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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 the: b, Y7 e$ S" m" o9 ?
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 humanmesenchymal 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 tumour7 _. a/ R" z) G( }1 i2 }* A- E0 o9 k! G/ n
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" b3 d' l4 R. a; P. U* s! Y: l
metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine0 W4 E6 j, i, ?# R8 h
receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by; f, ?' {* O) z
eliciting reversible changes in the phenotype of cancer cells.
The origins of the invasive and metastatic phenotypes of carcinoma3 I- j" _! [+ E( j0 P1 V* `8 ?
cells have been the subjects of intense investigation. Whereas some) E* I  u, R: r; h, S. P
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% V: ]+ j1 I3 ~- [4 e- g
cancer cells to paracrine signals that they receive from mesenchymal9 r0 @! f, |: X: R
cell types within the tumour-associated stroma. Although several" }& ?$ d* {9 K3 j) A
lines of evidence demonstrate the contributions of stromal cells to
primary tumour growth1, direct experimental demonstration of the1 k& |2 d3 V8 r2 ?/ ?' [# g. Q/ h5 g
influence of these various cells on the metastatic abilities of cancer' i$ z, I3 A* z5 ]
cells has been difficult to obtain. This is due, in part, to the complexity5 s( m4 v* k" B  ?1 ]; c0 K! \
of the mesenchymal cell types that are recruited into the stroma, and' d. w5 X1 H: T7 y2 A
to the elusive nature of the putative paracrine signals that are9 P3 y4 g- x5 s9 F( L
exchanged between the mesenchymal and epithelial compartments. @, M1 w3 N" C! @  O% U
of a tumour. Recent reports proposed that the bone-marrow-derived6 X1 s, t5 x- W7 S1 O# S4 f& R
mesenchymal stem cell (MSC) is a cell type that is recruited in large% ^& h6 [; K3 c, x1 C) a
numbers to the stroma of developing tumours2. To characterize better
the role of this stromal cell in tumorigenesis, we set out to determine
whether MSCs could supply contextual signals that serve to4 a1 `4 p9 ~5 X% y6 y9 h5 {# Z
promote cancer metastasis.8 d# d! ?2 B5 U/ \8 j" ~
Mesenchymal stem cells are pluripotent progenitor cells that contribute( o; n9 q9 @: q0 U& Q
to the maintenance and regeneration of a variety of connective
tissues, including bone, adipose, cartilage and muscle3. Although% n3 ~; ^4 x5 D6 d8 v9 Z! P
MSCs reside predominantly in the bone marrow, they are also distributed
throughout many other tissues, where they are thought to- z5 b) K3 t$ t8 `* S
serve as local sources of dormant stem cells4,5. The contributions of
MSCs to tissue formation become apparent only in cases of tissue1 O1 r! _& y9 T$ K) Y8 ]
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 the3 S( X4 |, O" [# v: q- u; Z: B6 Y
bone marrow, leading to the mobilization of multi-potent MSCs and
their subsequent recruitment to the damage site6. For example, MSCs
have been shown to contribute to the formation of fibrous scars after* w3 t4 x! F* S! {$ \
injury7.
The formation of breast carcinomas is often accompanied by a- f0 v* W; v' `) n8 ?8 D) h5 j, X
well-orchestrated desmoplastic reaction, which involves the recruitment
of a variety of stromal cells with both pro- and anti-tumorigenic
activities1. Such response closely resembles wound healing and scar
formation, and entails the constant deposition of growth factors,. g+ u" K; q$ m3 l% {
cytokines and matrix-remodelling proteins that render the tumour4 V4 U, H, F3 S8 O
site a ‘wound that never heals’8. This suggests that, similar to sites of9 X1 H) u9 G% l$ l
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: B  v2 ^9 z- R; M5 O
to generate fibroblastoid colony-forming units (CFU-F) in vitro3 s; D- K! B5 u6 S' j
(Supplementary Fig. 1a), a hallmark of MSCs3. The absence of such1 u0 c# f! V' r6 {( c, d2 [2 S
colonies from control Matrigel plugs or from neighbouring tissues. }3 G$ j8 v" J1 e5 _- _. @
(negative control; Supplementary Fig. 1a) suggested that endogenous7 q: [4 H$ z6 Q5 w2 V$ t" |+ J
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+ \( \. Q7 O- D  G  h* v& n
in which bone-marrow-derived human MSCs were allowed to! ^! y- n2 H0 r0 E
migrate towards media derived from MCF7/Ras or MDA-MB-231
cultures. We found that human MSCs migrated much more avidly4 r7 i0 q1 _4 F9 C# H( `
(,11-fold more) towards media derived from these cancer cells
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$ M1 Q8 i1 a0 i4 r/ a" Z- D3 S
or MDA-MB-231 human breast cancer xenografts localized specifically# `. Y4 V9 _; }( x: _- j) k/ O2 a
to the developing tumours, with no observable accumulation
in other tissues, such as the kidneys (Supplementary Fig. 1c), liver7 a2 l8 Y( Q3 T
and spleen (data not shown). Such findings indicated that MSCs
are specifically recruited by subcutaneous breast xenografts, and corroborated$ }, C: o/ L0 X; G
recent studies that described the localization of systemically5 s* N# o& x6 x, p& U4 }6 E
infused MSCs to other types of malignancy, such as gliomas9,10,0 @$ `2 g' Z1 u
colon carcinomas11,12, ovarian carcinomas13, Kaposi’s sarcomas14 and
melanomas15.MSCs enhance breast cancer metastasis
To investigate the functional consequences of the heterotypic interactions
between MSCs and mammary carcinoma cells, we established3 [" ~/ f% M' F  Y' x
a xenograft model in which GFP-labelled MCF7/Ras, MDA-MB-231," d$ m; H! ^5 w/ A* K0 ], R
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 MSCcontaining1 A0 g5 R. B, Q6 T. n$ Y) A
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.1 b: [5 `, Y! ]8 Y
We found that MSCs accelerated the growth of MCF7/Ras
tumours without affecting the kinetics of MDA-MB-231-, MDAMB-7 s. @2 d: @' ^5 p) A6 O$ R3 g( X% }8 F. c1 Q
435- or HMLER-containing tumours (Fig. 1a). More importantly,
whereas mice carrying tumours composed only of BCCs; q6 X( }! R, p
exhibited few microscopic metastases in the lungs (Fig. 1b, d), mice# r& @. J, x" e. F3 l
bearing the mixed MCF7/Ras1MSC, MDA-MB-2311MSC, MDAMB-  O# w/ _' d) H
4351MSC and HMLER1MSC tumours displayed a marked
increase in the numbers of micro- and macroscopic lung metastases: h7 p4 U7 h. z/ \
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs
of BCC1MSC-bearing mice compared to their BCC-control littermates; O/ G9 d) {- X" w" k+ J; B) e
revealed two-, three-, four- and sevenfold enhancements in, }9 {! }  Y3 W# s5 F9 [
the overall numbers of detectable HMLER, MDA-MB-435, MCF7/( |8 x# R" {& X/ v. D8 I
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c).
Furthermore, in contrast to the MDA-MB-231-bearing mice, the% _' z  U+ k" x0 A+ @3 y& H
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 enhanced1 a4 y  S& m7 H; c" k
metastatic potential after admixture of MSCs, we chose to focus3 i. R9 l4 v" y* p
further analysis on the MDA-MB-231 tumour model, because it
displayed the greatest relative increase in MSC-induced metastasis. |4 d5 B/ w# p' R5 H; Q
without any concomitant effect on either tumour cell proliferation
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall primary
tumour growth kinetics.1 j6 |$ m7 W0 ]! r) f' W( b
We note that admixture of other types of mesenchymal cells—8 ~" C8 m5 R; f4 t# @5 n6 H1 t
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 39 a+ F9 }+ l# {/ Z
and data not shown)—to MDA-MB-231 cancer cells before injection, {1 J4 O" P' [
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 indicated
that the metastasis-enhancing powers were a specific property9 R; d* `2 R9 L. a0 Q( k- c& h
of admixed MSCs or derivatives thereof.
Reversible metastasis  l  }+ V" C4 B/ |
Implantation of MSCs either contralaterally to MDA-MB-231 cells or3 l/ Y5 J) o  N* K5 O7 y2 `+ z
in nearby separate sites of injection did not affect the metastatic
potential of the resulting primary tumours (data not shown), indicating
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; p, n0 G1 R! {; {
carcinoma cells. Thus, the MSCs might favour the outgrowth of rare9 ^! V9 e) b" {  F
variants within the MDA-MB-231 cell populations that exhibit3 z, k+ n+ C+ z% L  H0 y) v
unusually high metastatic powers. Alternatively, the MSCs might3 b, \& }' M+ U, B2 h# @5 n
cause otherwise weakly metastatic MDA-MB-231 cells to acquire
enhanced metastatic abilities. This latter mechanism suggests the' c3 W( g8 E( s  I% L
possibility that the acquisition of the metastatic phenotype might
be reversible, in that carcinoma cells might revert to a lower metastatic
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)
as well as from their derived lung metastases (L-explants),, o/ i# l/ v+ h9 W
expanded in vitro, cleared from contaminating stromal components,$ P) C  x! C8 Q, ]; y8 @
and then re-injected into subcutaneous sites in host mice in order' y/ D+ B2 l/ C( W( O5 o: ?9 k! f1 X
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" @4 F9 I8 T- N; L6 w
a
c d  u* P1 D: C. R! U% p5 S% b: a& s
Days after injection
Tumour volume (mm3)
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( q0 a4 W/ J( G8 w  P( d
b
1 mm 1 mm& _! W4 Q5 b- c, B' N4 ~3 {. d
1 mm 1 mm
300 μm1 ]8 x  V5 }* V% i
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC
MCF7/Ras MCF7/Ras+MSC4 C: c. d# h- t2 d5 O
100 μm, q4 H9 n* k4 _, ^% o0 l8 h$ f: `( a& @
300 μm
100 μm
MDA-MB-231% u2 x) w# M  Q7 w. ~) I4 b  P7 T7 g
MDA-MB-435
MCF7/Ras
**
Metastasis index (fold)
05 I0 A0 ~7 c8 l! i) ?$ {% Z7 M  v
1) n* E/ i8 [  K) t# U+ o. K+ k6 Z
2% u7 f# p9 F9 ~. Z: N
3) c* C9 ~7 m/ l; x: }# J8 u0 x
4
52 d" s  ~) [3 d+ i) Y# b
6. a/ B' t5 h" S5 x; a  N6 n
7) h, V7 x. e& y0 M5 |  f
8( s! y5 g$ p4 `- J7 }- G1 P
9
*
**
**! I  o4 T& p$ v- X/ y4 _
MSC – + – + – + – +' M" b/ u+ P' `+ P( d
*/ |9 S1 @8 \& Z0 D$ i
HMLER! y9 ?% T) y) R2 K! ~' ~$ v+ j
700 1,200; q9 S& u# R# ~
1,000/ d: q; D, e) R  r7 A' i
800
600  `: I- v7 L3 F' W
400% N# e5 k# T8 f9 p- F
200
0
800
700
600
500: Q, U! x1 |) B5 C6 ^/ d( s
400
300
200
100, M1 {8 V0 Y5 P
0
3,500
0
500- i$ n* z' }2 T) s. G: i' R
1,000+ v4 C! X: ~6 E4 ]1 R
1,500
2,000) R5 }$ b5 T9 x6 C4 `5 Q
2,500
600 3,000/ w( o  z$ R# i- g  o# q7 |7 G) i
500
400
3009 A# A' a* D2 V: h) ^+ P* U
200
100% f; z- J7 U2 O+ z# F2 M9 R' U
0
0 21 31 38 49 56 63 70 78: A8 v. F9 ~" ?
MCF7/Ras alone
MCF7/Ras+MSC
MDA-MB-231 alone
MDA-MB-231+MSC+ G# v+ X  L. {$ ?$ G
MDA-MB-435 alone% e* u. S! q1 _, ~
MDA-MB-435+MSC$ b6 _- a8 F2 D+ }; J
HMLER alone1 z0 }# a& ^1 F  F! i2 F5 W
HMLER+MSC
HMLER HMLER+MSC
MCF7/Ras MCF7/Ras+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC3 V  B( t& a; Z( v
Figure 1 | MSCs promote breast cancer metastasis. a, Tumour volume. B( Z0 Y3 f' q# q. ]4 |+ W7 o! J
measurements (mean6s.e.m.) of 500,000 GFP-labelled BCCs injected( V: |& o6 a* e
subcutaneously into nude mice with or without 1.53106 MSCs.
Representative data from multiple experiments are shown. Diamonds, BCCs! H  O& ?, S+ h
alone, n55–7 mice per group; squares, BCCs plus MSCs, n55–8 mice per
group. b, Representative bright-field/fluorescence images of lungs of mice
bearing the indicated tumours. Cancer colonies are in green. MCF7/Rasbearing4 C* ?2 Y7 B4 J7 j
mice were killed at approximately day 150 to allow these tumours to; |- Y6 H, m! t- S- z8 @
grow to comparable sizes to their MCF7/Ras1MSC counterparts. c, The: [% h4 X/ C& f/ ~4 m5 W
lung metastasis indices pooled within each cohort of mice in a are expressed# j1 I( T/ a  \' r: |
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 onetailed9 `9 ?3 ^& `: z. I, r& s. `! X' Q. F
Student’s t-test. d, Representative haematoxylin-and-eosin-stained
sections of lungs of mice bearing the indicated tumours. Metastases are( r5 S# z8 v+ B- w5 t5 k
delineated by a dashed line.MSC-induced metastatic powers reflected a reversibly induced trait
of the MDA-MB-231 cells, and that the ability of these cells to metastasize8 E+ i9 ]% y% }0 t) c  F0 x
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. {, ?$ Y4 o6 H* I' C) u9 S
stable fashion.
The effects that the MSCs exerted on the BCCs might have$ S8 q. X% E* V
occurred within the site of primary tumour formation. Alternatively,$ y4 c# {/ G; ]+ z- C
the MSCs might have accompanied the metastasizing BCCs
to sites of metastasis formation. To distinguish between these two  f3 w1 q5 `) N8 D
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled" ~6 C# s3 c( Z
MDA-MB-231 cells and implanted the mixture subcutaneously in
host mice. We found that the tumour-derived lung metastases contained' J1 v6 V0 C, }- m+ {& I. j! K
green-labelled MDA-MB-231 cells but no detectable redlabelled
MSCs (or their derivatives; Supplementary Fig. 4a) when: I1 [" R8 n) m$ [
scored 4, 5 or 6 weeks after primary tumour implantation. The; k( Z. T( _# }! G6 S! c2 t6 o" p
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- X$ {% D: ?/ B8 l; k
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 prometastatic
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% ~8 y$ o$ o- w/ D: T- c$ E
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 cytokines,7 ]# ]; {$ i' E  d0 c. Y: q
chemokines and growth factors using the Luminex-based Bio-
Plex suspension array system (Fig. 3a). In some cases, the resulting3 t, Z3 @2 ]) {  U
a
b c d2 G0 f1 h8 k0 Z& U
Days after injection
Tumour volume: l' l( m3 H, T+ M
(× 100 mm3)) o6 J) l/ o: _/ {* ^1 k0 m" N
09 y! _9 Y9 z4 h+ a
52 ?" V8 o4 T% F- \; x
10* n5 s' P, s; f3 L
153 a: U5 [8 H8 z* q9 A8 g0 W% N! Q
20
25
14 17 21 24 28 31 34 38 42 45 48 66 71 77
Primary tumour explants# o8 ~/ N! `& K8 s
Lung explants
MDA-MB-231
MSC
+
Lung explants
Primary tumour
explants; R: b7 {' U2 ~+ v
Antibiotic% Z! I0 T/ A" W( T; g' [
BCC selection
T-explant
L-explant
T-explant
L-explant4 L1 Z- A, `$ Q
Tumour mass (g)
3.0& i8 O/ L9 K- K! K
2.00 _' M4 s, W: {% m# Q' j4 _0 _6 t
1.0. @) [+ I9 W3 F9 |( ^+ F$ R7 y
0 0
0.4, {) J* \/ [$ v" I; e7 C
0.8) P3 ]. m0 B' S. Q5 g1 A
1.2
1.6. A  D$ E# ~; G! L" G) V
2.0* x8 u' z# ?: e& }( G/ ^. z& f1 t! z
#
Metastasis index (fold)9 w. P' T, [. N
##, h1 O5 ]8 B( g+ _! Z* o
Figure 2 | MSC-induced increase in the metastasis of MDA-MB-231 cells
involves reversible mechanisms. a, BCCs were recovered from lung or. E9 }) u, U2 K8 ]5 m/ l
primary tumour tissues, cleared of stromal contaminants by culture in
blasticidin-containing media (5 mgml21), and re-injected as primary+ c% C3 ]$ R5 {# J7 l, c
subcutaneous tumours in recipient animals. b, Tumour growth+ Z, J0 R1 H8 Y  l9 K- p
(means6s.e.m.) of 500,000GFP-labelled lung-derived (L-explant) or primary
tumour-derived (T-explant) MDA-MB-231 cells inoculated subcutaneously.
Data shown are representative ofmultiple independent experiments in which
four different paired batches of L-explant and T-explant cultures were assayed; w8 W& Y  B  [9 w
in parallel. MDA-MB-231-T-explant (n58 mice); MDA-MB-231-L-explant
(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., `# m" f/ E- Y
a
CCL4
bFGF
VEGF+ L# \2 f* H9 D% N
IFN-γ
TNF-α
G-CSF8 v- p0 |3 T4 x2 Q
GM-CSF6 |; I0 S5 j0 _0 j4 J
CCL3$ Q" D0 T: X7 q
CCL59 Z! X/ @4 S& E6 b) x$ U/ Y8 W
MMP17 m; c! g4 J0 h* @4 @
MMP3. i- A5 M$ |- _7 k2 |4 q. K' G9 e) w
MMP9
MMP13
IL-1α
IL-1β3 y$ U% E' r0 H6 M: B/ W
IL-4
IL-55 P4 X# I6 |. U1 x0 x
IL-6
IL-7
IL-88 R! R6 k) A- J1 `, B: A% Q9 \( Q
IL-10
IL-120 t5 w9 {: d3 D3 n
IL-13
IL-17
IL-2- w. n+ }  ]$ h; O" S
TGF-β
Fold induction) K* F* v+ y+ P
MSC alone
MDA alone
MDA+MSC (2:1 ratio)
1 * * * * * * * * *
3
5. ^! e$ I/ `1 k' ?$ v( w2 S
70 V/ u" a( O* T5 c: W2 ^" e0 O
9; h9 u. I7 D# b2 K4 ^6 p
11+ `  h& r; ]/ D6 A8 E8 ]
13
60
b c0 t; D+ l/ @/ R0 g" r5 D
0.4 μm
2.0& u' c' j  x3 C1 i
1.8) {# O/ ]) `, I; B! t0 N0 R/ O
1.67 j/ `* ]0 l% V6 B; [
1.4) w9 Z/ @- g  f
1.2
1.09 z3 m- \# }& Q  q) u# j. B6 i
0.8
0.6
0.4
0.2& J, N' e+ G  d2 M+ Y& h
0 0
54 e0 [, }1 v! k! D
10
15
200 c$ W9 o% Y" t
25
30/ F  Q  d! v* Z: N
35
40
45+ K0 ?. e# p/ Q7 k" l2 i
50* T3 S6 n4 `, t' w% ~/ {# a
d1 d2 d3 d4
MDA alone
MSC alone0 I- A% n0 {8 B3 ^9 M
MDA+MSC
CCL5 levels (pg ml–1)9 L: _- l' y& }4 f3 P6 R
Co-culture
Fold CCL5 induction
MDA alone
MSC alone3 E9 u! T9 j9 t1 e
MDA+MSC
d
CCL5 (A.U. × 100)
30' A0 m! R' M5 R7 M: v( J
25
20( N8 w5 s/ t7 c& F4 g% R- S5 O2 W
15
10
5
0
+& [2 T$ U# H, N9 ^! f
MSC.c
+
MSC.18 E* X* g- U+ P7 B' h
+- K. H9 s" ?% s' A% Y  t  l* N
MSC.5( |; k& Y- o9 T* \) I& u
MDA
MSC) V" [; Q$ Q8 R; |! {9 d
MDA.1
MDA.c
MDA.55 D4 C; n' |7 t8 ^7 S0 L1 k" `% y
TC-MSC
MSC (from MDA tumour)
BCC (from MDA tumour)! R0 x0 _5 P0 H' @" v
Control (MDA/CCL5)2 F$ ~" C4 z# ]  R( }
CCL5
GAPDH
e5 @  K  d2 c, _9 C' n4 H7 y( C
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% m* C5 C2 g5 I5 x6 B+ x0 t
supernatants were measured by xMAP Bio-Plex cytokine arrays at day 3, and# m# T6 a: E# o/ f3 `
were normalized to the levels observed in the media of BCCs cultured alone." }& @  x$ Z& p6 m! u
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
MDA-MB-2311MSCcultures (1:3MDA:MSCs) at the indicated time points.
Data points representmeans6s.d. of quadruplicates. c,BCCs were separated" }" _; V3 ]9 v5 [& B( J
from co-cultured MSCs by a 0.4-mmmembrane. CCL5 levels were probed by% {6 _; n9 G, ?* E4 P
ELISA on the culture supernatants. Data are expressed as fold induction over/ W) }3 p* ]! ^$ Y
levels seen inMDA-MB-231 culture supernatants (mean6s.d. of triplicates).
d, CCL5 ELISA on the supernatants of MSC-siluc (MSC.c), MSC-siCCL5.10 P! W/ ?; u& ~
(MSC.1) and MSC-siCCL5.5 (MSC.5) co-cultured with MDA-MB-231-siluc  O: R4 k0 x& I- |+ E* h
(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; P) `5 @, I1 L
(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( X! m' Y4 t! W! |- i
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 or6 ?6 T# }3 r! Q7 S/ ?% u
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& x, E5 E  o3 ?, J/ y
between the MSCs and BCCs, as it accumulated to levels ,60-fold! @% m0 |6 c  J$ O: y5 k  \
higher than those produced by pure BCC cultures (Fig. 3a). This
cooperative induction of CCL5 was proportional to the numbers of4 }! R2 O9 U) H# h5 B( F- X1 Z
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was apparent
as early as the third day of co-culture (Fig. 3b). Moreover, this
induction required close physical contact between MSCs and cancer4 F& Q; A8 i0 z6 r& E  C7 c
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) R2 a; H% X7 Q5 I8 g# m
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). Importantly,. ^( ?; h+ Z! w2 t/ J8 Q. @$ f' C
however, subsequent co-culture of these MDA-MB-231.1 cells4 a  i8 P6 T' D0 B! y
with MSCs continued to allow accumulation of CCL5 in the culture
supernatants to levels that were comparable to those observed in the% @$ Y8 A% N/ e( B$ O# b
co-cultures of MSCs and control cancer cells (Fig. 3d). This suggested- l; n3 m6 U) Q3 G, X+ b
that the source of CCL5 was the admixed neighbouring MSCs.( N  r& _; x$ W9 M. }  K
Indeed, inhibition of CCL5 protein expression in MSCs using the2 R9 E  l) B+ w$ ?: N
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 coculture
of the two cell types. In support of this conclusion, analysis of6 H1 t& e5 N) D& ]9 O
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
transcription (RT–PCR) analysis of the RNA prepared from these coculture-
derived MSCs (Supplementary Fig. 5c), as well as from the6 ~. n/ l. T, j% R3 _* d
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after" J$ s' }- R) B% @$ z
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/ o2 t! |: {- @- K# p* F
been correlated with the severity of the disease, and localized CCL5# `, a3 P) w* `* Q6 o8 g7 O
protein expression was found to be elevated in invasive tumours
when compared to in situ ductal tumours or benign lesions16,17.
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 overexpressed
this chemokine in the MDA-MB-231 BCCs (Supplementary
Fig. 7a) and analysed its effects on cancer cell growth and" K# M8 Z6 d( s9 ?0 C0 R; n
tumorigenesis. The overexpressed CCL5 did not confer any proliferative
advantage on cultured cancer cells when compared with% d. {# c3 W6 o+ E  j( f: V
those lacking such overexpression (Supplementary Fig. 7b), and had
no effect on the ability of BCCs either to grow in an anchorageindependent
fashion in vitro (Supplementary Fig. 7c), or to form% E* V" k. f8 D: O3 Q( T' G
primary subcutaneous tumours in immunocompromised mice (Fig.4 N. e' r7 A6 [; Z+ C
4a). However, these tumours exhibited a ,5-fold enhancement in
their metastatic potential when compared with control tumours* _* n) F: n. l' n8 p& E% A7 y
lacking ectopic CCL5 (Fig. 4a). Similarly, overexpression of CCL5- r3 L! i+ N  f- J+ y" k1 f! u. S
in WI-38 fibroblasts sufficed to enable these cells to promote the5 t& I! O: w6 u6 Y( O1 g9 _
metastasis of admixed MDA-MB-231 BCCs (Fig. 4b), indicating that& ~: ^+ Z1 P) w: i" F
the actions ofCCL5 are responsible formuch, if not all, of the observed# |! x5 l# F; X2 D3 \
MSC-induced metastasis by the BCCs.) U" v+ q, b# z# F. t/ J# X
CCL5 promotes lung colonization, G8 c& I- g0 q& [/ S* R% k3 k
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,+ p' t+ k5 P/ X! j
CCL5 expression has been associated with increased tumour neovascularization,0 z9 H. y; C! t
suggesting that endothelial cells, which express a variety
of chemokine receptors, may also be attracted by CCL5 to sites of/ g4 ~0 O4 b6 T- h
tumour formation, thereby enhancing tumour angiogenesis20. Such  [2 `8 ?* b" T8 }9 |
observations suggest that CCL5 may contribute to breast cancer6 f  T7 E* d0 m
metastasis through the recruitment of a number of stromal cell types
to sites of primary tumour growth.: b3 }$ t& k* h+ s7 y2 W
However, immunohistochemical analyses indicated that the8 o7 {5 R! r* k( _
MDA-MB-231 control and CCL5-overexpressing MDA-MB-231& ]- i# q" G+ D+ d  [* C, N
(MDA-MB-231/CCL5) tumours exhibited comparable numbers of* C/ L! [& ?% C" @
tumour-infiltrating macrophages and had similar vessel densities (as
evident by F4/80 and MECA-32 staining for macrophages and. W8 }! p7 {6 }% f& M7 W2 }4 z+ o
endothelial cells, respectively; Supplementary Fig. 8). In addition,: [/ ]! D2 S) {( e$ Q
we found that ectopicCCL5 expression did not cause an accumulation: I) [% o8 m3 G0 B) N; a1 {2 B
of other stromal cells, such as SMA-positive cells, in the examined
tumours (Supplementary Fig. 8a). Together, these data indicated that4 D9 z, S9 Q4 t, t' D/ P
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 often6 b) K1 Y* c: i3 v# T. [, m1 }* p
facilitated by their transdifferentiation through the process termed
d f
Bcl-XL
Bcl-2
β-Actin) p) ~/ b( [  x1 K
S473-Akt
Motility
0.5%→10%
LY – – + +# q4 f, x7 Z) n' e: f" ^
g
Extravasated clusters (mean)7 B; t# N: x& g; e4 l
Migration (fold)
*
e( C- G& W9 k5 L2 L
0
MDA/vector& o7 O8 }% z" Z
MDA/CCL5
***
Invasion
10%→10%
Invasion( p$ o- ]: `7 Y; p* r; I! S/ c
0.5%→10%
Migration (fold)
**
0; h1 h: T4 h6 r* P1 @
0.52 y3 |6 X! B! o5 A2 q" s
1.06 B7 @  x) O& x. \( [) \" ^
1.5- M* b( g. k: C# i
2.08 _9 {+ I1 P# s" E! F& b
2.5
3.00 @$ j3 n3 T8 a5 g6 P
3.5
*
MDA/vector
MDA/CCL5) e  m3 H6 {7 @7 J1 ~; H
a b
*5 a* S1 X- U0 R
Nodules per lung (mean): k: H& ~7 \6 P1 z+ _, ^! A
c. }8 l: J0 _9 A6 {1 ?5 L
30' V) G* t! G" V+ N
252 {: \$ f# S! F
20' v: x1 t' g. x
15
10- F/ \0 D; F% Y
5) S! h( T. m6 i* y6 \  a" A
00 q& I. w- ~4 j
Tumour mass (mg)
30
25
20
15
103 K! }$ [6 n8 c' t, h
5+ R5 s  ?- C! B( R8 j
0 0. ?* }: l4 p' G
50
100' d% K1 \2 _* ^
150
2006 v4 x/ v7 j  j" h8 u; {3 _7 k+ d
250
0
50# x. G9 F6 I' [' A
100
150
200
250
300
3508 }4 |2 R- S9 W. s/ Q, ^3 v
Tumour mass (mg)) Z0 k' J: M- @1 f4 V) ~
Ctrl( W+ ~0 G1 O% z
CCL56 F" K  D9 F; C) ?" ^
Ctrl
CCL5
Ctrl
CCL5
Metastasis index (fold)7 M$ t' N! H* V  W) L
Metastasis index (fold)
03 _$ j4 p# d5 u
19 x3 K9 P5 O) }7 x
2
3
4$ f; h8 L" f8 `( G, E, V0 }
5. X' T. A! H  o3 o; m
* 6
0! d9 k* ~+ A% T
1! x' u& ^0 m" N- K6 `3 S6 Y4 ^
2' ?2 s, o  Z- y  ~# p8 }6 {+ X9 K; A
3. n! o" `( V9 o; f: M3 b
4
5
6' x$ f9 m. W& H& [
7
* 8& a* q* e" W4 P* w2 I' y' r
MDA/vector
MDA/CCL5; f8 M7 M* H9 @$ ^; I* `0 h) A2 A: j
MDA+WI-38/vector
MDA+WI-38/CCL5
10 z. t: F7 x/ ?0 [$ E5 W( p3 |
2
3
4/ v0 x/ b8 y: p* V. q
*
Figure 4 | CCL5 enhances breast cancer cell migration, invasion and- C' n1 i$ [% ~
metastasis. a, A total of 500,000 MDA-MB-231/vector (ctrl) or MDA-MB-1 O: Q) m: l9 o) u# R
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour6 E$ I) X8 O/ Y7 i+ g" N8 E' l
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. H* e3 P4 w* F3 x4 n- }( ~
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
250,000WI-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 onetailed0 a3 s; M/ B3 m& E6 H
Student’s t-test. c, A total of 800,000 indicated BCCs were introduced# Z% Z# V& b+ A( L, \
into the circulation of NOD/SCID hosts. GFP-positive cancer colonies in the: Z3 D- B( }% A
lungs were counted 6.5weeks later. Bars representmeans6s.e.m. (MDA-MB-
231 controls, n516 mice; MDA-MB-231/CCL5, n518 mice). Asterisk,- M1 A( o; q1 K# ^3 n7 B* z' R; o
P,0.01 in one-tailed Student’s t-test. d, Western blot analysis of lysates of: S% C3 U& k4 s* n
MDA-MB-231 control or MDA-MB-231/CCL5 cells. b-Actin was used as a
loading control. e, Transwell migration orMatrigel invasion assays on 50,000" N- V: n5 B# g8 K* ]' ]
MDA-MB-231 control orMDA-MB-231/CCL5 cells.Data are representative of  y6 M4 F: k0 j4 {; `4 D
multiple independent experiments and are expressed asmeans6s.d. Asterisk,
P,0.05; double asterisk, P,0.05; triple asterisk, P,0.01 in one-tailed1 `! T) s0 p! v" l) k* J" x
Student’s t-test. f, One million GFP-labelled BCCs were injected into the tail/ o$ }1 J$ w7 O0 \' _8 H
vein of NOD/SCID mice. Lungs were processed 48 h later and examined for
extravasated cells. Bars represent means6s.e.m. (MDA-MB-231 cells, n57" b  C! i5 c. M% V8 U* F5 J' V
mice; MDA-MB-231/CCL5, n510 mice). Asterisk, P,0.01 in one-tailed. A2 N/ ~* Z/ [/ O
Student’s t-test. g, Transwellmigrationassays on50,000MDA-MB-231 control8 O3 z+ i' R& F" q3 w! j2 c; a
orMDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;$ D( n# j# Q( n- H$ i
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.% F$ e' j4 _8 V0 ~+ d% S4 k# F' R' F: F
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 phenotypic4 v) I- [4 r2 {8 S4 T& @6 R
state of ‘partial EMT’, as they retain a distinctive epithelialmorphology1 ~# P1 I5 W1 W, l* I
in vitro and are still responsive to EMT-inducing stimuli in culture. In0 k% {* K6 Q5 e: l% C% e% M5 o, {
fact, we observed that ectopic CCL5 expression did not cause MDAMB-8 R( x  @3 M) F' k- q# x) ?
231 cells to undergo themorphological changes usually associated. z0 P2 B7 W" [1 X* O
with an EMT(Supplementary Fig. 9a), did not cause rearrangement of
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on$ n2 J0 o% O+ `2 X: n% K
the expression of mesenchymal markers closely associated with the9 y" t4 ~' e4 C+ t/ r* P
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c)+ i) n- p! e5 C$ B; `
and fibronectin (data not shown). These data suggested thatCCL5 does
not directly promote the EMT programme of MDA-MB-231 cells.* ~0 [2 C0 E- f2 e  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. R- U6 {' b# g; T& O. T
organs and the subsequent step of colonization. For that purpose,2 x2 Z+ `4 C5 b& p& R
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- L& L( o. W  }$ K
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: r" h' A# H7 X: b
of the invasion–metastasis cascade. We note once again that this
enhanced tissue-colonizing ability was not due to CCL5’s effects on5 b3 H: A: T0 O! q
cellular proliferation measured either in vitro (Supplementary Fig.5 [( w" \" y- K5 `1 V6 S
7b) or in vivo (Supplementary Fig. 7g, Ki67 staining).
Because improved colonization can be due to enhanced cellular; j. c/ e% C) }" N5 x
survival, we tested whether CCL5 protects against apoptosis.
Notably, we found that MDA-MB-231/CCL5 cells exhibited higher3 ]) Y& @2 \* D& b* o4 t  Z/ v
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& _$ J9 B' U* h( F$ S7 I
pro-apoptotic molecules such as BAX or BAD (data not shown).# b/ S7 y0 h& {3 M" y6 G! `2 f
Moreover, we found that overexpression of CCL5 had no effect on
the ability of MDA-MB-231 cells to withstand serum deprivation3 k. n% R& V+ n: s$ P( r
(Supplementary Fig. 7b), loss of substrate anchorage (Supplementary3 V4 Z  A9 b) d6 {% |9 h
Fig. 7d), or hyperoxia (data not shown). We also observed that) [* W* L5 J7 s3 v- Y
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;/ b$ z' ]- l! Q
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling2 Z$ r6 s8 a3 z
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohistochemical
analyses on control and CCL5-overexpressing tumours3 |$ \: z. T3 \; V# w- N$ V
revealed only minor differences in the levels of apoptotic CC3-1 O6 y8 I+ Q' r6 v: Q
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: ?2 `1 k7 `# k; O/ B
that the observed enhancement of lung colonization was not a consequence6 X6 d/ P% z0 l
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
against apoptosis, we tested whether the CCL5-enhanced lung colonization7 `- O4 c. r: o$ w/ [# i8 ?
could be due to an increased ability of MDA-MB-231/  u/ W$ D* V( v. e4 W2 L6 |
CCL5 cells to invade from the microvasculature into the lung. T/ D6 y! u/ I  K4 B4 V
parenchyma through the process of extravasation. Indeed, ectopic/ }4 |# y" N, _% {; d
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).; M' e3 @4 U  R4 \5 N
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein( k: G8 L6 m2 e& o7 P. }  `
injection—in order to remove most cells that remained within the
microvasculature of the lungs and thus had not extravasated—we- G" K. Q* \. I. s9 J
found twice as many deposits in the MDA-MB-231/CCL5-injected
group than their control-injected littermates (Fig. 4f). This indicates a, L/ [3 j  r) J% E0 H6 N- n' {. N9 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& c% U5 Y+ b# \5 ^/ L  Z( H
b silacZ
silacZ( ]- a9 _3 g1 j
si809
si809
si186# q- {, ~- N) Q! |( W: \
si186
CCR5
β-Actin$ S9 i( [# N- O& Y
a d
M
+0 d& p9 s6 M# q9 x. }+ a3 D0 \
MDA+MSC
+ IgG' q. r) t+ y# E, u7 ^( j& e6 f
+Anti-CCL5 Ab$ O0 r% W1 W2 z4 K  ], a5 l" j
MDA
+Anti-CCL5 Ab
MDA+MSC' j: c- u  L# m. S- j
MDA3 n: q$ Y* u$ T2 y
IgG: N4 d+ L5 }4 ~( t$ [. A
<: q6 n  I, M& m: E" H8 Z* i
<
<
<
<# |  ?4 o; J- C  N2 s
DAPI
CCR52 n' V1 _% A7 M5 H, ^- a7 j7 {% X
MSCs MDA MDA+MSC
DAPI DAPI
CCR5 CCR5
MSCs MDA MDA+MSC
*
c) G  z# ~, N- R( T9 u' Y7 `! k5 y* K
Metastasis index (fold)
Metastasis index (fold)/ D2 {+ T$ F) l: j4 e
*
Control siCCR5/ m, H- K2 I0 @% j; k
MSC – + + +0 \- p; i) b* }$ W# m
0
1
2
3. r- z. D' K' C7 F& |  ~3 f
4
5 e4 Q) [8 l6 V/ ~4 B( q. Q  |" y
+ + – –
Anti-CCL5
0
19 V) t7 v9 a5 Y0 m7 {/ N
2, Y0 M# y# P3 i+ a
3
4
5
6
– – + +
IgG
MDA
MDA+MSC
<
Figure 5 | CCL5–CCR5 interaction is essential
for the MSC-induced metastasis.( v) G/ l; D4 D2 |. X5 R$ U
a, Immunofluorescence analysis of CCR5/ `4 I6 k! O- s. \
distribution in MDA-MB-231 cells cultured with
MSCs. DAPI (for nuclei staining) is in blue;) T3 u* w" C* t! b* k) O  q
CCR5 detected in green. Arrowheads denote* B/ S: V. ~4 z) l; J3 _& U
MSCs. b, Western blot analysis showing CCR5
expression in MDA-MB-231/silacZ, MDA-MB-
231/siCCR5(809) and MDA-MB-231/) Q/ f0 M9 n6 ]$ M
siCCR5(186) lysates. b-Actin was used as a4 \# ]* L5 q* a* Q/ j. O  i! L
loading control. c, A total of 500,000 cells of the
MDA-MB-231 variants in b were co-mixed with' p1 E' l" p. A( k
1.53106 MSCs and injected subcutaneously into
nude mice. Mice were killed when tumours9 R8 u% z* F$ k. G
reached 1 cm in diameter and the metastasis, W- d" _8 m9 j; Y
index was calculated for each cohort (n55 per+ ?$ C) S8 ^' ?; h. f# ^8 x
group). Results represent means6s.e.m.;; _; j( _, ^' o
asterisk, P,0.05 using one-tailed Student’s, J, ]6 Y% J6 ~0 Q" v
t-test. d, Anti-CCL5 neutralizing antibody or) {3 l+ l7 \( U% R) O
control IgG was administered intraperitoneally
twice weekly in SCID mice bearing MDA-MB-231' u' N) b5 k+ n0 @$ q, f
(n59) or MDA-MB-2311MSC tumours
(n511). Representative lung pictures of the
indicated cohorts are shown. e, Lung metastasis
indices of mice in d. Data shown are% m# U8 ?6 ^5 E: Y- o/ V
representative of means6s.e.m. Asterisk,
P,0.05 in one-tailed Student’s t-test., m/ R3 s; k& S. l
kinase inhibitor LY294002. Drug concentrations that did not inhibit
the basal motility levels of MDA-MB-231 cells blocked the elevation* C8 \0 y+ \2 V4 R8 T5 N5 ?6 A0 S
of motility induced by ectopic CCL5 expression (Fig. 4g). These
results, when taken together, suggest that the observed CCL5-  R/ D, D* _& K. m/ a+ w; G
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.0 k0 E8 |3 m# j% d
Essential role for the CCL5–CCR5 loop6 E. l! b7 Z2 [6 c; |
CCL5 acts through three G-protein-coupled receptors, termed
CCR1, CCR3 and CCR5 (ref. 23). CCR5 has been determined to be) c# M4 Q0 g* c6 x6 e% d- d
the main receptor for CCL5 in MDA-MB-231 cells, as inhibition of its
surface expression through dominant-negative mutants abrogated% n! U5 V5 p9 @4 M- B9 G! P
the ability of these cells to respond to CCL5 chemotaxis24. We therefore
focused our efforts on evaluating the importance of the CCL5–; ^1 y" Y- j) o: ]& l; g
CCR5 interactions in MSC-induced metastasis.
We confirmed that CCR5 is expressed by MDA-MB-231 cells and* j/ s% R$ S& f7 K" U" Q, `
not by MSCs (Fig. 5a), supporting the notion that MSC-derived
CCL5 acts primarily in a paracrine fashion on MDA-MB-231 cells8 [3 j& F! i( D4 Y
in the BCC and MSC mixed cell populations described above. To: _! ]8 E8 t) w( \! v
probe whether the observed MSC-induced metastasis required$ G# _: q7 M# d8 W3 R
CCL5–CCR5 interactions, we inhibited CCR5 expression in MDAMB-
231 cells by more than 85% through shRNA knockdown (ref. 25$ r7 l  a, T; c4 {: S# f
and Fig. 5b), and mixed these cells with MSCs before implantation
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,5 a$ M( n+ _& E
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 intraperitoneal
injections of an anti-human CCL5 monoclonal antibody
also abrogated the MSC-induced metastasis by MDA-MB-231 cells8 V& _+ b) B2 W! s. ~% ^) P, u) d
(Fig. 5d, e). In addition, MSCs in which CCL5 expression was inhibited* H0 w$ k# Y. X/ z: V7 s  J+ q
by shRNA knockdown failed to promote metastasis of the% e, E2 V; ]6 F+ |
admixed MDA-MB-231 cells (data not shown). Taken together, these
results underscore the critical importance of the CCL5–CCR5 paracrine
interactions in enabling MSCs to induce metastasis of the9 [  f  t- X0 w) u& _7 h
MDA-MB-231 cells.
Discussion" K) y) a* q( C6 k3 m# d4 n2 L
Certain models of metastatic progression propose that cancer cell9 l* y4 P9 T; ]' V& f
invasion and metastasis from the primary tumour site are strongly
influenced by contextual signals emanating from the stroma of the' \& G2 y# ]2 G( ]
primary tumour. It follows that if carcinoma cells are subsequently
deprived of such signals, they may revert to an earlier phenotypic8 b% D2 D) R: A& \
state in which they no longer display the traits of high-grade malignancy.
Indeed, such a model has been proposed previously by others
on the basis of indirect evidence21. Here, we demonstrate that at least
one mesenchymal cell type, the MSC, can expedite tumour metastasis,- o- z5 {/ n; [0 z
and suggest that after primary human carcinomas recruit MSC3 T' I9 q8 h( K; _/ s+ y  g
populations into their midst, subsequent interactions between the- j7 X5 T" O2 j% g9 \6 j
MSCs (or their derivatives) and the BCCs endow the latter with
invasive and metastatic properties.( [" S5 F/ ^0 c0 N" m
Although the recruitment of labelled MSCs to tumour xenografts
has been established in a variety of experimental models of tumorigenesis,
there is currently no available way to quantify with any accuracy+ Y. h. D* A1 n- q& k
the number ofMSCs in actual human tumours, in part because no set; i: k- l( H9 {1 d2 w$ Q: J# Z5 l" k/ G
of markers has been identified that can uniquely stain these cells without* O9 h+ }4 h$ e  E- Q
concomitantly staining other mesenchymal types in the tumourassociated
stroma6. Our demonstration that the stroma derived from
tumour xenografts contained appreciable numbers of murine MSCs% v. P2 p" h& a5 v4 T6 N- b
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  U( o: r9 v" r" R4 H$ A& d" \9 h
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;- }( C( b$ m- ]% O0 V! }" b3 A# M
Fig. 6a). This suggested that, similar to tumour xenografts, human5 S# ?5 y% T8 D" _0 O2 {
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 cancers26 (SFT;3 D' K7 g1 y2 q. c! Q6 N! z. k
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free: M; m3 j: U3 c7 ^
stroma of primary invasive ductal carcinomas (Fig. 6d), specifically in0 g- ^6 o$ A5 ~$ l: l$ t$ a
the CD10-positive compartment27 (Fig. 6e). Collectively, these observations
argue strongly for a significant association between stromal6 C8 \3 y6 E5 d: L* C. _
CCL5 levels, MSCs and human invasive breast cancers.
c
STT1969B9 {# B8 \& L3 j8 {3 j- H- u% G1 _
STT31268 [- u! s- }( @' Y. u- Q- [
STT3124- J4 B! w' r; N8 I2 ]
STT656B
STT1968B  L6 w7 Q( V. X% M0 @5 ~
STT3122* t: n% p6 X5 H  T  I% T6 k
STT3053
STT1986B: u; @& o' F) d. p/ D0 k
STT8543 K/ @$ V" Q6 G" }
STT3125
STT1975
STT1987B
STT1079. J/ |/ Y1 X8 y" b
STT638
STT1774- W0 Y/ v/ z# o& Z2 P
STT1984# ~4 y6 Z/ H% ^3 b0 U* B
STT1737C
STT3068
STT3120, L# q. j, @( }$ s
STT850/ p2 }: R0 z) c) u0 v7 W
STT417B/ e0 v5 Y6 ]0 e  Z. P
STT31198 ^& B* E1 Y: L( h, c
STT1776( Y4 j1 Z. q7 q: ?& O% A5 G; ~( ]8 I
STT1777B
STT689B1 Y; o# `+ k) S/ W2 n
STT1971; K) [4 ?' B3 H1 k+ v8 n" O
STT5976 Y1 K/ \" m/ _$ N; j! \' }6 K
STT626
STT154
STT2774
STT1966
STT27761 t) @: O/ I$ y/ ]
STT2775! ^( g" _  _' V+ c8 G  x
STT27722 G8 c7 E2 s/ A+ m$ H2 x( ]2 i- j
STT1637; Y/ ^$ P( N4 b/ a& f% r6 N& j; I  q
STT1220C) E9 @1 S4 M+ a  Y
STT-094B-1
STT6758 |+ V( x$ e9 x' J
STT2770
STT695B% ]# P4 n0 V5 G; ^5 ]8 f: f! M! x: @
STT1771) O0 r+ H0 W- u' O7 t
STT17784 \% c' B/ o3 [# ]4 r& I
STT491
STT1823
STT200C. T. E. k3 c' M
STT741B
STT335C# ^9 H  P) B2 A/ B* ~
STT709B
STT516D
STT607B; P8 O$ ^; }" g% j+ O7 m  f% j$ X
STT680B7 f. S* L7 B7 O; S: F* ^
STT1148B
STT523B
STT526E5 @" J" T7 {8 n# |8 L" D
STT742F
e
CD132 L: f+ I" O. K' @. J
CD29
CD44+ F2 G9 w8 M6 c; A* T0 ^/ A
CD49e; c4 a; f1 N2 w8 F, A3 B* Z
CD54$ ]- {8 l7 e, K; D( }! c3 r
CD59
CD63
CD105
CD106
Nestin+ k; \1 u/ T! D7 s: {8 x
HAS2
IGF2( F: w' x' D$ G, `) O  a7 V9 g; v8 i
PLAU
TIMP15 y1 O. Q' C( ]  D
CAV1# w' v" b, Y  f# @7 q, g
IDC-7
T1126031 h2 b1 `* h2 C$ ^
T392303
Normal Invasive
CCL5% o# D, J4 N# S4 X. F* W5 B$ J
log2 ratios3 Q6 L, T: O+ a+ J+ C, g. A
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.0- H/ h6 A0 y6 u# W
log2 ratios- B4 o* c- _4 y% g9 t. o" B
d
–2
–18 z( H- s3 \" h5 v- d4 f
0 1 2
–2.0
–1.5$ A9 l: v, @( B/ y
–1.0# B5 B# R! r8 V" ~, v7 K
–0.5
0.0
DTF SFT
DTF  O) F$ t" r" N3 u2 n8 r3 ^2 x, e; q. p* Z
SFT8 |: S/ m  k5 B$ {+ P. X; v5 M0 O+ B0 I
a b
F8 \( C: f: o% q8 R' `6 b0 Z  k/ b: q
Figure 6 | Stromal fibroblastic cells of human invasive ductal carcinomas are4 m9 o' d% y4 z$ c+ a  p
rich in MSC markers and overexpress CCL5. a, SAGE TreeView display of
MSC markers expressed in stromal CD10-positive cells from invasive6 Q0 l# q  O" X# c& L! w
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
solitary fibrous tumours (SFT; blue outline, n513); narrow blocks are other
soft-tissue tumours (n532). c, Box plot showing that CCL5 expression is& o% K. f. f% b( N
higher (P50.004) in SFT than inDTF. The difference in log2 expression ratios
between SFT and DTF was tested with the Welch’s test. d, CCL5 Affymetrix" `5 ?4 ^* O5 A& Q: d" k
gene expression in the stroma of human invasive ductal cancers compared to$ E: W* b* c* z1 _
that in normal cancer-free breast tissue (indicated as ‘Normal’; see Methods).
e, CCL5 expression is mostly restricted to the CD10-positive fibroblastic cells- C! Q" b* L# ?, y4 g) v- Z- f; G8 A
derived from invasive ductal cancers. The heatmap shown is a cluster of  n# R/ V, E8 R- W3 O- _
CCL5.genelist obtained as in a.Details of thepurificationmethodologies of the, |; _8 m, z2 ^5 j# T0 |: h
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-8 M- ~7 {( V9 t
231 cell interactions, CCL5 seems to have an equally critical involvement$ E. y; x2 m! [6 J1 J$ f& `6 h
in the functional interaction of MSCs with MDA-MB-435) N: L: \7 d" M2 V! n, i
human BCCs. CCL5 levels accumulate synergistically when the two7 G% x; N- `0 K( w
cell types are co-cultured together (Supplementary Fig. 10a), and% d! w- g% ~; v# F3 _. e
MSCs in which CCL5 expression was compromised by shRNA knockdown1 k0 j1 @  r* c% l
failed to promote metastasis by MDA-MB-435 cells to which/ q( S% @' d& ]9 a$ F% ?( f/ o
theywere admixed (Supplementary Fig. 10b).With these facts in mind,
we point out that CCL5 does not seemto be involved in regulating the
MSC-induced metastasis of MCF7/Ras or HMLER cells, which may  t9 W* Z  I' L# S
depend on other paracrine factors such as VEGF and interleukin-8.+ _% P. N+ K! ^5 H7 V
Nevertheless, our observations highlight the recently discovered critical
roles of chemokine networks in malignant progression28,29 and suggest  ^& t2 ?$ q9 h
the possible utility of a variety of CCL5 analogues and CCR5 antagonists
currentlyused in anti-HIVtherapy30 in treatingmetastatic disease." M6 \, e$ F, n4 U& i" i5 c
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+ n  \3 g  u& }- ^* R* p$ u
(Fig. 2e). This indicated that acquisition of increased metastatic5 ], D# y, o; r* D" r6 }
powers by these tumour cells was reversible, and suggested that the+ E6 m8 ~8 P2 Q1 q& P  V/ ]$ a
maintenance of this phenotype depends on continuing contact with: E/ J9 t3 r0 R+ M% ~5 b- ~: g
stromal cells. If extended to other tumour types, the present results7 l1 B- a1 E: w
hold important implications for the molecular analysis of malignant
progression. They suggest that many of the cellular functions associated
with invasion and metastasis are often not expressed constitutively
by carcinoma cells, but rather only transiently in response to8 a6 x5 l- m3 q% I- w
contextual signals that tumour cells receive from their stromal microenvironment.
If so, analysis of the gene expression patterns of bulk
primary tumour populations may fail to detect the expression of key- c- t# K( v5 D/ C! B+ c0 l
genes mediating invasiveness and metastasis, if only because they are
being transiently expressed in minor subpopulations of cells within. L, b' y8 T" Y1 [( n
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 stromal
signals, rather than on the genes and proteins that directly mediate. X! w3 B3 U4 r( p/ W" H: S0 j1 `
the cellular phenotypes of invasion and metastasis.
METHODS SUMMARY2 [( Z! b2 \6 x4 c7 A9 U! h
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* l/ m3 B' v- ^# K
transwell chambers. Antibody treatment of tumour-bearing mice was conducted( {0 e9 Y4 W% N  P
by intraperitoneal injections. See Methods for detailed information regarding4 k# B4 y  J, B2 Q& X  _6 R
cell culture, viral infections, in vivo colonization and extravasation assays, RT–
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluorescence: g! L" ]- y1 f
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.

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