterisk, P , 0.01 in one-tailed
Student’s t-test.
b, A total of 500,000 MDA-MB-231 cells were admixed to
250,000 WI-38 fibroblast controls (WI-38/vector) or WI-38 fibroblasts
overexpressing CCL5 (WI-38/CCL5) and were injected subcutaneously in
NOD/SCID mice. Tumours (n 5 5 per group) were excised and weighed at
12 weeks. Masses shown represent mean 6 s.e.m. Lung metastasis indices are
expressed as fold increase (6s.e.m.) over controls. Asterisk, P , 0.01 in one-
tailed Student’s t-test.
c, A total of 800,000 indicated BCCs were introduced
into the circulation of NOD/SCID hosts. GFP-positive cancer colonies in the
lungs were counted 6.5 weeks later. Bars represent means 6 s.e.m. (MDA-MB-
231 controls, n 5 16 mice; MDA-MB-231/CCL5, n 5 18 mice). Asterisk,
P , 0.01 in one-tailed Student’s t-test.
d, Western blot analysis of lysates of
MDA-MB-231 control or MDA-MB-231/CCL5 cells. b-Actin was used as a
loading control.
e, Transwell migration or Matrigel invasion assays on 50,000
MDA-MB-231 control or MDA-MB-231/CCL5 cells. Data arerepresentative of
multiple independent experiments and are expressed as means 6 s.d. Asterisk,
P , 0.05; double asterisk, P , 0.05; triple asterisk, P , 0.01 in one-tailed
Student’s t-test.
f, One million GFP-labelled BCCs were injected into the tail
vein of NOD/SCID mice. Lungs were processed 48 h later and examined for
extravasated cells. Bars represent means 6 s.e.m. (MDA-MB-231 cells, n 5 7
mice; MDA-MB-231/CCL5, n 5 10 mice). Asterisk, P , 0.01 in one-tailed
Student’st-test.
g, Transwell migration assays on 50,000MDA-MB-231control
or MDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;
asterisk, P , 0.01 in one-tailed Student’s t-test.
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the epithelial-to-mesenchymal transition (EMT), in which cells shed
their epithelial characteristics and acquire instead a series of mesen-
chymal markers that enable their invasiveness and intravasation
21
.
Despite their lack of E-cadherin and their expression of detectable levels
of mesenchymal markers such as fibronectin (data not shown), the
MDA-MB-231 cells studied here exist in an intermediary phenotypic
state of ‘partial EMT’, as they retain a distinctive epithelial morphology
in vitro and are still responsive to EMT-inducing stimuli in culture. In
fact, we observed that ectopic CCL5 expression did not cause MDA-
MB-231 cells to undergo the morphological changes usually associated
with an EMT (Supplementary Fig. 9a), did not cause rearrangement of
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on
the expression of mesenchymal markers closely associated with the
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c)
and fibronectin (data not shown). These data suggested that CCL5 does
not directly promote the EMT programme of MDA-MB-231 cells.
We proceeded to explore an alternative possibility: that CCL5
expression affected some of the later, critical steps of the invasion–
metastasis cascade, namely the lodging of cancer cells in secondary
organs and the subsequent step of colonization. For that purpose,
MDA-MB-231/CCL5 cells were injected intravenously into host
mice, and the lungs of these hosts were examined ,6 weeks later
using fluorescence microscopy. These experiments revealed that
CCL5-overexpressing cells indeed had a significant ,1.8-fold
advantage over their control counterparts in colonizing the lungs
(Fig. 4c), suggesting that CCL5 exposure has effects on later steps
of the invasion–metastasis cascade. We note once again that this
enhanced tissue-colonizing ability was not due to CCL5’s effects on
cellular proliferation measured either in vitro (Supplementary Fig.
7b) or in vivo (Supplementary Fig. 7g, Ki67 staining).
Because improved colonization can be due to enhanced cellular
survival, we tested whether CCL5 protects against apoptosis.
Notably, we found that MDA-MB-231/CCL5 cells exhibited higher
levels of the Ser 473-phosphorylated, activated form of Akt, but
exhibited no difference in the levels of other pro-survival proteins,
such as Bcl-XL or Bcl-2 (Fig. 4d), or a reduction in the levels of
pro-apoptotic molecules such as BAX or BAD (data not shown).
Moreover, we found that overexpression of CCL5 had no effect on
the ability of MDA-MB-231 cells to withstand serum deprivation
(Supplementary Fig. 7b), loss of substrate anchorage (Supplemen-
tary Fig. 7d), or hyperoxia (data not shown). We also observed that
ectopic CCL5 expression did not protect MDA-MB-231 cells from
doxorubicin-induced apoptosis monitored using western blots for
cleaved caspase-3 (CC3) and cleaved PARP (as markers of apoptosis;
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohisto-
chemical analyses on control and CCL5-overexpressing tumours
revealed only minor differences in the levels of apoptotic CC3-
positive cancer cells among the examined groups (Supplementary
Fig. 7g, h). Together, these observations suggested that CCL5 does
not exert any detectable pro-survival functions in vitro or in vivo, and
that the observed enhancement of lung colonization was not a con-
sequence of significant anti-apoptotic activities of CCL5.
Akt serves as a key relay switch for upstream signals that promote
both cell survival as well as cellular motility
22
. Because CCL5-induced
Akt phosphorylation did not correlate with enhanced protection
against apoptosis, we tested whether the CCL5-enhanced lung col-
onization could be due to an increased ability of MDA-MB-231/
CCL5 cells to invade from the microvasculature into the lung
parenchyma through the process of extravasation. Indeed, ectopic
expression of CCL5 enhanced the motility of MDA-MB-231 cells
through permeable Boyden chamber membranes by ,1.5-fold as well
as the invasion of these cells through Matrigel layers by ,1.6 or ,2.5-
fold in either high or low serum conditions, respectively (Fig. 4e).
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein
injection—in order to remove most cells that remained within the
microvasculature of the lungs and thus had not extravasated—we
found twice as many deposits in the MDA-MB-231/CCL5-injected
group than their control-injected littermates (Fig. 4f). This indicates a
clear effect of CCL5 on cancer cell extravasation.
Finally, we investigated the role of Akt in mediating the actions of
CCL5 on cellular motility by using the phosphatidylinositol-3-OH
b
silacZ
silacZ
si809
si809
si186
si186
CCR5
β-Actin
ad
M
+
MDA+MSC
+ IgG
+Anti-CCL5 Ab
MDA
+Anti-CCL5 Ab
MDA+MSC
MDA
+ IgG
<
<
<
<
<
DAPI
CCR5
MDAMSCs MDA+MSC
DAPI DAPI
CCR5 CCR5
MDAMSCs MDA+MSC
*
c
Metastasis index (fold)
Metastasis index (fold)
*
Control
siCCR5
MSC + + +
0
1
2
3
4
5
e
++
Anti-CCL5
0
1
2
3
4
5
6
0
1
2
3
4
5
6
++
IgG
MDA
MDA+MSC
<
Figure 5
|
CCL5
CCR5 interaction is essential
for the MSC-induced metastasis.
a
, Immunofluorescence analysis of CCR5
distribution in MDA-MB-231 cells cultured with
MSCs. DAPI (for nuclei staining) is in blue;
CCR5 detected in green. Arrowheads denote
MSCs.
b, Western blot analysis showing CCR5
expression in MDA-MB-231/silacZ, MDA-MB-
231/siCCR5(809) and MDA-MB-231/
siCCR5(186) lysates. b-Actin was used as a
loading control.
c, A total of 500,000 cells of the
MDA-MB-231 variants in
b were co-mixed with
1.5 3 10
6
MSCs and injected subcutaneously into
nude mice. Mice were killed when tumours
reached 1 cm in diameter and the metastasis
index was calculated for each cohort (n 5 5 per
group). Results represent means 6 s.e.m.;
asterisk, P , 0.05 using one-tailed Student’s
t-test.
d, Anti-CCL5 neutralizing antibody or
control IgG was administered intraperitoneally
twice weekly in SCID mice bearing MDA-MB-231
(n 5 9) or MDA-MB-2311MSC tumours
(n 5 11). Representative lung pictures of the
indicated cohorts are shown.
e, Lung metastasis
indices of mice in
d. Data shown are
representative of means 6 s.e.m. Asterisk,
P , 0.05 in one-tailed Student’s t-test.
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kinase inhibitor LY294002. Drug concentrations that did not inhibit
the basal motility levels of MDA-MB-231 cells blocked the elevation
of motility induced by ectopic CCL5 expression (Fig. 4g). These
results, when taken together, suggest that the observed CCL5-
enhanced lung colonization could be ascribed, in significant part,
to its ability to promote extravasation and/or motility of cancer cells
at sites of dissemination rather than promoting the survival and/or
proliferation of these cells.
Essential role for the CCL5
CCR5 loop
CCL5 acts through three G-protein-coupled receptors, termed
CCR1, CCR3 and CCR5 (ref. 23). CCR5 has been determined to be
the main receptor for CCL5 in MDA-MB-231 cells, as inhibition of its
surface expression through dominant-negative mutants abrogated
the ability of these cells to respond to CCL5 chemotaxis
24
. We there-
fore focused our efforts on evaluating the importance of the CCL5–
CCR5 interactions in MSC-induced metastasis.
We confirmed that CCR5 is expressed by MDA-MB-231 cells and
not by MSCs (Fig. 5a), supporting the notion that MSC-derived
CCL5 acts primarily in a paracrine fashion on MDA-MB-231 cells
in the BCC and MSC mixed cell populations described above. To
probe whether the observed MSC-induced metastasis required
CCL5–CCR5 interactions, we inhibited CCR5 expression in MDA-
MB-231 cells by more than 85% through shRNA knockdown (ref. 25
and Fig. 5b), and mixed these cells with MSCs before implantation
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,
achieved using either of two different shRNA constructs, abrogated
the ability of MSCs to enhance the metastasis of MDA-MB-231 cells
(Fig. 5c). Furthermore, neutralization of CCL5 protein using intra-
peritoneal injections of an anti-human CCL5 monoclonal antibody
also abrogated the MSC-induced metastasis by MDA-MB-231 cells
(Fig. 5d, e). In addition, MSCs in which CCL5 expression was inhi-
bited by shRNA knockdown failed to promote metastasis of the
admixed MDA-MB-231 cells (data not shown). Taken together, these
results underscore the critical importance of the CCL5–CCR5 para-
crine interactions in enabling MSCs to induce metastasis of the
MDA-MB-231 cells.
Discussion
Certain models of metastatic progression propose that cancer cell
invasion and metastasis from the primary tumour site are strongly
influenced by contextual signals emanating from the stroma of the
primary tumour. It follows that if carcinoma cells are subsequently
deprived of such signals, they may revert to an earlier phenotypic
state in which they no longer display the traits of high-grade malig-
nancy. Indeed, such a model has been proposed previously by others
on the basis of indirect evidence
21
. Here, we demonstrate that at least
one mesenchymal cell type, the MSC, can expedite tumour meta-
stasis, and suggest that after primary human carcinomas recruit MSC
populations into their midst, subsequent interactions between the
MSCs (or their derivatives) and the BCCs endow the latter with
invasive and metastatic properties.
Although the recruitment of labelled MSCs to tumour xenografts
has been established in a variety of experimental models of tumorigen-
esis, there is currently no available way to quantify with any accuracy
the number of MSCs in actual human tumours, in part because no set
of markers has been identified that can uniquely stain these cells with-
out concomitantly staining other mesenchymal types in the tumour-
associated stroma
6
. Our demonstration that the stroma derived from
tumour xenografts contained appreciable numbers of murine MSCs
indicates that significant steady-state levels of thesecellsare maintained
in developing tumours. Interestingly, the use of CD10—one of the
markers associated with human MSCs—to purifycells from the stroma
of human primary invasive breast carcinomas yielded a population of
cells that expresses a number of other markers collectively used to
characterize human MSCs (for example, CD44, CD105 and CD106;
Fig. 6a). This suggested that, similar to tumour xenografts, human
carcinomas also acquire significant numbers of MSCs. Furthermore,
we note that CCL5, which is prominent in the stromal gene expression
signature associated with poor prognosis of breast cancers
26
(SFT;
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free
stroma of primary invasive ductal carcinomas (Fig. 6d), specifically in
the CD10-positive compartment
27
(Fig. 6e). Collectively, these obser-
vations argue strongly for a significant association between stromal
CCL5 levels, MSCs and human invasive breast cancers.
c
STT1969B
S
T
T
3126
ST
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3124
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TT6
56
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STT
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96
8
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STT3122
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STT854
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1
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9
75
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87
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STT1079
ST
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7
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17B
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9
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STT1777B
STT689B
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71
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9
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STT626
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196
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STT2776
ST
T277
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2F
e
CD13
CD29
CD44
CD49e
CD54
CD59
CD63
CD105
CD106
Nestin
HAS2
IGF2
PLAU
TIMP1
CAV1
IDC-7
T112603
T392303
Normal Invasive
CCL5
log
2
ratios
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.0
log
2
ratios
d
–2
–1
0
1
2
–2.0
–1.5
–1.0
–0.5
0.0
DTF SFT
DTF
SFT
ab
F
Figure 6
|
Stromal fibroblastic cells of human invasive ductal carcinomas are
rich in MSC markers and overexpress CCL5. a
, SAGE TreeView display of
MSC markers expressed in stromal CD10-positive cells from invasive
tumours
27
. b, Soft-tissue tumours were ranked by CCL5 expression
26
,fromlow
(green) to high (red). Wide blocks indicate expression ratios of tumours
classified as desmoid-type fibromatosis (DTF; yellow outline, n 5 10) or
solitary fibrous tumours (SFT; blue outline, n 5 13); narrow blocks are other
soft-tissue tumours (n 5 32).
c, Box plot showing that CCL5 expression is
higher (P 5 0.004) in SFT than in DTF. The difference in log
2
expression ratios
between SFT and DTF was tested with the Welch’s test.
d, CCL5 Affymetrix
gene expression in the stroma of human invasive ductal cancers compared to
that in normal cancer-free breast tissue (indicated as ‘Normal’; see Methods).
e, CCL5 expression is mostly restricted to the CD10-positive fibroblastic cells
derived from invasive ductal cancers. The heatmap shown is a cluster of
CCL5.genelist obtained as in
a. Detailsof the purification methodologies of the
various groups indicated in
a, d and e are found in ref. 27.
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Although we have focused here on CCL5 in the MSC–MDA-MB-
231 cell interactions, CCL5 seems to have an equally critical involve-
ment in the functional interaction of MSCs with MDA-MB-435
human BCCs. CCL5 levels accumulate synergistically when the two
cell types are co-cultured together (Supplementary Fig. 10a), and
MSCs in which CCL5 expression was compromised by shRNA knock-
down failed to promote metastasis by MDA-MB-435 cells to which
they were admixed (Supplementary Fig. 10b). With these facts in mind,
we point out that CCL5 does not seem to be involved in regulating the
MSC-induced metastasis of MCF7/Ras or HMLER cells, which may
depend on other paracrine factors such as VEGF and interleukin-8.
Nevertheless, ourobservations highlight therecently discovered critical
roles of chemokine networks in malignant progression
28,29
and suggest
the possible utility of a variety of CCL5 analogues and CCR5 antago-
nists currently used in anti-HIV therapy
30
in treating metastatic disease.
Notably, we have observed that MSCs induce the metastasis of cells
to the lung that are, on isolation and re-injection into recipient mice,
no more metastatic than their predecessors in the primary tumour
(Fig. 2e). This indicated that acquisition of increased metastatic
powers by these tumour cells was reversible, and suggested that the
maintenance of this phenotype depends on continuing contact with
stromal cells. If extended to other tumour types, the present results
hold important implications for the molecular analysis of malignant
progression. They suggest that many of the cellular functions assoc-
iated with invasion and metastasis are often not expressed constitu-
tively by carcinoma cells, but rather only transiently in response to
contextual signals that tumour cells receive from their stromal micro-
environment. If so, analysis of the gene expression patterns of bulk
primary tumour populations may fail to detect the expression of key
genes mediating invasiveness and metastasis, if only because they are
being transiently expressed in minor subpopulations of cells within
such tumours. Additionally, attempts at determining the metastatic
propensities of tumours may need to be focused on the genes and
proteins that confer responsiveness of primary tumour cells to stro-
mal signals, rather than on the genes and proteins that directly medi-
ate the cellular phenotypes of invasion and metastasis.
METHODS SUMMARY
Cells labelled with GFP or ds-red, or harbouring various overexpression or
shRNA constructs, were generated by viral transduction followed by FACS
enrichment or antibiotic selection. Xenograft experiments were conducted in
nude or NOD/SCID mice and metastasis was estimated using fluorescence
microscopy. The levels of cytokines, growth factors and chemokines were
assessed by immunoassays. Migration and invasion assays were conducted using
transwell chambers. Antibody treatment of tumour-bearing mice was conducted
by intraperitoneal injections. See Methods for detailed information regarding
cell culture, viral infections, in vivo colonization and extravasation assays, RT–
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluore-
scence determinations, western blotting, and antibodies used.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 13 April; accepted 14 August 2007.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank F. Reinhardt for assistance in animal studies, A. Lu
for technical help, J. Yao for SAGE data analysis and the MIT Comparative
Pathology Laboratory for immunohistochemical analyses. We are grateful to
A. Bernad, X.-F. Qin, D. Baltimore and W. Hahn for providing constructs. We would
also like to thank R. Hynes, T. Jacks and R. Goldsby for discussions. A.E.K. is a fellow
of the Susan G. Komen Breast Cancer Foundation. R.A.W. is an American Cancer
Society Research Professor and a Daniel K. Ludwig Cancer Research Professor. This
research is supported by grants from the Breast Cancer Research Foundation
(R.A.W.), the Ludwig Trust (R.A.W.), the Susan G. Komen Breast Cancer
Foundation (R.A.W.) and the Dana-Farber/Harvard Cancer Center Specialized
Program of Research Excellence (SPORE) in Breast Cancer (A.E.K., R.A.W. and
K.P.).
Author Contributions A.E.K. conceived and designed this study, and performed
most experiments; R.A.W. supervised research; A.E.K. and R.A.W. wrote the
manuscript; A.B.D. and R.T. provided human MSCs; A.B.D. helped in in vivo CCL5
neutralization; A.S. helped in the Luminex screens; A.P.V. and M.W.B. provided
technical support in tissue culture, ELISA, western blot, RT
PCR and soft-agar
analyses; G.W.B. performed CCL5 analysis on soft tumour expression data; A.L.R.
obtained and classified the clinical specimens; K.P. fractionated the clinical
samples and performed SAGE analyses; and A.L.R. performed the microarray
analysis on sorted stroma.
Author Information The clinical microarray data on the sorted stroma is deposited
at http://www.ncbi.nlm.nih.gov/geo, GSE8977. Reprints and permissions
information is available at www.nature.com/reprints. Correspondence and
requests for materials should be addressed to R.A.W. (weinberg@wi.mit.edu).
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METHODS
Cell lines. The MCF7/Ras
31
and MDA-MB-231 (ATCC HTB-26) cancer cells
were infected with pWZL-blasticidin-GFP-expressing retroviral particles and
grown in Dulbecco’s modified Eagle medium supplemented with 10% calf serum,
100 units ml
21
penicillin, 100 mgml
21
streptomycin and 2 mM L-glutamine
(complete medium) at 37 uCin5%CO
2
. The MDA-MB-435 and human mam-
mary epithelial cells HMLER
32
were infected with pRRL3-GFP-expressing lenti-
virus and grown in complete medium or MEGM media with bovine pituitary
extract, respectively. Bone-marrow-derived human MSCs were isolated from hip
aspirates of healthy volunteers, propagated as previously described
33
, and used
between the 4th and 8th passages. Three different batches of MSC cultures derived
from three different donors were assayed and exhibited consistent results. MSCs
expressing GFP or ds-red were generated by lentiviral (pRRL3-GFP) or retroviral
ds-red (Clontech) particles. WI-38 human embryonic lung fibroblasts (ATCC
CCL-75) were grown in Dulbecco’s modified Eagle medium supplemented with
10% calf serum and were used before the 20th passage. The MDA-MB-231 cells
overexpressing human CCL5 (MDA-MB-231/CCL5) were generated by retroviral
infection of parental cells with pLZ-CCL5-IRES-gfp
24
viral particles. Control
cultures were infected with pLZ-IRES-gfp retrovirus. WI-38 fibroblasts overex-
pressing CCL5 were generated by retroviral infection of parental cells with pWZL-
Blasticidin-CCL5-expressing viral particles. MDA-MB-231 cells lacking CCR5
expression were generated by lentiviral infection of parental cells with FG12-
siCCR5(809) or FG12-siCCR5(186) viral particles
25
. MDA-MB-231 cells infected
with the control FG12 vector harbouring shRNA against bacterial lacZ (FG12-
silacZ) were used as a control cell line. All infected cells were enriched for GFP
expression using FACS.
Animal studies. All mouse studies were performed under the supervision of
MIT’s Division of Comparative Medicine and were done in accordance with
protocols approved by the Institutional Animal Care and Use Committee.
Athymic female nude (NCR nude, nu/nu) mice were purchased from Taconic
Laboratories and NOD/SCID mice were bred in-house. Animals were housed
under pathogen-free conditions and were given autoclaved food and water ad
libitum. For xenograft experiments, cancer cells were implanted alone, or were
mixed with MSCs or WI-38 fibroblasts and injected subcutaneously into recipi-
ent animals as previously described
32
. Nude mice were used at ,10–13 weeks of
age and received sub-lethal 400 rad of c-radiation using a dual
137
caesium source
18–24 h before injection. Female NOD/SCID mice were used at 12–14 weeks of
age. Tumours were measured twice weekly using precision calipers. Tumour
volume was calculated as 4/3pr
3
where r is the estimated radius. Tumours were
dissected out at the end of the experiments and weighed.
CFU-F studies. Tumour xenografts were implanted in recipient NOD/SCID
females and allowed to grow for 4 weeks. Tumours were then excised, treated
with collagenase and the GFP-negative mouse stroma was isolated from the GFP-
positive cancer cells using FACS. CFU-F culture assays were performed on the
sorted mouse stroma as standard (Stem Cell Technologies). Colonies were
stained 14 days later using Giemsa stain and enumerated under light microscopy.
Quantification of lung metastasis. Mice were killed using CO
2
asphyxiation and
entire lungs were removed, washed in PBS, and placed in ice-cold Hank’s buffer
(HBSS, Gibco). Excised lungs were immediately dissected into their various
lobes under bright-field microscopy and examined under fluorescence micro-
scopy within 12 h of excision. This circumvents tissue autofluorescence (typically
apparent ,24–36 h after necropsy), which greatly masks the GFP signal from the
disseminated cancer cells. Lung metastasis burden was estimated as the number
of GFP-positive colonies observed under fluorescence microscopy. The lung
metastasis index for each mouse was calculated as the ratio of the number of
GFP-positive colonies observed in the lungs divided by the mass of the primary
tumour (in grams). Indices were pooled within each cohort and were expressed
as mean 6 s.e.m.
Immunoassays. The levels of cytokines, growth factors and chemokines in the
culture media were assessed by xMAP Bio-Plex cytokine array (Bio-Rad Life
Sciences) using a Luminex 100 plate reader (Bio-Rad Life Sciences) according
to the manufacturer’s protocols. The levels of CCL5 in the various culture super-
natants were measured using enzyme-linked immunosorbent assay (ELISA;
R&D systems).
RT–PCR analysis. GFP-labelled MSCs were admixed to MDA-MB-231 cells and
implanted subcutaneously in recipient NOD/SCID mice. Four weeks later,
tumours were excised, and GFP-labelled MSCs were purified using FACS and
processed for RT–PCR analysis for CCL5 using the following forward and reverse
primers: CCL5-left, 59-TGCAGAGGATCAAGACAGCA-39, and CCL5-right,
59-GAGCACTTGCCACTGGTGTA-39. RT–PCR on cultured cells was per-
formed as standard.
Migration assays. Cancer cells were seeded in the upper well of a 24-well trans-
well Boyden chamber (8 m m pore size; Costar) and migration was assessed 18 h
later. For MSC migration assays, MSCs were layered in the upper well of a 24-well
transwell chamber and allowed to migrate towards cell-free media derived from
MCF7/Ras or MDA-MB-231 cells placed in the bottom wells. Membranes were
processed as standard. Migrating cells were stained with crystal violet and
counted using bright-field microscopy.
Extravasation assays. GFP-labelled cancer cells were infused into the circulation
of recipient NOD/SCID mice through tail-vein injection. Forty-eight hours later,
mice were anaesthetized and their thoracic cage opened. Texas-red lectin (to
visualize blood vessels; from Lycopersicon esculentum, Vector) was then intro-
duced into the left cardiac ventricle, followed by 4% PFA and then 20 ml of cold
PBS. Frozen lung tissue was prepared and sections were processed for fluore-
scence microscopy as standard.
Anchorage-independent growth assays. We carried out soft agar assays as
described previously
34
.
Western blot analyses. Western blotting was done using standard protocols.
We used primary antibodies against phosphorylated S473-Akt (4051, Cell
Signaling), Bcl-XL (2762, Cell Signaling), Bcl-2 (2872, Cell Signaling), b-actin
(ab8224, Abcam), CCR5 (ab21653, Abcam), cleaved caspase-3 (9664, Cell
Signaling), PARP (9542, Cell Signaling), N-cadherin (205606, Calbiochem)
and vimentin (V9, NeoMarkers). We used goat antibodies to mouse (115-035-
146) and to rabbit (111-035-144) conjugated with horseradish peroxidase as
secondary antibodies (Jackson Immunoresearch), and developed the blots using
ECL (Dura, Pierce).
Immunohistochemistry. Immunohistochemical analyses were performed on
formalin-fixed, paraffin-embedded tissues. Sections (4-mm thick) were depar-
affinized, re-hydrated and subjected to antigen retrieval procedures as described
previously
35
.
Immunofluorescence. Cells were plated on 0.2% gelatin-coated coverslips in
complete medium overnight, washed in PBS, permeabilized in 0.1% Triton-
X100, blocked in 1% BSA/10% serum, fixed in 3.6% PBS-buffered para-
formaldehyde, and processed for indirect immunofluorescence analyses as
standard.
Anti-CCL5 treatment. MDA-MB-231 cancer cells alone or admixed with MSCs
were injected subcutaneously into recipient mice. Anti-CCL5 (32 mg per mouse;
AF-278-NA, R&D) or control isotype IgG (32 mg per mouse) antibodies were
injected into the peritoneum 48 h after tumour implantation and twice weekly
for the duration of the experiments (10 weeks).
Anoikis assays. Cancer cells were starved in 1% IFS/DME for 24 h, then trypsi-
nized and suspended in serum-free media. Tubes were continuously rotated at
37 uC for the duration of the experiments. Viable cells were counted using the
Trypan blue exclusion assay.
TUNEL assays. Apoptosis quantification analysis was performed using the
TUNEL assay as recommended by the manufacturer (Roche), with the exception
that the GFP-labelled MDA-MB-231/control and MDA-MB-231/CCL5 cells
were fixed for 60 min in ice-cold methanol (instead of 15 min in 3.6% para-
formaldehyde) to quench the GFP fluorescence.
Gene expression analysis. Expression ratios (relative to reference mRNA) and
classes of soft-tissue tumours were obtained from ref. 26 via NCBI GEO
(GSE4305). CCL5 expression was calculated as the mean of probes for
IMAGE:1325655 and IMAGE:840753. For Affymetrix gene expression analysis,
RNA extraction, cRNA synthesis and hybridization to Human Genome U133
Plus 2.0 Arrays were performed as described previously
36
. Raw expression data
obtained using Affymetrix GENECHIP software was normalized and analysed
using DNA-Chip Analyser (dChip) custom software (W. H. Wong and C. Li,
http://www.dChip.org/). Array probe data were normalized to the mean expres-
sion level of each of the three CCL5 probes across stromal samples prepared
from a set of 15 normal and 7 IDC (invasive ductal carcinoma) specimens.
Leukocyte- and endothelial cell-free stroma was isolated as previously
described
27
. Comparisons between ‘Normal’ and ‘Tumour’ (IDC breast stroma)
were performed using the dChip ‘Compare Sample’ function. SAGE data were
obtained from http://cgap.nci.nih.gov/SAGE/AnatomicViewer and performed
as previously described
27
. Data were normalized, log-transformed and clustered
using average linkage uncentred analysis. Detailed purification methodologies
and sample identification numbers have been previously published
27
.
31. Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas
promote tumor growth and angiogenesis through elevated SDF-1/CXCL12
secretion. Cell 121, 335
348 (2005).
32. Elenbaas, B. et al. Human breast cancer cells generated by oncogenic
transformation of primary mammary epithelial cells. Genes Dev. 15, 50
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(2001).
33. Lodie, T. A. et al. Systematic analysis of reportedly distinct populations of
multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue
Eng. 8, 739
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35. Kuperwasser, C. et al. Reconstruction of functionally normal and malignant
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