Mesenchymal stem cells within tumour stroma promote breast cancer metastasis - 藏经阁干细胞之家



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发表于 2012-5-17 17:15 |只看该作者 |倒序浏览 |打印

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the6 K: F% V! d6 W0 {3 s' J
tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour1 W' E! g4 N6 I
pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived humanmesenchymal stem cells,
when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their% S; `  k& T& \  X, X
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.
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 alterations
specified by the genomes of cancer cells, alternative views propose
that metastatic traits are acquired through exposure of epithelial& I  \! ^, c5 f+ P% j0 s
cancer cells to paracrine signals that they receive from mesenchymal
cell types within the tumour-associated stroma. Although several8 m" W8 {' @; a) Z2 K( V. W0 c5 Q. |
lines of evidence demonstrate the contributions of stromal cells to8 v: _! U. r6 L5 l5 K& Z; w
primary tumour growth1, direct experimental demonstration of the( j, m2 H2 s. E, \  o# z
influence of these various cells on the metastatic abilities of cancer- q& q+ ^% S3 w5 g4 X$ d$ l
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 tumours2. To characterize better9 p5 O$ _: g& i" ]1 U* N
the role of this stromal cell in tumorigenesis, we set out to determine
whether MSCs could supply contextual signals that serve to  h1 j; B. E2 }* e9 u& b
promote cancer metastasis.
Mesenchymal stem cells are pluripotent progenitor cells that contribute
to the maintenance and regeneration of a variety of connective
tissues, including bone, adipose, cartilage and muscle3. Although
MSCs reside predominantly in the bone marrow, they are also distributed
throughout many other tissues, where they are thought to
serve as local sources of dormant stem cells4,5. The contributions of* r* f) K0 G# d& l# r9 r
MSCs to tissue formation become apparent only in cases of tissue/ t/ Q, I1 G6 n9 F- v7 U, _5 w
remodelling after injury or chronic inflammation. These conditions& i5 h, i& v/ g. |  o; @" |2 B
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 and0 A6 [  B' M, D$ d* I8 J0 \" _8 E
their subsequent recruitment to the damage site6. For example, MSCs$ Z! E2 o7 w7 W
have been shown to contribute to the formation of fibrous scars after
injury7.% Z5 q+ X# A9 T, H
The formation of breast carcinomas is often accompanied by a+ Y& ]9 F; S. x. L/ P/ Z
well-orchestrated desmoplastic reaction, which involves the recruitment
of a variety of stromal cells with both pro- and anti-tumorigenic- S7 t0 K  n/ B) W# ]' m3 l, D1 Z
activities1. Such response closely resembles wound healing and scar. ?8 x* o  `7 q
formation, and entails the constant deposition of growth factors,
cytokines and matrix-remodelling proteins that render the tumour$ `( Q/ U9 e4 g  }, Z
site a ‘wound that never heals’8. This suggests that, similar to sites of
injury, actively growing tumours recruit MSCs through the release of( q# x6 T( A) m  T( z; t3 }
various endocrine and paracrine signals. Indeed, as we have found,
mouse stroma prepared from developing human MCF7/Ras or9 `! {" Y- w0 J0 D8 j7 l
MDA-MB-231 breast cancer xenografts is rich in cells with an ability. M  g3 W. b0 [% M+ u
to generate fibroblastoid colony-forming units (CFU-F) in vitro
(Supplementary Fig. 1a), a hallmark of MSCs3. The absence of such
colonies from control Matrigel plugs or from neighbouring tissues8 B2 o2 t* ]; K4 ~
(negative control; Supplementary Fig. 1a) suggested that endogenous
murine MSCs localize specifically to sites of neoplasia.9 ^1 U% X# o+ L' u
To investigate whether human breast cancer cells also have the
ability to attract human MSCs, we established a transwell assay2 ^6 ^8 D2 P0 P$ C
in which bone-marrow-derived human MSCs were allowed to, d/ U# H" L8 Y  _* B! _$ F. O
migrate towards media derived from MCF7/Ras or MDA-MB-2319 |! W+ \  d& r% y9 Q6 h
cultures. We found that human MSCs migrated much more avidly( f' j- P7 H+ h) D7 U
(,11-fold more) towards media derived from these cancer cells$ [, U4 E. Y# d7 k
than towards control media (Supplementary Fig. 1b). More importantly,
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 specifically
to the developing tumours, with no observable accumulation' E' `+ U9 m9 x* j; H, V
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 corroborated
recent studies that described the localization of systemically& h5 f4 Q% `: d% v' g% |
infused MSCs to other types of malignancy, such as gliomas9,10,
colon carcinomas11,12, ovarian carcinomas13, Kaposi’s sarcomas14 and, Z/ W0 M. u$ j2 |, n& U( ~
melanomas15.MSCs enhance breast cancer metastasis
To investigate the functional consequences of the heterotypic interactions, z% Z& G* U4 ?( ~# L
between MSCs and mammary carcinoma cells, we established
a xenograft model in which GFP-labelled MCF7/Ras, MDA-MB-231,0 ]! e6 {$ ]) C4 C
MDA-MB-435 and HMLER (see Methods) human breast cancer. @' L. s9 I- G! M
cells (BCCs) were mixed with bone-marrow-derived human MSCs. Z) H9 L) ~% z7 E* U  F/ T2 b
(hereafter referred to as MSCs) and injected subcutaneously into5 c, J* ]3 v$ v3 p8 d0 q) s8 H
immunocompromised mice. The growth kinetics of the MSCcontaining& g- P+ A5 f& Q9 n
tumours (BCCs plus MSCs) were compared to those of! Q) d/ n5 g$ {: f( `* W" D4 y
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/Ras4 Z/ G8 V* f; R$ E8 Y/ z/ O% l8 d
tumours without affecting the kinetics of MDA-MB-231-, MDAMB-' M& E0 F" X* j2 y1 R! J
435- or HMLER-containing tumours (Fig. 1a). More importantly,- G$ W9 O6 v! K
whereas mice carrying tumours composed only of BCCs4 s, _0 a5 D/ ]
exhibited few microscopic metastases in the lungs (Fig. 1b, d), mice
bearing the mixed MCF7/Ras1MSC, MDA-MB-2311MSC, MDAMB-& L( k3 X4 i0 c5 J! N
4351MSC and HMLER1MSC tumours displayed a marked
increase in the numbers of micro- and macroscopic lung metastases7 n; C4 A5 s% j: D0 @/ R/ w
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs) |* }7 h2 V5 X' c/ p$ z) G
of BCC1MSC-bearing mice compared to their BCC-control littermates6 \- j0 @2 F* m  u
revealed two-, three-, four- and sevenfold enhancements in, G9 c8 T: N  e, d& C4 w
the overall numbers of detectable HMLER, MDA-MB-435, MCF7/8 a* f4 D1 R( c+ l; N( `
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c).
Furthermore, in contrast to the MDA-MB-231-bearing mice, the1 U: `  B7 A, g5 ~0 g
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$ L7 s9 @7 k3 l2 y# `* Z, I! @
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  ^; B$ P5 w* ]+ W1 @* K5 |
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall primary
tumour growth kinetics.! X+ ~, X5 w  `! F$ v- z
We note that admixture of other types of mesenchymal cells—( W/ ]4 ^" ^) j4 |
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 3) r; @& y* X0 v- j
and data not shown)—to MDA-MB-231 cancer cells before injection- c  D/ n; W0 ?7 h& U9 C4 k
into host mice did not result in either enhanced growth kinetics9 b; H1 P! B/ i
(Supplementary Fig. 3a, b) or increased numbers of lung metastases7 O, ?4 \# `; s; Z3 L5 u/ B6 M
(Supplementary Fig. 3c, d). Taken together, these observations indicated
that the metastasis-enhancing powers were a specific property
of admixed MSCs or derivatives thereof.$ _' B! Y: G1 R2 n2 y# G' T/ `
Reversible metastasis8 F# H& J- }5 y, g* V2 N( Z3 q
Implantation of MSCs either contralaterally to MDA-MB-231 cells or: s, r; N% u) {4 W, Q. `; ]
in nearby separate sites of injection did not affect the metastatic
potential of the resulting primary tumours (data not shown), indicating* o8 {( H8 g! \+ Y+ ?
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' Y1 `; ^$ }2 u/ o+ W6 w0 k8 \
carcinoma cells. Thus, the MSCs might favour the outgrowth of rare
variants within the MDA-MB-231 cell populations that exhibit( S0 A. {2 B& p+ @8 u5 @
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% }) s6 ]/ v! B/ o8 ?& ?3 V4 D& ]
possibility that the acquisition of the metastatic phenotype might8 K3 L* [! f+ r
be reversible, in that carcinoma cells might revert to a lower metastatic3 f( a4 M! t$ N+ T  F1 ]& t4 b
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 (Texplants)  d' F. e6 w- _4 P: \# ?
as well as from their derived lung metastases (L-explants),
expanded in vitro, cleared from contaminating stromal components,# }: v/ h1 l$ |1 e1 \  ~
and then re-injected into subcutaneous sites in host mice in order9 _$ v+ E' M, r5 i8 u
to evaluate their respective metastatic powers (Fig. 2a). Although& o5 X! I; r; w  @3 A: I- u% c
the growth rate of the resulting L-explant primary tumours was4 z: s) ]( _4 o. I& v6 o
marginally enhanced compared to their T-explant counterparts
(Fig. 2b, c), these L-explant cells were no more metastatic than the! ^3 N2 F/ r( X
parental T-explant cancer cells (Fig. 2d). This suggested that the3 M% {" N% z) h5 u) X
a
c d
Days after injection
Tumour volume (mm3)7 ?5 h( N; m& h8 }
10 17 24 31 38 46 72 80 89 13 19 26 33 40 47 54 61 68 75 81 10 19 24 31 38 46
b
1 mm 1 mm
1 mm 1 mm
300 μm! }: N* _$ E$ u* H9 n
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC3 L* N# X3 k" o/ x' j! D, A. v' y
MDA-MB-435 MDA-MB-435+MSC  Y* K$ L' v' O2 C+ _3 u: k. s) J8 N
MCF7/Ras MCF7/Ras+MSC# s! C( ^  g  I6 c3 j% F, s
100 μm
300 μm* ~6 q9 |$ z' M" _1 Q
100 μm* h5 Y  @' h' @/ Y# ]
MDA-MB-231
MDA-MB-435
MCF7/Ras3 d5 H. M( }* z* T- S
**
Metastasis index (fold)
0
11 J) ^' X" W0 Q" d/ n
2
3! O2 Z7 c# |6 F" t* L
4
5
6
7) a. ?( S# i1 U4 P5 X
8
9
*  q. G) w2 Y6 F. P( Q4 z; s
**
**! C3 A0 I9 @/ s4 G5 B
MSC – + – + – + – +  j8 ?, `- c, {9 x
*/ G: y4 u! D9 h1 O- A/ _4 y5 a
HMLER
700 1,200
1,0006 \& n; A/ K$ a8 l3 ?3 _
800
600
400) X9 _) L& E5 B
2000 {( r4 Q0 f- E  f0 O& x
0/ O4 t+ n4 o2 t& a, d6 m
800/ `1 q+ i( J4 _" v. L% r; v9 z
700: h2 h. ]7 H4 f% D* M. T
600
500
400
300( r' J* N! o& v- R
2006 ], }3 E, Y. i; L) [: }
100' U; d; v, S/ e  L+ ~6 A# V
0
3,500' N0 F; b) c) M2 F! M/ [
08 r5 b( r3 N/ G& f
5003 L$ |) o& P' R2 T
1,000
1,5009 n' g" K% l8 \& I
2,000% j2 _8 {) T- p. Q4 _- p- b; e
2,5003 {0 M. C8 R1 I3 a/ i
600 3,000! X0 ~) G) F+ ?
500
400
300  n5 b6 H# I# j& x
200& b/ P  q, |0 G; N. Y( @
100; `( F% X& V- ]
03 K% D) [& t2 y' r. R+ ?4 t
0 21 31 38 49 56 63 70 78/ G) y7 ^$ W% y% ]9 S  k
MCF7/Ras alone4 n$ |! [$ T! Z, z/ x0 m( q
MCF7/Ras+MSC
MDA-MB-231 alone
MDA-MB-231+MSC$ V* J1 t: M! V% `! G8 k
MDA-MB-435 alone
MDA-MB-435+MSC- d5 j% g  }* O* n2 u
HMLER alone5 q" S& y. d' Y+ z" ~& C
HMLER+MSC6 N* L3 L7 d# C- V) F7 Q2 W
HMLER HMLER+MSC, \7 E. n5 B8 b+ n; c& W% V- r# i
MCF7/Ras MCF7/Ras+MSC0 o& h8 w" Z. R, N: g4 ]
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC" B" c0 P' d3 d4 F* P* J6 A
Figure 1 | MSCs promote breast cancer metastasis. a, Tumour volume6 a) O; H* H, N$ `
measurements (mean6s.e.m.) of 500,000 GFP-labelled BCCs injected7 z: t8 @- \' T( K
subcutaneously into nude mice with or without 1.53106 MSCs.
Representative data from multiple experiments are shown. Diamonds, BCCs
alone, n55–7 mice per group; squares, BCCs plus MSCs, n55–8 mice per, i- T, H8 x6 J: D" p
group. b, Representative bright-field/fluorescence images of lungs of mice
bearing the indicated tumours. Cancer colonies are in green. MCF7/Rasbearing
mice were killed at approximately day 150 to allow these tumours to
grow to comparable sizes to their MCF7/Ras1MSC counterparts. c, The" [' Q# Z- x0 T1 Q( j3 G- u
lung metastasis indices pooled within each cohort of mice in a are expressed& k; C+ t+ M' }7 M
as fold increase (6s.e.m.) over controls. Data shown are representative of% U' N1 b6 C  `, b: f
multiple repeats. Asterisk, P,0.01, double asterisk, P,0.05 using onetailed
Student’s t-test. d, Representative haematoxylin-and-eosin-stained
sections of lungs of mice bearing the indicated tumours. Metastases are4 k0 R9 _# P5 p) O6 u% c  s4 o3 C
delineated by a dashed line.MSC-induced metastatic powers reflected a reversibly induced trait- T7 G; w% @( U  Z
of the MDA-MB-231 cells, and that the ability of these cells to metastasize
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 a8 Z% p6 ]! G* r2 [( m, A3 Z
stable fashion.
The effects that the MSCs exerted on the BCCs might have
occurred within the site of primary tumour formation. Alternatively,6 |8 j0 L; B1 a' N9 D
the MSCs might have accompanied the metastasizing BCCs
to sites of metastasis formation. To distinguish between these two2 F! }+ g5 z  M1 F
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled
MDA-MB-231 cells and implanted the mixture subcutaneously in& V, Z+ E- e0 R' d) }5 W
host mice. We found that the tumour-derived lung metastases contained
green-labelled MDA-MB-231 cells but no detectable redlabelled  w* R: a" l/ g* U
MSCs (or their derivatives; Supplementary Fig. 4a) when/ ?1 e* y0 |$ S' h
scored 4, 5 or 6 weeks after primary tumour implantation. The) V( s0 C) a  d' v  K' j' V' Q
absence of red-labelled MSCs from the lung metastatic sites cannot1 r4 k$ F; m  d' p" n: Q
be ascribed to an inhospitable lung parenchyma, as MSCs that lodge; f" W9 T  ~0 f
in the lungs of recipient animals after tail-vein infusion survive in that
environment for ,6 weeks after injection (Supplementary Fig. 4b).5 `* `( S/ I. ?4 @0 X% ?% [9 g2 ]
Hence, it appeared that the admixed MSCs do not migrate in large
numbers to the sites of metastasis, and that they exerted their prometastatic- C4 x+ @1 V* }3 f2 ~1 O
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+ R# Q/ b7 z! m# o3 c
metastasize. To understand this crosstalk better, in vitro co-cultures$ l/ `- D% R, Y& q
of MDA-MB-231 breast cancer cells and MSCs were established and
their conditioned media were screened for the levels of various cytokines,6 @, W# K+ S# O& h) S  d( t
chemokines and growth factors using the Luminex-based Bio-& M5 P+ i5 |$ M2 D1 j2 }  j- b
Plex suspension array system (Fig. 3a). In some cases, the resulting
a
b c d
Days after injection
Tumour volume
(× 100 mm3)( f' v3 g, z% }% `
0
53 p: @1 l4 Q1 ~; k
10
15/ P0 ^# a" e3 z7 h7 i
20. g: E9 S% Z1 [* _3 T' M5 ~4 a
25
14 17 21 24 28 31 34 38 42 45 48 66 71 77
Primary tumour explants
Lung explants
MDA-MB-231. T1 h+ J' q( v( ^2 U' r
MSC" A: u2 b5 {2 t! @7 h7 X
+
Lung explants
Primary tumour7 [9 k& U. j7 u" B: u
explants$ _& e" R2 |: t. T/ L# Q8 }( i4 w
Antibiotic+ B. N6 M5 T% F2 S% U; b* J
BCC selection
T-explant
L-explant
T-explant- k) i; |3 x- Q
L-explant% L5 \  D  _& [/ ]) G0 J) t  `4 c
Tumour mass (g)
3.0& {$ M9 C$ l  Q" Z, F' y: L/ w
2.01 r% q  m: [+ g$ S6 `+ e" W
1.0
0 0
0.4. |  z. H0 x$ Y  {# ]% P, p: l
0.80 _6 b3 R. q" e) b
1.2
1.6
2.0+ M. @: U8 _4 u& @0 p1 M
#
Metastasis index (fold)" h0 u* K* _! y5 Y2 E6 _9 P
##
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 mgml21), and re-injected as primary5 A8 Y! L0 `4 M
subcutaneous tumours in recipient animals. b, Tumour growth
(means6s.e.m.) of 500,000GFP-labelled lung-derived (L-explant) or primary
tumour-derived (T-explant) MDA-MB-231 cells inoculated subcutaneously.- d8 b. g# S; y, }1 J
Data shown are representative ofmultiple independent experiments in which
four different paired batches of L-explant and T-explant cultures were assayed! l5 `' m2 \( A) \
in parallel. MDA-MB-231-T-explant (n58 mice); MDA-MB-231-L-explant% K+ Y3 V4 H; Q2 L9 v. O
(n510mice). c,Masses (means6s.e.m.) of tumours in b.Hash,P.0.4 using
one-tailed Student’s t-test and indicates no statistical significance. d, Lung
metastasis index of mice in c. Doublehash, P.0.3 using one-tailed Student’s
t-test and indicates no statistical significance.2 [/ ~% N- o1 o/ [8 o
a& V" P" O- F6 f7 g0 v$ G6 Y
CCL4  c( P. M5 ~# I
bFGF
VEGF
IFN-γ
TNF-α& q  s. ]/ I. {8 O% T, S3 D# F* ]2 [
G-CSF) C4 A) `8 v( h. E# ~. U9 b, C$ p7 P
GM-CSF
CCL34 A/ m* a3 I; V( ~% m0 s8 S! z
CCL51 M  [2 V$ k) y7 ?7 X7 ~
MMP1
MMP3
MMP9
MMP13$ P& Y2 i2 t. c2 E; w' V/ B+ G
IL-1α/ v8 ^" H6 Z* O/ E2 W
IL-1β
IL-4
IL-5( k  F' F2 M! C; q; b0 ]
IL-6, _% _6 ]% V2 b5 j
IL-7
IL-8+ L+ ?$ n3 G$ U1 q. J# u, N. S. s, t
IL-10- d1 L; Y/ P+ m7 z
IL-12+ ]' s! E; ~8 v7 `  {* [" z# `
IL-13# r! j4 `7 X$ e6 P1 S; S7 ?% N
IL-17- v* i7 m4 S( g& k4 k& Q/ G
IL-2
TGF-β4 g' w% c- G' V+ }8 E+ \2 H
Fold induction
MSC alone
MDA alone
MDA+MSC (2:1 ratio)
1 * * * * * * * * *+ @- ^+ `6 M) |2 H
3
5
75 [& W$ K) K* K! _
9
11! H5 M5 I( d1 Y4 p0 T& a8 {8 Q- ^4 R
138 ~( Z; o6 N* {  C" ]
604 n0 m3 R$ ^# Y  I  a) Z
b c
0.4 μm
2.01 p# G+ x9 i) f/ }
1.86 {" t6 `. \4 s0 R! J. v
1.64 J, x  L- N, f8 E- r, b" }# Y$ ]' D
1.4
1.2# C9 I: [8 ~7 e- z
1.06 t+ g% @2 v( M2 ^* h5 X! T
0.8
0.6! m6 P2 @' z, m4 d) X* y% ?
0.4
0.28 X" N/ V- q0 G% a6 |
0 0
5
10
152 `  o1 j2 e: j7 r6 ]5 C# i
20. K; |# j) B- c# k
25( c- B! P- }! F) P" a
302 w+ \* s* J7 F+ Z% l
35+ V2 w" F8 [: G7 @
40" Z2 V8 R  g* a7 q# G7 A
45
508 ~" _% a5 m7 g* g$ M' _
d1 d2 d3 d47 V# U% g$ W' a! Y4 c  o
MDA alone
MSC alone
MDA+MSC
CCL5 levels (pg ml–1)) M) e+ H" @8 x7 J: n' O
Co-culture
Fold CCL5 induction
MDA alone
MSC alone$ E7 |. J7 u4 H6 O* A  Q# y
MDA+MSC
d8 _2 \) g# \* y* n
CCL5 (A.U. × 100)
30& D0 i' A5 U6 N4 W9 V- z0 K
25
20
15
10
5
0* [2 W# k+ r1 ]  c: C2 ~) {- \. E
+
MSC.c  E1 M( e9 F7 \, Q- O( |
+
MSC.13 r- \8 q' p; U) S- c; s
+
MSC.5* |2 J) a3 d  ^
MDA
MSC- }5 s  g+ R! H; z( L, v
MDA.12 J# G; X. j7 a# b) k, E' {# K, W
MDA.c& L, q/ u. \$ n$ C
MDA.58 X5 e' b, ]! ~
TC-MSC. B; R8 A7 R( d8 r
MSC (from MDA tumour)7 i1 j5 D$ D2 V5 v) E: i
BCC (from MDA tumour)
Control (MDA/CCL5)2 v* J, X, _! {; Q
CCL5
GAPDH! r: S' R( b3 c: `  L* J7 G9 R
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
completemedia 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 induction6s.d. of triplicates. Asterisk indicates
undetectable levels. b, CCL5 ELISA on the media of MDA-MB-231,MSCs, or: o2 K- _4 @/ ]: R, i6 Z, a
MDA-MB-2311MSCcultures (1:3MDA:MSCs) at the indicated time points.6 k5 r3 u4 ^$ v, h4 b1 v
Data points representmeans6s.d. of quadruplicates. c,BCCs were separated
from co-cultured MSCs by a 0.4-mmmembrane. CCL5 levels were probed by4 L; z' K, y" g" d6 Z# H7 o
ELISA on the culture supernatants. Data are expressed as fold induction over( S/ @& X7 A# m5 V' Q" p) l" F1 l0 M
levels seen inMDA-MB-231 culture supernatants (mean6s.d. of triplicates).% P, s/ v! O5 Q- I
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-siluc0 ^/ A9 j, U& @/ [2 g8 y6 h4 ]& O
(MDA.c), MDA-MB-231-siCCL5.1 (MDA.1), or MDA-MB-231-siCCL5.5
(MDA.5). Data are expressed as means6s.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" g& h8 J* }% ^+ C3 C5 G6 ]6 i, C
implantation. Tissue-cultured MSCs (TC-MSC) and MDA-MB-231/CCL5
cells were used as controls. GAPDH was used for equal loading.levels of certain released factors (for example, interferon-c or; }) G* _# l& }" R% u
tumour-necrosis factor-a) reflected the additive contributions of$ V+ O! C5 `, j& O
the two cell types when cultured on their own. Notably, the levels
of only one cytokine, CCL5, reflected a synergistic interaction+ b/ y8 u" b& z0 w! S5 K
between the MSCs and BCCs, as it accumulated to levels ,60-fold. _* Q. L, I& q8 y  o+ [3 n# u
higher than those produced by pure BCC cultures (Fig. 3a). This
cooperative induction of CCL5 was proportional to the numbers of: g4 n* J% g0 T. W
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was apparent2 R; a8 k% u, h, e0 g& @; J
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; m1 g# S- l* \% A$ _8 |
separated by a permeable membrane (Fig. 3c).! K9 S+ s+ I* U  M
We undertook to determine the source of the CCL5 produced% d0 F" z/ I* e
under conditions of co-culture. To do so, we stably reduced the
expression of CCL5 in MDA-MB-231 cells by.80% using short* a+ q3 g) t1 B
hairpin (sh)RNA (variant siCCL5.1; Supplementary Fig. 6). Importantly,
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.- g! Z6 s1 R% a! b& l
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; w8 B5 Q/ I9 r( o" s6 q4 E+ S
that the MSCs were the major source of the CCL5 observed on coculture8 h+ E! j& d3 P! t7 q6 V2 T3 E& L
of the two cell types. In support of this conclusion, analysis of! o9 @2 T. C0 ]* e3 w
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 induction
of CCL5 in the culture of MSCs, but not that of BCCs (Supplementary
Fig. 5b). Finally, polymerase chain reaction with reverse. v4 g4 q. w5 f, D
transcription (RT–PCR) analysis of the RNA prepared from these coculture-* y9 x( I% c9 k" q. y- D) J; ~- Z$ J
derived MSCs (Supplementary Fig. 5c), as well as from the0 L) a- O& f" v$ b6 Y
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after, n5 G9 s) ^" W; Q' M
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" M6 V+ s8 s/ A' I! n
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- Y: x& U* j& A0 V/ e
when compared to in situ ductal tumours or benign lesions16,17.
However, the precise contributions of CCL5 to cancer development, @& e/ e+ E  B4 b! T# H/ k0 r6 r, Y6 i
and progression are poorly understood. To investigate further the
possible causal role of CCL5 in cancer cell metastasis, we overexpressed8 N5 W4 K- w6 Y: }
this chemokine in the MDA-MB-231 BCCs (Supplementary" L$ L& k+ x  [8 u
Fig. 7a) and analysed its effects on cancer cell growth and
tumorigenesis. The overexpressed CCL5 did not confer any proliferative; y/ q7 Z) l  |+ h
advantage on cultured cancer cells when compared with" j* A8 d: x' G3 v" a- L
those lacking such overexpression (Supplementary Fig. 7b), and had! h7 E" }* f, l! k8 ^
no effect on the ability of BCCs either to grow in an anchorageindependent- F* Q( X' X  y
fashion in vitro (Supplementary Fig. 7c), or to form
primary subcutaneous tumours in immunocompromised mice (Fig.6 q8 E6 \7 V- d$ n
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 ofCCL5 are responsible formuch, if not all, of the observed) A) n" G4 H  ^% T
MSC-induced metastasis by the BCCs.
CCL5 promotes lung colonization
Previous reports have described an important role for CCL5 as a5 C& O% M) ~/ Z! e! [- L
chemoattractant for stromal cells, such as macrophages, that express
one of the receptors for CCL5, CCR5 (refs 18, 19). Furthermore,+ o+ i1 k: }  L3 k; `$ U* d
CCL5 expression has been associated with increased tumour neovascularization,
suggesting that endothelial cells, which express a variety( E4 @) W. L6 F
of chemokine receptors, may also be attracted by CCL5 to sites of* z! a$ u0 _! T$ r2 J/ Q
tumour formation, thereby enhancing tumour angiogenesis20. Such/ f6 N! ]. h. G7 w! L) u% z$ t# O
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.8 S% x0 d2 L4 M" y2 b5 y
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. S" ?( U/ k5 F: ?7 O
evident by F4/80 and MECA-32 staining for macrophages and/ R, @; N/ z  D7 A' W
endothelial cells, respectively; Supplementary Fig. 8). In addition,
we found that ectopicCCL5 expression did not cause an accumulation
of other stromal cells, such as SMA-positive cells, in the examined& F6 o' C% C- J7 d+ c
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
d f
Bcl-XL
Bcl-2
β-Actin
S473-Akt
Motility  d2 j6 ]8 W4 K( c
0.5%→10%' F: Z, S2 I( k6 g7 q% F' m
LY – – + +
g# J- |7 P8 c& q6 ~! l* X: V
Extravasated clusters (mean)
Migration (fold), x# D+ H, ]: U# K- s4 `+ F
*% Y) g+ E6 A. B' ?  T9 F! m& G
e' w; X+ D6 t$ ?# h8 {# [
0
MDA/vector, g# R; R+ h/ n+ G( W9 `
MDA/CCL5( F$ G. q+ ^. z) K
***
Invasion3 d$ ~% W# J; y% M
10%→10%
Invasion' D- z' |: d, Z
0.5%→10%
Migration (fold)
**
0/ G3 @) M2 W) ?7 y
0.5! K" a1 @: W- T- w3 e5 W3 e4 [. Y( K7 g
1.0
1.5' H. c% |# m( b
2.0: Z+ H5 O0 F' X: p5 v
2.5
3.0
3.5
*
MDA/vector% |' ~. \7 z! s1 d6 G
MDA/CCL5
a b" X4 n3 B3 w* H, [
*9 y0 t* E5 ~2 |
Nodules per lung (mean)- d6 a  D! ?; g
c
30
258 s' z0 b8 _" }& X$ u, g
201 y  u; L, Y! ~) [. H- B. L
15
10
58 X! ]) h0 i/ S' }% g
0: ?" v: T' ?" p' J
Tumour mass (mg)7 o& C8 H0 ~5 H0 S) S3 i+ d- _' _2 P
30) ^7 R/ m1 i: N0 x
25: ]! O4 B! }+ @; l
20' u& P+ ~1 V! |$ }, R6 w' p! R/ O4 ?
151 m: z) i9 G. d
10+ B* n) C' L  A5 G9 k
5
0 0
501 E: I- B2 O) \& y0 \
100
150
200) ]3 Z! B5 G7 Y+ ?+ g) Q5 @
250
0
50& u% M+ |  L2 V# n; B" i
100
1504 N$ I  T5 [. H) Z
2005 K5 z( ]# e! C4 v' d
250
3004 l/ @7 d  t; p) P# m
350
Tumour mass (mg)
Ctrl
CCL51 C0 Y* h) ?4 n' F2 c5 {6 E; q2 z
Ctrl
CCL5
Ctrl" e* [. Q& r: J& c1 [
CCL5
Metastasis index (fold)/ M& b% o6 Y; D' A9 d, y  g* P, d
Metastasis index (fold)6 B1 E$ `# r$ @# b  f
0
14 J- S7 C' Q/ q* _5 p. `
2
3/ d$ n$ @* Q6 x. E, e- t
4
59 f) C" E2 ]  p2 @8 U1 X
* 6) b# {+ r: K& k/ Y
0
1: V- _* o* s' T# b
2
33 o5 F8 ^0 y& d  h  g; @
4) K% @; P2 `" w! N9 O9 O6 k
5- ]- [. K8 A% v1 r1 J! v% u
6( A0 P) A% H9 l3 V
74 {" S: s) s0 O
* 8
MDA/vector
MDA/CCL51 G6 l7 E, L* T" `* g1 h
MDA+WI-38/vector3 K  p* K) r; P9 D( h* c$ p
MDA+WI-38/CCL5$ L1 [8 y9 V" R( d! r; F5 o8 S% Y
1# F. Q  I2 B6 _6 E4 b0 D+ _% B
2
3
4% H: |1 r2 H( y* }1 n
*
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-3 o( D1 U4 A3 [
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour
masses (mean6s.e.m., n56 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  y  G1 |( D% O% z- Z" _
Student’s t-test. b, A total of 500,000 MDA-MB-231 cells were admixed to
250,000WI-38 fibroblast controls (WI-38/vector) or WI-38 fibroblasts
overexpressing CCL5 (WI-38/CCL5) and were injected subcutaneously in
NOD/SCID mice. Tumours (n55 per group) were excised and weighed at
12weeks. Masses shown represent mean6s.e.m. Lung metastasis indices are
expressed as fold increase (6s.e.m.) over controls. Asterisk, P,0.01 in onetailed
Student’s t-test. c, A total of 800,000 indicated BCCs were introduced1 p9 ~! b$ l' F
into the circulation of NOD/SCID hosts. GFP-positive cancer colonies in the
lungs were counted 6.5weeks later. Bars representmeans6s.e.m. (MDA-MB-
231 controls, n516 mice; MDA-MB-231/CCL5, n518 mice). Asterisk,5 e  r% H. T4 I+ X% k6 L7 b
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/ a0 j$ W, A% m, }1 K( l5 y2 O" w+ F
loading control. e, Transwell migration orMatrigel invasion assays on 50,000
MDA-MB-231 control orMDA-MB-231/CCL5 cells.Data are representative of  ]0 a& R: y3 I3 H) \
multiple independent experiments and are expressed asmeans6s.d. Asterisk,/ d6 w, n6 T0 Z& P' ?
P,0.05; double asterisk, P,0.05; triple asterisk, P,0.01 in one-tailed4 l4 e1 }8 f8 a( H4 Q( g5 C
Student’s t-test. f, One million GFP-labelled BCCs were injected into the tail3 @) G2 [; r5 X' s0 ^/ J
vein of NOD/SCID mice. Lungs were processed 48 h later and examined for' F( M9 c& I/ z2 b
extravasated cells. Bars represent means6s.e.m. (MDA-MB-231 cells, n57) [- t9 |4 p+ P9 H
mice; MDA-MB-231/CCL5, n510 mice). Asterisk, P,0.01 in one-tailed. O( m: y, A0 }3 y3 I
Student’s t-test. g, Transwellmigrationassays on50,000MDA-MB-231 control% w' v  _" c; G( v- \# {
orMDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-; R: g* F% G* P
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;2 w7 E: z, _3 U$ d
asterisk, P,0.01 in one-tailed Student’s t-test.the epithelial-to-mesenchymal transition (EMT), in which cells shed
their epithelial characteristics and acquire instead a series of mesenchymal
markers that enable their invasiveness and intravasation21.8 O: W1 `+ N) w2 W2 ^
Despite their lack of E-cadherin and their expression of detectable levels; D3 V* U7 `* E2 n5 p$ v! x3 `
of mesenchymal markers such as fibronectin (data not shown), the/ ^- H: t2 \* l8 b. a
MDA-MB-231 cells studied here exist in an intermediary phenotypic
state of ‘partial EMT’, as they retain a distinctive epithelialmorphology+ k1 ~- d( }# x3 V2 L
in vitro and are still responsive to EMT-inducing stimuli in culture. In
fact, we observed that ectopic CCL5 expression did not cause MDAMB-7 e' `4 d/ w! ^
231 cells to undergo themorphological changes usually associated
with an EMT(Supplementary Fig. 9a), did not cause rearrangement of( e( ?7 F; U9 l$ N6 F9 _4 q3 z: F
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on; }8 ^3 n* [$ B0 h0 l, w
the expression of mesenchymal markers closely associated with the
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c)" E* b: v; @9 s& w  m
and fibronectin (data not shown). These data suggested thatCCL5 does2 A2 x9 Q$ O9 u* o" c- C
not directly promote the EMT programme of MDA-MB-231 cells.9 j& J, ~, d  S. j) a" H
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/ |. w! v: }& y& b
organs and the subsequent step of colonization. For that purpose,
MDA-MB-231/CCL5 cells were injected intravenously into host$ B% H% g! c5 K1 U: p+ L
mice, and the lungs of these hosts were examined ,6 weeks later) h6 I. N9 s6 w$ X' T% S4 k5 y+ Y5 i
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  a+ z- l: `) l0 n5 c
(Fig. 4c), suggesting that CCL5 exposure has effects on later steps% E$ R$ }' e' K) o& i
of the invasion–metastasis cascade. We note once again that this
enhanced tissue-colonizing ability was not due to CCL5’s effects on  b8 \* _2 z' v0 j+ ~# X) L
cellular proliferation measured either in vitro (Supplementary Fig.! N) D6 C- _1 c% S, h3 P8 o
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" M# ]& U  _+ h5 c
levels of the Ser 473-phosphorylated, activated form of Akt, but
exhibited no difference in the levels of other pro-survival proteins,- C. I9 u* d# Q
such as Bcl-XL or Bcl-2 (Fig. 4d), or a reduction in the levels of3 s5 Z1 \# x& ^  P5 C7 R
pro-apoptotic molecules such as BAX or BAD (data not shown).
Moreover, we found that overexpression of CCL5 had no effect on2 O: x9 a# p& }: n# c
the ability of MDA-MB-231 cells to withstand serum deprivation9 }: w* T( X# f: b/ \# T- k8 [
(Supplementary Fig. 7b), loss of substrate anchorage (Supplementary
Fig. 7d), or hyperoxia (data not shown). We also observed that$ I' e/ P2 m% q2 {; e
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;' P* Q2 ?, m% k; j( ]" n
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling, |3 U6 B/ ~, X
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohistochemical
analyses on control and CCL5-overexpressing tumours3 N% A: u$ {/ Q1 _* V( T
revealed only minor differences in the levels of apoptotic CC3-2 x& _3 u- D! t' k+ y
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 consequence7 F, d3 Q& {- a- L  Q* O
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 motility22. Because CCL5-induced
Akt phosphorylation did not correlate with enhanced protection& T) z8 W, `- F4 F
against apoptosis, we tested whether the CCL5-enhanced lung colonization! D# v5 V1 N( b0 {7 ~, H
could be due to an increased ability of MDA-MB-231/! d9 w+ G) R2 ^
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-, g8 C# [9 z1 B# J4 j/ g8 _$ s
fold in either high or low serum conditions, respectively (Fig. 4e).
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein3 i' C  ~: b" W0 a
injection—in order to remove most cells that remained within the! q; D) q& c6 T, {
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% q* x& U5 d/ i! ~
clear effect of CCL5 on cancer cell extravasation.
Finally, we investigated the role of Akt in mediating the actions of; ~4 i  v0 s" U
CCL5 on cellular motility by using the phosphatidylinositol-3-OH' \, b& C- {  O0 R; n+ }
b silacZ
silacZ
si809
si809
si186
si186
CCR51 {& W! ]6 A4 f  H  [
β-Actin# x7 q5 J* E+ v* R' O7 `
a d. u6 }9 ?9 X8 X- h  L
M
+
MDA+MSC
+ IgG
+Anti-CCL5 Ab) Z# t; S' ~* Z6 K, C6 Y9 p: B
MDA
+Anti-CCL5 Ab& R4 w; T. d& O% M) ?6 X( M. s% t
MDA+MSC, {6 q. g! c: k4 B) l0 \4 h! {7 j
MDA7 c$ l& S3 X: [9 g. q
IgG
<1 K5 l% b5 V/ R4 }
<6 p% G# m: @- n' E0 a- R
<9 g0 g4 e  D1 Z- b' U8 t
<
<
DAPI
CCR5
MSCs MDA MDA+MSC) f+ e, v: T3 {9 ]+ a7 q
DAPI DAPI/ }4 d9 X3 ~! ?8 d1 |- _
CCR5 CCR51 k% c' S) S/ k- Y- i. h6 v/ n
MSCs MDA MDA+MSC
*
c- A; s. i  Q6 l2 U' ?3 R
Metastasis index (fold)
Metastasis index (fold)) o; z& J- Z9 }4 p( P: Q& `
*. I" u1 g- r. d& P
Control siCCR55 ?% C9 t& d" G; k3 K- f
MSC – + + +" w, b2 d! w5 F& A
0
1
2
3
4$ z, \, q2 O. m$ T
5 e
+ + – –
Anti-CCL5
02 g% z6 |. F, J8 ?
1. }' x$ q/ W- s  g( T+ s
2
36 }6 \6 h7 |# M* Q! S% i
40 h8 d! f, A- G7 n8 U
5
6
– – + +4 v' x  G. c! Q' ]8 T8 C
IgG! R  \; r7 O2 }2 @5 x1 [# b
MDA! O7 p& S' W3 b' u6 g8 P' ~; H% i
MDA+MSC
<
Figure 5 | CCL5–CCR5 interaction is essential5 c" t# G" K* W& R( G# M
for the MSC-induced metastasis.
a, Immunofluorescence analysis of CCR53 d! u( Z- c$ I" S6 _' ?
distribution in MDA-MB-231 cells cultured with
MSCs. DAPI (for nuclei staining) is in blue;- O' ^9 K. a  W& ]5 [9 V
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 a4 b' C, o% |7 w! ?
loading control. c, A total of 500,000 cells of the
MDA-MB-231 variants in b were co-mixed with% f' _& o% I5 q$ a/ z% b
1.53106 MSCs and injected subcutaneously into8 H6 k: z- p' t' T  W/ A7 z
nude mice. Mice were killed when tumours
reached 1 cm in diameter and the metastasis$ _, Y' `& K' q3 ]/ r' G% i
index was calculated for each cohort (n55 per9 G: O0 ?( ^% q$ P: r3 }4 z
group). Results represent means6s.e.m.;
asterisk, P,0.05 using one-tailed Student’s
t-test. d, Anti-CCL5 neutralizing antibody or  C9 H- F/ u2 b7 H
control IgG was administered intraperitoneally; k" k2 F4 t9 X0 n: G
twice weekly in SCID mice bearing MDA-MB-231# b- p; }4 F! g8 w( |+ T0 P
(n59) or MDA-MB-2311MSC tumours
(n511). Representative lung pictures of the% ?- t1 \0 i& K# ^
indicated cohorts are shown. e, Lung metastasis2 J) [3 F7 \. {( D7 ^
indices of mice in d. Data shown are. g# f& O+ Z* B* [- U+ d
representative of means6s.e.m. Asterisk,, o/ K: Q2 m) h! W. i* _% `
P,0.05 in one-tailed Student’s t-test.$ H9 W/ C# F) p( H6 f! d7 j4 ]
kinase inhibitor LY294002. Drug concentrations that did not inhibit
the basal motility levels of MDA-MB-231 cells blocked the elevation' W3 b. I4 {' x- D6 D$ ^$ p  N
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 loop1 ~; O4 e2 a5 S1 Z- r4 L% I
CCL5 acts through three G-protein-coupled receptors, termed3 V9 Q, `, @$ _% x8 Z9 n, @. ~
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 chemotaxis24. We therefore
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 MDAMB-$ R' B: l$ N& J* p
231 cells by more than 85% through shRNA knockdown (ref. 25
and Fig. 5b), and mixed these cells with MSCs before implantation5 V2 v! W$ M& h! `
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,# w- B2 p2 _$ D: h
achieved using either of two different shRNA constructs, abrogated: W3 ~3 k9 T0 f  }, i8 _
the ability of MSCs to enhance the metastasis of MDA-MB-231 cells6 |+ t' ]# a9 }3 L
(Fig. 5c). Furthermore, neutralization of CCL5 protein using intraperitoneal
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 inhibited6 J# x7 P* E# v* e% Z% A
by shRNA knockdown failed to promote metastasis of the$ M1 |4 M7 r: O
admixed MDA-MB-231 cells (data not shown). Taken together, these$ O9 O- _- \0 _8 ?/ A' g: V3 h
results underscore the critical importance of the CCL5–CCR5 paracrine1 K, O( ^, T6 O3 [& P
interactions in enabling MSCs to induce metastasis of the4 a( q# ?6 w! C+ @) z3 D, f
MDA-MB-231 cells./ R9 n) T* @0 V* I
Discussion5 [6 i5 K# b4 R' M. s' w4 C
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: h% e: R5 n% k/ G9 {: K: ]( P
primary tumour. It follows that if carcinoma cells are subsequently
deprived of such signals, they may revert to an earlier phenotypic4 A3 G2 Q# V; P/ l8 V  O" _1 z
state in which they no longer display the traits of high-grade malignancy.1 G7 m: I9 j. B- x0 y# O6 G& e: l; ~
Indeed, such a model has been proposed previously by others
on the basis of indirect evidence21. Here, we demonstrate that at least! F2 s0 p. Z& `7 A$ h/ R
one mesenchymal cell type, the MSC, can expedite tumour metastasis,
and suggest that after primary human carcinomas recruit MSC6 g& r* G- z7 h2 I# a
populations into their midst, subsequent interactions between the+ `9 B; S# u( c& c
MSCs (or their derivatives) and the BCCs endow the latter with. i( ?) O1 F! _! y
invasive and metastatic properties.! P9 V# @2 Y. h( d( D. F' u
Although the recruitment of labelled MSCs to tumour xenografts, L/ E9 N, ^5 g
has been established in a variety of experimental models of tumorigenesis,+ k( z( ?. X& G
there is currently no available way to quantify with any accuracy2 @6 M7 [2 ]: \
the number ofMSCs in actual human tumours, in part because no set- k/ s. W/ p$ T  _8 N
of markers has been identified that can uniquely stain these cells without
concomitantly staining other mesenchymal types in the tumourassociated
stroma6. Our demonstration that the stroma derived from+ N+ }! y" g  e) ?$ O
tumour xenografts contained appreciable numbers of murine MSCs8 r" A1 H& |: L* t
indicates that significant steady-state levels of these cells aremaintained
in developing tumours. Interestingly, the use of CD10—one of the
markers associated withhumanMSCs—to purify cells fromthe 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,9 v7 ~" B& E" l
we note that CCL5, which is prominent in the stromal gene expression4 v" V0 x8 V8 X! H" |- Y) Z7 I
signature associated with poor prognosis of breast cancers26 (SFT;' T: g+ B6 U7 _% @: ~5 W
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free
stroma of primary invasive ductal carcinomas (Fig. 6d), specifically in, ?5 G# t, B: y" [" \& ^) [
the CD10-positive compartment27 (Fig. 6e). Collectively, these observations' H% y' b" N) T) p7 R2 m5 C! x5 w
argue strongly for a significant association between stromal
CCL5 levels, MSCs and human invasive breast cancers.
c# U  {( m$ e; z; U% [
STT1969B
STT3126
STT31242 S: o- s# D; B
STT656B, X1 G4 o& W# ~
STT1968B
STT3122
STT30538 v. h5 W: r- u/ I7 u7 X
STT1986B2 Y% F0 O  {* m
STT854
STT3125) _. f% R3 G8 J2 L& S9 ~
STT1975
STT1987B
STT10792 v6 Y2 K3 Z. C$ v
STT638" C) D% L, }; X4 v, N5 g- a6 _2 I
STT1774
STT1984, w. E- x' R* P; c) E% u( J
STT1737C( C" I: w" R  a" E; }+ U/ k; _
STT30684 i7 a" ]' N5 i. ^& g! J
STT31200 i! j: b# \5 t. ^
STT8504 N, ?' x0 ?: D+ K0 o( A
STT417B
STT3119; L, }6 B* L8 F
STT1776
STT1777B
STT689B
STT1971/ B  R, q- B1 C
STT597
STT626% ~! y+ t+ _+ a6 t( T6 P
STT154
STT2774& ^% q; n: B- V9 G. W
STT19668 I. w3 \; y) ?* e! \# v
STT2776+ }2 y6 k. c0 K! C  O7 q0 U  S
STT2775: k$ \' _! \% }. Q
STT2772! v# @" J8 v2 N! ?  m0 _3 h
STT1637
STT1220C' k% x* H5 n9 y7 n  ?7 r
STT-094B-1: Q: ]8 L: P8 B3 ?
STT675
STT2770
STT695B
STT1771; [( F7 _9 M& v! w7 s: b5 b# _
STT1778( Y( i% @; y) ^. R. Y" U
STT4910 o; `% A; W* D( O1 L
STT18238 d% ~& T; c$ Y
STT200C
STT741B
STT335C  Q% x4 q3 m0 s5 M( }. Z
STT709B
STT516D8 L+ V$ q5 E% I/ A6 Y( A
STT607B0 K& E3 ]8 q5 L% @6 |& {
STT680B9 \& m! f* _4 J2 E/ g9 {, h0 Y
STT1148B
STT523B5 E1 C  G. e5 J7 x
STT526E
STT742F3 g/ g# X9 B; n8 X: T# J
e, ~* H+ x) ~5 W$ @: I
CD13( i: W" \0 l/ _) w; e# u, t; A
CD292 ]8 `% ?6 m6 R/ F) s2 Z8 c
CD44
CD49e
CD54
CD59
CD63. d# O/ d1 n- B2 s2 T+ x
CD1050 s" L) ^, G0 D
CD106
Nestin
HAS23 L" y& q3 }& c- u
IGF2. a0 H3 v% d1 Y, s- [: P1 f. c9 ?
PLAU5 r1 }+ p% J- c. b' g+ f& U0 s2 I
TIMP19 p1 c& A* t7 _- a8 e/ M( M' {
CAV1
IDC-7
T112603
T392303
Normal Invasive
CCL58 Y* m% h0 m; ~
log2 ratios
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.00 R/ Y+ n& ~" b8 s3 d
log2 ratios( l. M. F0 y; j! E
d
–2
–1
0 1 2
–2.0
–1.5& e- b9 t& V7 c% u
–1.0  S* J' T' c* [# l& V5 a8 a
–0.5
0.0
DTF SFT! r9 g4 d  R6 ~& E: P& i$ v
DTF, f- P$ E  \: L- M# F$ _0 V
SFT3 K( Y+ C3 G" L; X
a b
F
Figure 6 | Stromal fibroblastic cells of human invasive ductal carcinomas are7 P- {0 J  V( U' g+ D! Y) d9 c# _
rich in MSC markers and overexpress CCL5. a, SAGE TreeView display of
MSC markers expressed in stromal CD10-positive cells from invasive6 u; p* C4 p7 O
tumours27. b, Soft-tissue tumourswere ranked byCCL5 expression26, fromlow
(green) to high (red). Wide blocks indicate expression ratios of tumours
classified as desmoid-type fibromatosis (DTF; yellow outline, n510) or/ ^4 H5 ^4 ]) |: H+ r& r8 M
solitary fibrous tumours (SFT; blue outline, n513); narrow blocks are other
soft-tissue tumours (n532). c, Box plot showing that CCL5 expression is
higher (P50.004) in SFT than inDTF. The difference in log2 expression ratios. _! M% S6 ?0 Y" W* F& v
between SFT and DTF was tested with the Welch’s test. d, CCL5 Affymetrix/ k0 q7 N  A- j
gene expression in the stroma of human invasive ductal cancers compared to3 c: j3 N- [4 l+ M
that in normal cancer-free breast tissue (indicated as ‘Normal’; see Methods).
e, CCL5 expression is mostly restricted to the CD10-positive fibroblastic cells  y2 e% R8 O% q' B0 M) L
derived from invasive ductal cancers. The heatmap shown is a cluster of3 A  R+ f  `" E" C4 i) U1 d
CCL5.genelist obtained as in a.Details of thepurificationmethodologies of the$ w1 S6 P: \' I% t, n1 U
various groups indicated in a, d and e are found in ref. 27.Although we have focused here on CCL5 in the MSC–MDA-MB-4 l. W1 q$ g) v; f3 M
231 cell interactions, CCL5 seems to have an equally critical involvement: f. v. J8 f7 F* B' y4 q, M
in the functional interaction of MSCs with MDA-MB-435/ o4 j3 I  k) n0 p
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 knockdown1 _8 l& G6 M8 \" m0 k$ W
failed to promote metastasis by MDA-MB-435 cells to which
theywere admixed (Supplementary Fig. 10b).With these facts in mind,
we point out that CCL5 does not seemto be involved in regulating the9 E8 g! K2 }6 _
MSC-induced metastasis of MCF7/Ras or HMLER cells, which may) d- Z$ O7 Q5 k. J5 |( s
depend on other paracrine factors such as VEGF and interleukin-8.
Nevertheless, our observations highlight the recently discovered critical4 F4 P; |- k0 H3 z( ~% t7 ~  F
roles of chemokine networks in malignant progression28,29 and suggest4 v' w! V, b! k, {- A+ ]( l
the possible utility of a variety of CCL5 analogues and CCR5 antagonists# O: c  N! M6 f& w7 o3 H
currentlyused in anti-HIVtherapy30 in treatingmetastatic disease.) Q( d, W2 s. P6 _1 ^
Notably, we have observed that MSCs induce the metastasis of cells' E) z- z4 D' O/ K3 p; d
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  X/ Z. B* V  S
maintenance of this phenotype depends on continuing contact with6 A: C3 q4 e$ F; F3 }+ J7 K; a0 S
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 associated) ^% r. P; b  ]8 d, D
with invasion and metastasis are often not expressed constitutively* W5 Q. h9 s6 p) Y8 u) q
by carcinoma cells, but rather only transiently in response to0 K- x; l+ _# t0 D* S
contextual signals that tumour cells receive from their stromal microenvironment.
If so, analysis of the gene expression patterns of bulk- ~  M) B$ e  ]% X% w: H
primary tumour populations may fail to detect the expression of key2 Y9 L& @" D' \
genes mediating invasiveness and metastasis, if only because they are
being transiently expressed in minor subpopulations of cells within" X7 u. r1 [( f2 r
such tumours. Additionally, attempts at determining the metastatic# g1 }& r. w; A2 z3 l3 x  |5 R
propensities of tumours may need to be focused on the genes and) |$ X) V! l+ q* h0 f+ O7 n
proteins that confer responsiveness of primary tumour cells to stromal" m' v! l! I4 z  u8 Z, V3 ]
signals, rather than on the genes and proteins that directly mediate
the cellular phenotypes of invasion and metastasis.
METHODS SUMMARY6 J) \2 Q0 ]: T) V) t3 U7 @
Cells labelled with GFP or ds-red, or harbouring various overexpression or/ C$ {9 `& o8 n/ Q0 r
shRNA constructs, were generated by viral transduction followed by FACS5 |. T( D, J* P0 {5 Z) A
enrichment or antibiotic selection. Xenograft experiments were conducted in/ ]0 l- C5 b2 Z8 A& G0 h
nude or NOD/SCID mice and metastasis was estimated using fluorescence/ e+ z7 H; A5 F9 T3 Z( {
microscopy. The levels of cytokines, growth factors and chemokines were
assessed by immunoassays. Migration and invasion assays were conducted using2 c& W8 k* W4 `, s7 i, i9 R
transwell chambers. Antibody treatment of tumour-bearing mice was conducted9 k" L# t3 x9 B; C5 T6 _  t
by intraperitoneal injections. See Methods for detailed information regarding8 Z5 ^% f& Y6 s) x2 D
cell culture, viral infections, in vivo colonization and extravasation assays, RT–/ X) u) C* ^% w6 `
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluorescence$ N: M7 [) Q9 W& [' a5 Q
determinations, western blotting, and antibodies used.
Full Methods and any associated references are available in the online version of' H8 l; L& \- I, C# Y" V
the paper at www.nature.com/nature.

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