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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, T  E& r, L  _' ]
tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour' V2 l3 S' c0 I! n( e
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' F! L% s3 I0 w+ ]
metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour4 a' K% _. H" B6 L2 Q' v
xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from4 q! s# Y/ d) t3 a4 I( T. g- Q
mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and' T) R( r' b5 L2 l- ^5 P. |* i
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.* |% u. q, y" E! ~5 Z; V
The origins of the invasive and metastatic phenotypes of carcinoma
cells have been the subjects of intense investigation. Whereas some$ p# j6 P, Y# e& F+ D0 _
current models depict these phenotypes as cell-autonomous alterations0 W4 [% f& S( P5 n! g" I1 n
specified by the genomes of cancer cells, alternative views propose) |: C' w% y- c8 k$ ]
that metastatic traits are acquired through exposure of epithelial
cancer cells to paracrine signals that they receive from mesenchymal
cell types within the tumour-associated stroma. Although several3 u/ u" N) `& W: K$ s
lines of evidence demonstrate the contributions of stromal cells to
primary tumour growth1, direct experimental demonstration of the
influence of these various cells on the metastatic abilities of cancer
cells has been difficult to obtain. This is due, in part, to the complexity
of the mesenchymal cell types that are recruited into the stroma, and* d" Y6 c2 x2 c8 y1 u
to the elusive nature of the putative paracrine signals that are, t% M3 y' e$ [8 p% a* b8 N
exchanged between the mesenchymal and epithelial compartments0 K+ {1 v# k! f- Y# {" n* w7 a4 \
of a tumour. Recent reports proposed that the bone-marrow-derived2 c. z6 {" o2 v0 u: G  I) m8 L! }
mesenchymal stem cell (MSC) is a cell type that is recruited in large5 C6 b: U" s( @' m* K6 C
numbers to the stroma of developing tumours2. To characterize better+ y! C5 u% R; W' k/ a/ j
the role of this stromal cell in tumorigenesis, we set out to determine9 `1 }" L3 L2 A1 j) I
whether MSCs could supply contextual signals that serve to
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' o7 r( N: O4 U; @( J) f& @) w
MSCs reside predominantly in the bone marrow, they are also distributed, A9 c) A' c! s2 f9 V1 E0 N
throughout many other tissues, where they are thought to4 ]' q+ f1 ]$ V3 A9 c
serve as local sources of dormant stem cells4,5. The contributions of' o7 |+ e/ `/ [7 H
MSCs to tissue formation become apparent only in cases of tissue
remodelling after injury or chronic inflammation. These conditions
are typically accompanied by the release of specific endocrinal signals
from the injured or inflamed tissue that are then transmitted to the
bone marrow, leading to the mobilization of multi-potent MSCs and  O/ o7 ~: R5 x" M. u" E- Y
their subsequent recruitment to the damage site6. For example, MSCs
have been shown to contribute to the formation of fibrous scars after) l% S/ N: C6 f
injury7.
The formation of breast carcinomas is often accompanied by a' w$ a: Z3 }: f7 s5 l
well-orchestrated desmoplastic reaction, which involves the recruitment9 H& p9 _) {1 N- \0 y
of a variety of stromal cells with both pro- and anti-tumorigenic
activities1. Such response closely resembles wound healing and scar1 D! p# G/ B8 x+ }  R# C# h
formation, and entails the constant deposition of growth factors,
cytokines and matrix-remodelling proteins that render the tumour
site a ‘wound that never heals’8. This suggests that, similar to sites of$ H) y  a" V+ f; j
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 or8 U3 C1 `8 o" W2 X. r
MDA-MB-231 breast cancer xenografts is rich in cells with an ability
to generate fibroblastoid colony-forming units (CFU-F) in vitro
(Supplementary Fig. 1a), a hallmark of MSCs3. The absence of such. a$ f$ Q9 U' ~8 e% n
colonies from control Matrigel plugs or from neighbouring tissues
(negative control; Supplementary Fig. 1a) suggested that endogenous
murine MSCs localize specifically to sites of neoplasia.
To investigate whether human breast cancer cells also have the
ability to attract human MSCs, we established a transwell assay
in which bone-marrow-derived human MSCs were allowed to- j' B& e) D$ x. P
migrate towards media derived from MCF7/Ras or MDA-MB-231
cultures. We found that human MSCs migrated much more avidly
(,11-fold more) towards media derived from these cancer cells3 a- b9 M# `8 z5 g0 }* I7 Z' i8 G4 [
than towards control media (Supplementary Fig. 1b). More importantly,1 q7 g9 {7 `4 C6 T
green fluorescent protein (GFP)-labelled human MSCs
infused into the venous circulation of mice bearing MCF7/Ras9 w0 m" s, l0 i) E
or MDA-MB-231 human breast cancer xenografts localized specifically
to the developing tumours, with no observable accumulation
in other tissues, such as the kidneys (Supplementary Fig. 1c), liver% g! _+ z. F! ]' b% k- z
and spleen (data not shown). Such findings indicated that MSCs
are specifically recruited by subcutaneous breast xenografts, and corroborated' b7 |( x- ^0 A6 d
recent studies that described the localization of systemically9 E$ |. B9 Z: \, P8 Q- f
infused MSCs to other types of malignancy, such as gliomas9,10,
colon carcinomas11,12, ovarian carcinomas13, Kaposi’s sarcomas14 and, M, t  Y* b( m0 c
melanomas15.MSCs enhance breast cancer metastasis! ~- w& E+ t, Z4 n" h9 W, b
To investigate the functional consequences of the heterotypic interactions
between MSCs and mammary carcinoma cells, we established
a xenograft model in which GFP-labelled MCF7/Ras, MDA-MB-231,
MDA-MB-435 and HMLER (see Methods) human breast cancer
cells (BCCs) were mixed with bone-marrow-derived human MSCs9 c5 m. V- l* S5 Z9 z, L+ }3 Q. a
(hereafter referred to as MSCs) and injected subcutaneously into( S1 z, N) F( _) a; R  t
immunocompromised mice. The growth kinetics of the MSCcontaining
tumours (BCCs plus MSCs) were compared to those of! y) W7 v( E- W  o9 O
BCCs injected alone (BCCs) over the subsequent 8–12 weeks, after
which the histopathology of the resulting tumours was studied.) Y) {6 ~# O9 N. E5 {& `
We found that MSCs accelerated the growth of MCF7/Ras
tumours without affecting the kinetics of MDA-MB-231-, MDAMB-
435- or HMLER-containing tumours (Fig. 1a). More importantly,
whereas mice carrying tumours composed only of BCCs
exhibited few microscopic metastases in the lungs (Fig. 1b, d), mice
bearing the mixed MCF7/Ras1MSC, MDA-MB-2311MSC, MDAMB-) u2 b1 Q9 _2 D6 k0 U
4351MSC and HMLER1MSC tumours displayed a marked8 _% F4 t' F3 G2 H# C
increase in the numbers of micro- and macroscopic lung metastases# n6 U4 }; s3 x2 \% r
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs% r/ s4 ]6 V) k8 \
of BCC1MSC-bearing mice compared to their BCC-control littermates
revealed two-, three-, four- and sevenfold enhancements in
the overall numbers of detectable HMLER, MDA-MB-435, MCF7// O5 F8 u; }. w! A
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c).  j$ y9 p: V8 e; C; j
Furthermore, in contrast to the MDA-MB-231-bearing mice, the2 \% H5 X, v$ r$ p) z5 {- T! B
MDA-MB-2311MSC-bearing mice showed metastases to various0 P  g1 a$ G& b  G
other tissues, including the mammary glands (Supplementary
Table 1). Although all four of the tested cell lines exhibited enhanced" m+ f$ {# @( N0 ~
metastatic potential after admixture of MSCs, we chose to focus4 F5 i' U2 p* @
further analysis on the MDA-MB-231 tumour model, because it" [( X# J! @; T9 ^& n
displayed the greatest relative increase in MSC-induced metastasis7 e: f2 m$ n! O: d$ N
without any concomitant effect on either tumour cell proliferation
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall primary! w+ s0 h7 u& L% ~- c- F6 }: l
tumour growth kinetics.
We note that admixture of other types of mesenchymal cells—5 C9 l6 Z# b  p- V# [- k' }
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 3
and data not shown)—to MDA-MB-231 cancer cells before injection
into host mice did not result in either enhanced growth kinetics
(Supplementary Fig. 3a, b) or increased numbers of lung metastases
(Supplementary Fig. 3c, d). Taken together, these observations indicated
that the metastasis-enhancing powers were a specific property9 M( {! ^. A" L3 U, m) B
of admixed MSCs or derivatives thereof.8 S! [4 E. n( B, ^+ f4 r
Reversible metastasis
Implantation of MSCs either contralaterally to MDA-MB-231 cells or
in nearby separate sites of injection did not affect the metastatic
potential of the resulting primary tumours (data not shown), indicating3 `' s- @+ c7 f4 s) N; U. h
that MSCs could enhance cancer metastasis only when they+ d# V. `+ O# h* N; K
were in close proximity to the engrafted BCCs. This influence might
be ascribed to various effects that MSCs exert on the commingled
carcinoma cells. Thus, the MSCs might favour the outgrowth of rare0 O! y( t+ v, O# R* b
variants within the MDA-MB-231 cell populations that exhibit
unusually high metastatic powers. Alternatively, the MSCs might
cause otherwise weakly metastatic MDA-MB-231 cells to acquire
enhanced metastatic abilities. This latter mechanism suggests the
possibility that the acquisition of the metastatic phenotype might; I# a) z$ O4 x: B
be reversible, in that carcinoma cells might revert to a lower metastatic. L! x* `  C- Q. ]! B; w
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),
expanded in vitro, cleared from contaminating stromal components,0 B! v$ X3 F+ s7 ]% x, w0 W/ i2 y
and then re-injected into subcutaneous sites in host mice in order
to evaluate their respective metastatic powers (Fig. 2a). Although
the growth rate of the resulting L-explant primary tumours was; I+ R2 J0 i# `4 J" M
marginally enhanced compared to their T-explant counterparts6 F' t: Z9 ?. G
(Fig. 2b, c), these L-explant cells were no more metastatic than the  \8 t4 o. }" J0 R
parental T-explant cancer cells (Fig. 2d). This suggested that the
a
c d
Days after injection
Tumour volume (mm3)( X* V2 t; |: b7 e# D/ G1 X
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  k  i9 q6 q+ n% ^1 ^7 Q5 ]
1 mm 1 mm
1 mm 1 mm
300 μm
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC8 [: T$ U- p1 `. k4 t2 h+ J
MDA-MB-435 MDA-MB-435+MSC1 N3 B' N- G  `5 [# I/ I3 c7 V
MCF7/Ras MCF7/Ras+MSC6 o  ~5 z* y0 A  y
100 μm
300 μm
100 μm
MDA-MB-231
MDA-MB-435) C! }1 L2 J* Z0 n
MCF7/Ras
**
Metastasis index (fold): @$ O; C7 a; Q. `" t) D
08 O9 w' t: b; k; g# k
1
2  w) g! l' Y7 E$ k+ }8 Z
3
4
5) L) l  Y+ o9 z$ `( V* m! K. `8 t
68 o9 C' L# W9 Z3 S0 ?! f5 p1 Y
74 A$ U3 D, @6 \4 |( a2 ~) K: l! T
8
97 F2 P. y2 M0 Z3 @& H
** z* \7 l9 c3 p" U% @4 m) `: l. A
**
**
MSC – + – + – + – +$ u% D5 ]4 s: ]- m
*
HMLER% [: p' y, q: r1 U" k% q, L  a
700 1,200
1,0006 W' k% q& n! O% L& z
800
600
400
200/ o6 X) X: ^7 a
0
8007 N1 u) R! ?) D  g# j2 t
700* h( |. _( u2 S3 R, n# n7 h
6005 w8 b7 f! ~) u! E3 b
500
400( P# y/ o/ G7 A& O* z9 Y: ]
300
200
100- Y% Q* ~: a  I$ q( o
0! m7 q! L" P  Z$ |7 P
3,500
0: u! M8 K  p! X
500
1,0001 @2 q" n! T8 ]- G
1,5005 M+ U* E- U  F4 Z& v! y
2,000$ j7 Z3 B9 ~  x! M% u2 g
2,500. f3 h: V/ q: f: o0 G
600 3,000. C8 [4 C5 k( m* C: x! c
500  J( b3 Y& D4 }3 c% F
400( _! N- z6 h7 g' d1 s7 c
300
200
1007 @6 G% u4 U& |* \1 d
06 D- p8 m0 v$ j
0 21 31 38 49 56 63 70 78
MCF7/Ras alone7 e6 ?8 v) P1 [+ w$ Q5 ?- g% W. Q
MCF7/Ras+MSC! ?& U. v/ t# x; H3 Z( `' e6 F' y
MDA-MB-231 alone: }# X5 O! L7 v
MDA-MB-231+MSC, W- r1 u1 U# {  t" O
MDA-MB-435 alone
MDA-MB-435+MSC( ^* \/ s  q# n; u
HMLER alone! {3 Y8 r; S0 V5 j9 V
HMLER+MSC
HMLER HMLER+MSC& S3 u* k6 q* Z4 O6 m8 m
MCF7/Ras MCF7/Ras+MSC3 E% ?! J/ h0 H; a( o4 j
MDA-MB-231 MDA-MB-231+MSC# y0 n$ @1 P3 e7 g- C: a6 [1 r
MDA-MB-435 MDA-MB-435+MSC# s: a0 T1 V; t/ ^$ s5 Y% z
Figure 1 | MSCs promote breast cancer metastasis. a, Tumour volume
measurements (mean6s.e.m.) of 500,000 GFP-labelled BCCs injected
subcutaneously into nude mice with or without 1.53106 MSCs.
Representative data from multiple experiments are shown. Diamonds, BCCs( G6 d! V% r3 R/ L9 M
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/Rasbearing
mice were killed at approximately day 150 to allow these tumours to8 y4 ~$ k, G$ b/ H+ }
grow to comparable sizes to their MCF7/Ras1MSC counterparts. c, The4 S6 Q* e. d+ R* Y5 Y/ U% K! E( Y
lung metastasis indices pooled within each cohort of mice in a are expressed, A/ x) n, i( 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 onetailed
Student’s t-test. d, Representative haematoxylin-and-eosin-stained
sections of lungs of mice bearing the indicated tumours. Metastases are1 f0 I7 q1 p7 k0 }" t5 m& X
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 metastasize
to the lungs was a consequence of their ‘education’ by MSCs in9 K7 N# n2 N. ]" a& T" p* p7 o
the primary tumour rather than the selection of rare variants of2 ^8 X* E/ o& n8 v' I, I4 W
MDA-MB-231 cells that display elevated metastatic potency in a& h/ @/ J: e2 @3 v
stable fashion.0 g' k" f3 i! C0 I
The effects that the MSCs exerted on the BCCs might have3 Y5 P0 P; S5 X1 g
occurred within the site of primary tumour formation. Alternatively,
the MSCs might have accompanied the metastasizing BCCs! \/ B' e9 {, r4 K$ b
to sites of metastasis formation. To distinguish between these two/ m& U$ j; x5 u, I0 e
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled, ?  X3 Z1 j6 [8 f
MDA-MB-231 cells and implanted the mixture subcutaneously in
host mice. We found that the tumour-derived lung metastases contained% i! D3 C! d4 W" s) S/ e
green-labelled MDA-MB-231 cells but no detectable redlabelled
MSCs (or their derivatives; Supplementary Fig. 4a) when
scored 4, 5 or 6 weeks after primary tumour implantation. The9 d* }/ C  x$ v9 P8 m1 K9 ?# l& a
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 that5 p( G% {# I# f' {; q
environment for ,6 weeks after injection (Supplementary Fig. 4b).* o& h1 N' v5 y4 n, t
Hence, it appeared that the admixed MSCs do not migrate in large! x  a, ]% @& h1 t2 W# T# S
numbers to the sites of metastasis, and that they exerted their prometastatic
effects on BCCs in the context of primary tumours.( ?& o% O( D& K  h, j. r$ F" E
CCL5 in MSC-induced metastasis, J4 @) M7 ]! E* m
The aforementioned observations indicate that MSCs supply locally! M' U1 i* h4 Z. y2 m
acting paracrine cues that induce BCCs within primary tumours to$ q* E3 E: c) F4 R) G; y6 v! c
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,
chemokines and growth factors using the Luminex-based Bio-
Plex suspension array system (Fig. 3a). In some cases, the resulting( \. e; j; Y0 t. o/ S/ h: A1 G4 @! r
a
b c d
Days after injection
Tumour volume) b8 g$ L( B6 v% d. g
(× 100 mm3)
0
5
103 X. f+ O, `1 D
15' ?# _& c; o8 v1 d/ A' L
20
25. h" g8 W7 B) V
14 17 21 24 28 31 34 38 42 45 48 66 71 77
Primary tumour explants
Lung explants  v/ y- d3 y+ |2 }$ P# G" K
MDA-MB-231
MSC
+
Lung explants
Primary tumour8 J, u' I( y/ l  _
explants1 P& o. h3 r( e& t, L. r' Z1 A
Antibiotic
BCC selection
T-explant& q3 E1 }  q/ c* C
L-explant0 `; \; S7 H) z  t) u9 X7 m3 k
T-explant& v6 z! m, t) `# F$ _
L-explant, d9 F+ T, N; T/ U& k/ [
Tumour mass (g)
3.0& ~, {! K' Q. ^# d
2.0
1.0, n# Z! w( f- e/ }6 y6 X1 |# I
0 0
0.4
0.8
1.2" k" {2 p( j. H1 j  A; J+ l
1.6# n( i+ b' j& S. L7 v, L5 P6 G
2.0' V( ?; }+ x9 Y& r9 ~
#5 F( u% l: t" \, K% N2 [7 y
Metastasis index (fold)
##
Figure 2 | MSC-induced increase in the metastasis of MDA-MB-231 cells9 C, l8 P  V8 q6 z
involves reversible mechanisms. a, BCCs were recovered from lung or
primary tumour tissues, cleared of stromal contaminants by culture in6 V4 G) m: F% S" H3 o) A
blasticidin-containing media (5 mgml21), and re-injected as primary
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.) d# U; |5 p" Y0 k
Data shown are representative ofmultiple independent experiments in which
four different paired batches of L-explant and T-explant cultures were assayed7 _2 {( ]# ~/ @4 f* W# f! v! `
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 using8 C4 K; ^  Z* Z: a+ v5 A
one-tailed Student’s t-test and indicates no statistical significance. d, Lung: Q# x5 |% D1 T! P+ ?
metastasis index of mice in c. Doublehash, P.0.3 using one-tailed Student’s
t-test and indicates no statistical significance.
a8 Q/ z* v1 w" E! M
CCL4. E9 S. ]) T0 L
bFGF/ h8 [& T4 ~0 j; t. z
VEGF
IFN-γ
TNF-α1 ~& x" o2 k0 u  i( E+ ]) P" _$ S
G-CSF/ h3 g; R1 y6 ]* U: m! d! {7 V
GM-CSF- f4 I. R- n7 M7 z
CCL3
CCL5# _: U9 i* K$ h7 v
MMP1
MMP3% r! f* e( P2 F* G( \# [
MMP9* j- @6 |% v' f8 ~+ W
MMP13
IL-1α4 P) f+ M/ W. M1 }6 b2 ~! q
IL-1β5 s; }1 f6 k. F1 G" Y. K& e4 L
IL-4
IL-5* L% M/ F- _% X2 U& n7 F+ o- I7 U! d
IL-6
IL-72 G0 ^4 W8 s+ c& y6 D1 z- t4 g4 k
IL-8
IL-10) u! r" R$ U7 [0 e7 e
IL-12
IL-13
IL-17
IL-2
TGF-β
Fold induction
MSC alone
MDA alone
MDA+MSC (2:1 ratio)2 P6 ]' c# e6 `8 X* d
1 * * * * * * * * *6 m! f% X/ V2 K; o1 l5 u3 ?
3
5
75 _" C' c3 o* T2 o, H6 @, v$ R; [
9, I- _& z2 b1 q$ ^& z2 ~& E) ]
11. ?$ `. _: U( p9 M6 z2 r1 M
13/ n6 }5 T0 L, e2 r
60
b c# v/ @1 r9 c1 T% Z" O0 P  C
0.4 μm
2.0
1.88 h1 |  L; |. x4 Y3 I& G$ C
1.6
1.4
1.21 A! s& h" ^- o7 d- ~1 ~/ m
1.0
0.8
0.66 h& W2 v" F1 O4 e0 v
0.4
0.2
0 0
5: ^- A9 O; W# O
10
156 m! }  d3 `% W* S* t$ d5 q; z
20. l3 u5 `8 S7 n! {: ^+ c
25$ R( ~0 r% L, U! {
30
354 [' h- w  Q" G6 p" O
40
45
50
d1 d2 d3 d4; z9 y, v7 [" V8 K& ?; W2 `! f
MDA alone
MSC alone
MDA+MSC
CCL5 levels (pg ml–1)* H* K. ~1 o6 x. Z8 v3 y
Co-culture* A7 ~9 T" }$ W% i/ m" G; {
Fold CCL5 induction. ~$ A  [, c/ I" O. ^
MDA alone) `9 K) A/ z$ c. j7 }8 |
MSC alone
MDA+MSC: ?# w( M' F( b; m( h
d
CCL5 (A.U. × 100)+ T! s$ n, s4 M, q5 l
30% A$ H# a9 w& H2 L8 K  N: j$ T
25) Q0 d; d5 Z+ M9 V
20! R0 s4 A3 @* J3 q% e: {$ }
15
10
5
0
+  G8 S# G; X  B0 H1 U
MSC.c
+- z' y/ R4 N8 V& g# \
MSC.1) E+ g# e7 P( U
+; I0 X9 p. }1 E( d/ }
MSC.5
MDA
MSC5 j! R3 f2 u6 v4 A0 |+ H! Z0 P
MDA.1
MDA.c
MDA.56 W! I: m/ O- J' \( H/ f6 _# |! H
TC-MSC. ?7 x  ~7 R: V
MSC (from MDA tumour)1 ]# ^* {! @5 m: ^) n, K
BCC (from MDA tumour)
Control (MDA/CCL5)
CCL5
GAPDH
e- v* E5 l$ ^0 Q$ k5 G
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 culture4 X' q" g% F8 k; P3 l+ R+ i
supernatants were measured by xMAP Bio-Plex cytokine arrays at day 3, and, l; V/ U  c. m- O: V9 z
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
MDA-MB-2311MSCcultures (1:3MDA:MSCs) at the indicated time points.# c% ]2 J( P/ P# I4 F/ J' _5 H8 E  j
Data points representmeans6s.d. of quadruplicates. c,BCCs were separated
from co-cultured MSCs by a 0.4-mmmembrane. CCL5 levels were probed by3 m' I! C* [: ?6 o
ELISA on the culture supernatants. Data are expressed as fold induction over
levels seen inMDA-MB-231 culture supernatants (mean6s.d. of triplicates)." ^. J+ `9 }: p' S  }" q# R6 p( a
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-siluc3 C( n2 K% U: H
(MDA.c), MDA-MB-231-siCCL5.1 (MDA.1), or MDA-MB-231-siCCL5.5% ~$ M7 H  f9 M9 h5 q
(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  t4 }5 V* j6 _5 s& W  [
GFP–MSC1MDA-MB-231 tumours (3:1 ratio) 4 weeks after tumour
implantation. Tissue-cultured MSCs (TC-MSC) and MDA-MB-231/CCL5) N/ g1 b5 b3 F1 j1 n! B2 k: C' _
cells were used as controls. GAPDH was used for equal loading.levels of certain released factors (for example, interferon-c or8 b) k5 Y: y2 S( N
tumour-necrosis factor-a) reflected the additive contributions of# n1 C) b' k' T! G. [
the two cell types when cultured on their own. Notably, the levels
of only one cytokine, CCL5, reflected a synergistic interaction
between the MSCs and BCCs, as it accumulated to levels ,60-fold# A1 y2 I/ |! c! q! H; i& _
higher than those produced by pure BCC cultures (Fig. 3a). This# X' W, }% T( `0 U1 r4 d
cooperative induction of CCL5 was proportional to the numbers of
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was apparent+ R9 |4 u4 d5 M& y" J6 d
as early as the third day of co-culture (Fig. 3b). Moreover, this3 m. s( k# g, K( x) T& m1 s5 P% g
induction required close physical contact between MSCs and cancer3 r* j9 d2 `4 V  l+ e3 E
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  S$ G8 B; J$ u0 D+ H# h! K; t: `
under conditions of co-culture. To do so, we stably reduced the
expression of CCL5 in MDA-MB-231 cells by.80% using short+ A0 ?2 ]2 \3 ?
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 the6 B# c/ L6 @! d8 R9 O& E. T
co-cultures of MSCs and control cancer cells (Fig. 3d). This suggested/ ~1 h! r/ V3 D* S! _
that the source of CCL5 was the admixed neighbouring MSCs.
Indeed, inhibition of CCL5 protein expression in MSCs using the
same shRNA hairpin vector (MSC.1; Fig. 3d) resulted in more than& U3 D# g1 M3 t# d1 D& H; ^! f
75% reduction of CCL5 protein levels in the co-cultures, indicating# X9 A$ V0 E( M: j9 s
that the MSCs were the major source of the CCL5 observed on coculture* u2 E2 r+ z6 y; b, c1 f* d
of the two cell types. In support of this conclusion, analysis of
CCL5 levels in the media of MSCs or MDA-MB-231 cells separated' n8 w  N7 z- O
from one another after 3 days of co-culture indicated a strong induction0 w0 T- O4 l) J% y* H
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-* ~; @$ o/ l( J# Y8 Y' y
derived MSCs (Supplementary Fig. 5c), as well as from the' j7 E" I1 K& z8 l
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after' i: t& ?9 a9 T2 Q
tumour implantation (Fig. 3e), indicated a strong accumulation of
CCL5 messenger RNA, suggesting that an active signal transduction' p  @' H1 r+ J* Q+ t1 t; {4 ^
pathway is triggered in MSCs by the nearby BCCs.- A$ T$ G$ L0 }+ ^
A series of observations has linked CCL5 signalling and cancer. For, I3 X- z7 P6 n# p
example, CCL5 levels in the plasma of breast cancer patients have- \7 G0 ~3 I3 W% H* Q
been correlated with the severity of the disease, and localized CCL5
protein expression was found to be elevated in invasive tumours
when compared to in situ ductal tumours or benign lesions16,17.! _& `4 T; {2 m! y* ^9 A  T( O
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 overexpressed0 E: s4 a6 T0 ^5 n6 u! j
this chemokine in the MDA-MB-231 BCCs (Supplementary
Fig. 7a) and analysed its effects on cancer cell growth and5 _1 L; Z/ \( h5 z: k; q
tumorigenesis. The overexpressed CCL5 did not confer any proliferative
advantage on cultured cancer cells when compared with9 X9 u2 y7 g. ~! g4 q
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  B7 c& C: g1 g
primary subcutaneous tumours in immunocompromised mice (Fig.
4a). However, these tumours exhibited a ,5-fold enhancement in
their metastatic potential when compared with control tumours
lacking ectopic CCL5 (Fig. 4a). Similarly, overexpression of CCL5
in WI-38 fibroblasts sufficed to enable these cells to promote the
metastasis of admixed MDA-MB-231 BCCs (Fig. 4b), indicating that
the actions ofCCL5 are responsible formuch, if not all, of the observed6 p6 h; \& C1 Q; K5 d
MSC-induced metastasis by the BCCs.9 i2 e. }* }7 f% g7 d" t* g$ B" N
CCL5 promotes lung colonization2 m- W6 h* k  _) @3 E; U3 V1 g1 \
Previous reports have described an important role for CCL5 as a- }" D1 i, f3 ?6 M. ?
chemoattractant for stromal cells, such as macrophages, that express
one of the receptors for CCL5, CCR5 (refs 18, 19). Furthermore,1 i, q. c% s5 Q( c$ A* x- T3 @; h8 @
CCL5 expression has been associated with increased tumour neovascularization,% T7 e$ _* l& j" _$ X
suggesting that endothelial cells, which express a variety
of chemokine receptors, may also be attracted by CCL5 to sites of
tumour formation, thereby enhancing tumour angiogenesis20. Such
observations suggest that CCL5 may contribute to breast cancer  t( Y! l6 a7 F' e
metastasis through the recruitment of a number of stromal cell types
to sites of primary tumour growth.
However, immunohistochemical analyses indicated that the
MDA-MB-231 control and CCL5-overexpressing MDA-MB-231$ \; o# M" l- z* N$ P4 x
(MDA-MB-231/CCL5) tumours exhibited comparable numbers of
tumour-infiltrating macrophages and had similar vessel densities (as/ @9 [$ Y" o8 V/ X
evident by F4/80 and MECA-32 staining for macrophages and
endothelial cells, respectively; Supplementary Fig. 8). In addition,
we found that ectopicCCL5 expression did not cause an accumulation: S4 r9 Z& R7 V7 J6 W8 a
of other stromal cells, such as SMA-positive cells, in the examined: B8 i6 `$ O( L5 b- H6 A; Q
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.; t# C2 o7 k( t8 U) z. j1 W
Invasion and metastatic dissemination of carcinoma cells are often
facilitated by their transdifferentiation through the process termed- U# \) p& t  j# b. y+ ^; D
d f3 a# H8 Y7 d! p" l! u
Bcl-XL
Bcl-2
β-Actin
S473-Akt, ~/ T% R  @8 t! E% Z
Motility
0.5%→10%
LY – – + +
g
Extravasated clusters (mean)- ?* w, h* e. r; E$ O+ t
Migration (fold), w! t0 z  g/ u( \! z! X
*
e
0
MDA/vector
MDA/CCL52 J2 d4 ~/ _0 q" ~9 g
***. q( C' ^3 e! I8 V
Invasion
10%→10%
Invasion+ J- n* X% n7 u% j' U  D
0.5%→10%+ D" ^* u1 a0 I& t2 E
Migration (fold)
**
08 X5 e# l4 K0 y/ T5 v' h, ]" C/ A
0.5
1.0* b6 b: |5 z3 E4 m
1.5
2.0
2.54 g& `1 }3 W* D* |# F
3.0
3.54 a8 h6 N+ B$ s6 S& E0 @, N
*5 v+ \+ X4 t. M1 F- F' ~
MDA/vector2 G( ~6 d  Q; s9 |  X  Y
MDA/CCL5
a b6 c9 m2 i$ `% L: ]( w# w* e
*$ G) j3 t  O3 b  w
Nodules per lung (mean)1 B& G7 j+ [; Y" q" i
c
30/ ~. ~0 R- l) L" z9 b5 t4 D% E% ?
25
207 z' r9 H& s& x1 c, i/ g
15
10
5
05 s* z3 g+ F0 m5 r& D" M
Tumour mass (mg)
300 X, ?2 q$ b$ q8 v
25% H. G; _0 j7 ]
20
15
10
5
0 0/ ]! {/ [8 a! b* Z4 h3 k9 F6 U8 m/ Z
504 s5 I; `9 L2 G1 N  b
100
150
200
250+ u$ P0 ]* ~9 [  g1 p
0
50* F! B/ u7 m) e2 ?: S1 M: f
100/ `4 K/ w5 j: g! V' b
150. d7 ~+ J# I% R2 I* h' N. C* ^; y
200' Y% ?+ m* @2 G
250
300
350
Tumour mass (mg)6 l2 z/ M$ x  g% M. g9 t* d% v( ?
Ctrl1 f0 ?, d& Y& @) n0 T
CCL54 j/ e5 w" C3 C/ j
Ctrl# ?% N1 F! V+ S2 F! J
CCL5' ?* _# X: Y7 h# l; \, e
Ctrl
CCL5/ a, M$ w# \- R
Metastasis index (fold)) G1 c4 @6 q2 ~. v; _; O( A* z+ B
Metastasis index (fold)
03 u& x) }! u6 H, X
12 r: ?2 X$ f# a6 ~8 t
2% k0 X9 x  V4 {  l
3
4
5. v3 }2 k2 q. s+ U, ~% \
* 6
04 r3 C- Z, I. Z: |+ H) ?
1
2, o" ~. f2 d: V2 K8 ^2 O
3& K0 {& r9 U; M$ h$ K4 J
49 b( C/ k8 Y$ l% o% N7 l6 R
5
6( o. O" \7 m/ K$ K! y7 Q1 s9 E1 q" K
7
* 8
MDA/vector
MDA/CCL5( t$ S6 x1 z% ]2 [4 f
MDA+WI-38/vector5 }+ a+ O) U  ^% r+ \& ?  y2 f
MDA+WI-38/CCL5
1& \% s8 y7 J. Y8 S& u
27 u5 O  v, J) c1 k7 e
3
4- G0 R" E5 m$ x: s" f
*# C( z( X: T8 b; ?6 T
Figure 4 | CCL5 enhances breast cancer cell migration, invasion and
metastasis. a, A total of 500,000 MDA-MB-231/vector (ctrl) or MDA-MB-
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour
masses (mean6s.e.m., n56 each group) were taken at 10 weeks. Lung3 L; {! b3 @8 ^) s( ?4 s$ U
metastasis indices are expressed as fold increase (6s.e.m.) over controls. Data7 {! r) ?: {; h0 o1 P8 h
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( b/ J: ?8 ~. c# G' }  t
overexpressing CCL5 (WI-38/CCL5) and were injected subcutaneously in
NOD/SCID mice. Tumours (n55 per group) were excised and weighed at5 `% Y8 d4 _$ j
12weeks. Masses shown represent mean6s.e.m. Lung metastasis indices are$ u" k) K0 A0 x" ^9 B9 o) f
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 introduced4 E- z7 m/ {" l6 p7 c; w$ S
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-" A* ]% y6 L' N! D' Z2 c
231 controls, n516 mice; MDA-MB-231/CCL5, n518 mice). Asterisk,
P,0.01 in one-tailed Student’s t-test. d, Western blot analysis of lysates of+ [# g$ d* w/ G: g* m' z
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
MDA-MB-231 control orMDA-MB-231/CCL5 cells.Data are representative of' Z7 }( J8 L3 s8 m
multiple independent experiments and are expressed asmeans6s.d. Asterisk,7 n; O; q' {' n( I
P,0.05; double asterisk, P,0.05; triple asterisk, P,0.01 in one-tailed5 D5 h, x; n( u) j. G  F* i% _
Student’s t-test. f, One million GFP-labelled BCCs were injected into the tail* N% ], J5 q* r% h' @9 D  t
vein of NOD/SCID mice. Lungs were processed 48 h later and examined for0 a7 J; F( c& z
extravasated cells. Bars represent means6s.e.m. (MDA-MB-231 cells, n57# E4 f; ?# p; }. |
mice; MDA-MB-231/CCL5, n510 mice). Asterisk, P,0.01 in one-tailed! H9 i3 |& E5 r
Student’s t-test. g, Transwellmigrationassays on50,000MDA-MB-231 control
orMDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-$ C% j% e( z" a7 ?7 O
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;* i$ }& q8 R; }" E# N1 M( ?
asterisk, P,0.01 in one-tailed Student’s t-test.the epithelial-to-mesenchymal transition (EMT), in which cells shed  q( ?7 \! t8 A% a
their epithelial characteristics and acquire instead a series of mesenchymal: g( u4 v8 f' X1 a
markers that enable their invasiveness and intravasation21.4 z; x% o8 |: y
Despite their lack of E-cadherin and their expression of detectable levels. \$ P  E6 g* X( [6 p8 p; U
of mesenchymal markers such as fibronectin (data not shown), the/ @0 y) l/ \( r# m0 e
MDA-MB-231 cells studied here exist in an intermediary phenotypic$ h$ X* S  L) p( d! W7 Z
state of ‘partial EMT’, as they retain a distinctive epithelialmorphology
in vitro and are still responsive to EMT-inducing stimuli in culture. In
fact, we observed that ectopic CCL5 expression did not cause MDAMB-( H7 _* @7 l' l" X3 H# ~) K
231 cells to undergo themorphological changes usually associated) s( d; d5 x! |" ^3 I
with an EMT(Supplementary Fig. 9a), did not cause rearrangement of* A# c' T; v$ g& n8 H5 T
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on
the expression of mesenchymal markers closely associated with the6 c: X$ Y" z( j
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c). j, u* T3 Q9 c4 z, ~
and fibronectin (data not shown). These data suggested thatCCL5 does* Q8 f- o8 O; p
not directly promote the EMT programme of MDA-MB-231 cells." x+ ~, ?3 S+ \3 F: Q- t
We proceeded to explore an alternative possibility: that CCL5  s8 Q( h2 l. T! z
expression affected some of the later, critical steps of the invasion–/ [' _% K  l9 u) N+ ~
metastasis cascade, namely the lodging of cancer cells in secondary" O* {5 ~. {/ d# ]
organs and the subsequent step of colonization. For that purpose,( F: X# I4 J: a6 [) T% H- C" `
MDA-MB-231/CCL5 cells were injected intravenously into host. Z% G9 ~4 {3 Z! ^- u
mice, and the lungs of these hosts were examined ,6 weeks later
using fluorescence microscopy. These experiments revealed that: f9 B! h2 n' w2 z1 _/ c
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, N. |" c# q0 i/ v2 p. Y
of the invasion–metastasis cascade. We note once again that this+ \$ Z4 R0 a) t7 U+ z
enhanced tissue-colonizing ability was not due to CCL5’s effects on
cellular proliferation measured either in vitro (Supplementary Fig.
7b) or in vivo (Supplementary Fig. 7g, Ki67 staining).4 w! W" o4 `0 f5 o# l- o
Because improved colonization can be due to enhanced cellular: j1 Q5 [8 Z2 L4 ~
survival, we tested whether CCL5 protects against apoptosis.. w! X: ^# K$ b! N
Notably, we found that MDA-MB-231/CCL5 cells exhibited higher7 @4 k' Z' v' {# s
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* |$ b* T1 a8 n$ o+ D2 e7 s) T7 J$ a
pro-apoptotic molecules such as BAX or BAD (data not shown).
Moreover, we found that overexpression of CCL5 had no effect on0 ~9 J5 O6 B- O0 X" N% c
the ability of MDA-MB-231 cells to withstand serum deprivation! h  N. x# X7 X1 [' A% y4 h
(Supplementary Fig. 7b), loss of substrate anchorage (Supplementary
Fig. 7d), or hyperoxia (data not shown). We also observed that( T7 r4 Z  o0 Y; {) b4 C# T
ectopic CCL5 expression did not protect MDA-MB-231 cells from, ]) \3 [3 q' N% W" `
doxorubicin-induced apoptosis monitored using western blots for
cleaved caspase-3 (CC3) and cleaved PARP (as markers of apoptosis;
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling9 V) E, P/ }+ j; N; e8 j; }
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohistochemical. G9 t7 L4 `  P% ~5 w* c/ D" U
analyses on control and CCL5-overexpressing tumours
revealed only minor differences in the levels of apoptotic CC3-4 ?, I! x/ h0 v: Z! U
positive cancer cells among the examined groups (Supplementary3 M6 R: ]2 y  v/ s. [
Fig. 7g, h). Together, these observations suggested that CCL5 does
not exert any detectable pro-survival functions in vitro or in vivo, and$ a& U$ ^# e  Y
that the observed enhancement of lung colonization was not a consequence
of significant anti-apoptotic activities of CCL5.0 O0 ^7 T! F+ X$ }! l
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# g% a3 Y" b4 H  J( h! k
against apoptosis, we tested whether the CCL5-enhanced lung colonization% S/ j0 w3 |% Z6 Y
could be due to an increased ability of MDA-MB-231/- I' l3 Y/ h0 D
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% F: V% Z7 o8 }# t5 @' H- \$ o6 P
through permeable Boyden chamber membranes by,1.5-fold as well) a. H/ j) Z# l4 x0 w
as the invasion of these cells through Matrigel layers by,1.6 or,2.5-4 w3 R3 G7 j# [! S. g1 T5 ~
fold in either high or low serum conditions, respectively (Fig. 4e).
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein2 H2 S/ R; m& k0 X  {: i: D
injection—in order to remove most cells that remained within the
microvasculature of the lungs and thus had not extravasated—we
found twice as many deposits in the MDA-MB-231/CCL5-injected! g: V6 |3 Q1 b, D# ^' v( b
group than their control-injected littermates (Fig. 4f). This indicates a4 `3 h" G0 C* H* }* l
clear effect of CCL5 on cancer cell extravasation.
Finally, we investigated the role of Akt in mediating the actions of
CCL5 on cellular motility by using the phosphatidylinositol-3-OH
b silacZ
silacZ, m, F3 Y0 s2 b2 I
si809
si8091 W3 X6 x# A1 c- s: p8 A
si1866 |' y6 I; j6 P, k" g% S* T
si186/ V1 y' {% M1 x' w; {  S1 t: J
CCR5
β-Actin
a d8 h8 x$ v$ ?! x  o8 f( k
M. {# ?2 z# `4 C8 {: [% f* Q+ P2 r0 l' v8 z
+; i8 t  `; ]2 V* x% g
MDA+MSC+ i$ a# B1 {) Q5 ^! u! z2 h4 P. y
+ IgG; U+ i3 e2 z* X- x  d# \( p, r6 E
+Anti-CCL5 Ab
MDA9 {3 ]- Y% D7 H) `* N; W
+Anti-CCL5 Ab
MDA+MSC
MDA1 X& l, j9 r1 E3 {  }% `/ ?- t$ W
IgG
<) H7 N5 [. \" U8 V5 x) E( H
<7 _6 g% x! C, f# a4 ^, A
<
<2 S/ {( ?' A$ M- F4 Z% u6 ]- b& o
<
DAPI
CCR5, ~! _/ A, _& t% J- F3 \
MSCs MDA MDA+MSC3 o% h" r: d5 Q7 _7 c" G5 I7 \
DAPI DAPI9 @& T, t# b, [- ]3 }/ r
CCR5 CCR5
MSCs MDA MDA+MSC0 a; ?1 s: U, P8 N
*
c
Metastasis index (fold)
Metastasis index (fold)$ o  g7 Y2 \" W: s! {! I7 _
*
Control siCCR5
MSC – + + +
0
19 ^% {7 q7 x) g& n0 ~; Z! f+ X! \/ X  B
27 E2 f8 H) q3 y6 @$ h
3
4
5 e
+ + – –
Anti-CCL5
0
1) H7 k' V2 n2 D
2
3
4
5+ @. H" T: A" l, r+ ?. \/ h
6
– – + +
IgG) w$ M3 ~0 i; F4 T. a" c
MDA
MDA+MSC, d/ `% h5 `  V* J6 L
<9 j1 N" F# M8 t, r9 L6 W5 P
Figure 5 | CCL5–CCR5 interaction is essential
for the MSC-induced metastasis.
a, Immunofluorescence analysis of CCR5- p* p* q  v( r& K4 x* C
distribution in MDA-MB-231 cells cultured with6 r5 `: m2 |0 d: V
MSCs. DAPI (for nuclei staining) is in blue;
CCR5 detected in green. Arrowheads denote
MSCs. b, Western blot analysis showing CCR52 S. d  X! ~- l( o  F$ E8 B
expression in MDA-MB-231/silacZ, MDA-MB-
231/siCCR5(809) and MDA-MB-231/' R5 R2 Y3 {( M8 V$ N/ X
siCCR5(186) lysates. b-Actin was used as a
loading control. c, A total of 500,000 cells of the
MDA-MB-231 variants in b were co-mixed with  C& O2 g7 ~6 F% `, b
1.53106 MSCs and injected subcutaneously into9 h$ a' ?2 X& ^7 v
nude mice. Mice were killed when tumours
reached 1 cm in diameter and the metastasis
index was calculated for each cohort (n55 per) @( T; ^& S9 E! M2 l
group). Results represent means6s.e.m.;
asterisk, P,0.05 using one-tailed Student’s
t-test. d, Anti-CCL5 neutralizing antibody or4 {8 Q& J3 s% I2 U4 q# K" h: ?
control IgG was administered intraperitoneally
twice weekly in SCID mice bearing MDA-MB-231" b- j. R' C  q4 q! g
(n59) or MDA-MB-2311MSC tumours7 G) s" g/ {" K$ q
(n511). Representative lung pictures of the" K' ]1 R  b' ~6 h
indicated cohorts are shown. e, Lung metastasis: o1 z- v- X7 B( S; T/ J* E
indices of mice in d. Data shown are
representative of means6s.e.m. Asterisk,& `# \. R. l3 }7 \
P,0.05 in one-tailed Student’s t-test.
kinase inhibitor LY294002. Drug concentrations that did not inhibit
the basal motility levels of MDA-MB-231 cells blocked the elevation  S! |, ~  S( U3 @& m- @' x. K
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# A, R' a/ M7 W0 a
at sites of dissemination rather than promoting the survival and/or9 s0 l3 h! i/ O6 D/ }
proliferation of these cells.- \, h2 V& s; @
Essential role for the CCL5–CCR5 loop2 l" ^8 q! _8 e. r: R* @4 G
CCL5 acts through three G-protein-coupled receptors, termed) _) O3 F0 n2 L/ d, V
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- R! M2 J2 n, Y% f" S9 W5 M, H9 `
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) S6 ]( @: {" R1 X6 c" J- l5 r' H
not by MSCs (Fig. 5a), supporting the notion that MSC-derived- c6 O7 z1 u3 A$ w: d
CCL5 acts primarily in a paracrine fashion on MDA-MB-231 cells
in the BCC and MSC mixed cell populations described above. To; y! r1 U; R7 |- |
probe whether the observed MSC-induced metastasis required
CCL5–CCR5 interactions, we inhibited CCR5 expression in MDAMB-0 s3 [3 f' Y- Q, E1 R
231 cells by more than 85% through shRNA knockdown (ref. 25, P% y7 {# ]1 u
and Fig. 5b), and mixed these cells with MSCs before implantation
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,5 Q" f0 F$ Y. F* O# ]
achieved using either of two different shRNA constructs, abrogated
the ability of MSCs to enhance the metastasis of MDA-MB-231 cells7 c2 k- I9 Y1 h
(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 Y/ K+ D: [, f, c
(Fig. 5d, e). In addition, MSCs in which CCL5 expression was inhibited) t; q, F7 L+ Q7 W4 W
by shRNA knockdown failed to promote metastasis of the7 Q, Y/ r% X. D
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 the
MDA-MB-231 cells." F# W; }: T* Z- `5 g1 c6 d
Discussion
Certain models of metastatic progression propose that cancer cell- r+ a# c' i  K3 a# y5 {2 K( b- {; m
invasion and metastasis from the primary tumour site are strongly' U, g' ?7 \& |
influenced by contextual signals emanating from the stroma of the
primary tumour. It follows that if carcinoma cells are subsequently
deprived of such signals, they may revert to an earlier phenotypic
state in which they no longer display the traits of high-grade malignancy.
Indeed, such a model has been proposed previously by others0 A9 K' G% a; Y2 Q. b
on the basis of indirect evidence21. Here, we demonstrate that at least
one mesenchymal cell type, the MSC, can expedite tumour metastasis,
and suggest that after primary human carcinomas recruit MSC
populations into their midst, subsequent interactions between the
MSCs (or their derivatives) and the BCCs endow the latter with
invasive and metastatic properties.
Although the recruitment of labelled MSCs to tumour xenografts' D2 Y( u6 J, z% E+ X) Y) N
has been established in a variety of experimental models of tumorigenesis,
there is currently no available way to quantify with any accuracy
the number ofMSCs in actual human tumours, in part because no set
of markers has been identified that can uniquely stain these cells without9 |4 G7 c  A0 L! C$ `7 t! _8 T7 m
concomitantly staining other mesenchymal types in the tumourassociated+ b- |  j7 B+ {2 ^) F
stroma6. Our demonstration that the stroma derived from  _0 W9 \# Q1 m! z. ?. _% K
tumour xenografts contained appreciable numbers of murine MSCs
indicates that significant steady-state levels of these cells aremaintained
in developing tumours. Interestingly, the use of CD10—one of the2 ~# s6 X0 K1 U$ b8 z- T& {* f
markers associated withhumanMSCs—to purify cells fromthe stroma
of human primary invasive breast carcinomas yielded a population of. C1 P, e9 u# ]: C6 c9 u4 P
cells that expresses a number of other markers collectively used to
characterize human MSCs (for example, CD44, CD105 and CD106;# h( _* _/ i4 s* ~! q
Fig. 6a). This suggested that, similar to tumour xenografts, human/ d% F8 E9 p5 o0 J) N
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;1 b& t* J5 b1 {6 [! y
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free9 P. \- |7 {; Z6 H7 o0 H& l; r
stroma of primary invasive ductal carcinomas (Fig. 6d), specifically in+ i% E+ d  A/ B5 Z1 e0 T
the CD10-positive compartment27 (Fig. 6e). Collectively, these observations
argue strongly for a significant association between stromal
CCL5 levels, MSCs and human invasive breast cancers.* Y; J5 A$ {+ ?1 w9 H5 d0 t
c% J  {( d8 k+ s+ w& L# K
STT1969B
STT31260 A$ U6 w/ |) B& v* C/ ]
STT31243 Z* b9 r9 v9 f
STT656B: _8 d* B% f4 _8 k  t
STT1968B" a8 A$ f& z2 ~6 m9 o
STT3122
STT3053
STT1986B
STT8543 G+ E  Y9 H3 @) O
STT3125: h9 `, Z2 e+ g+ o6 y- {
STT1975
STT1987B9 {( L5 k' F) T- {4 r
STT1079
STT638( E1 r. F3 s# I, W& W: A6 y2 [/ I4 t
STT17743 d& e! O! f8 \# {
STT1984
STT1737C1 r( j& S; M5 u, D3 X" `4 H
STT3068
STT3120* Q+ Q! ?6 }, w8 {
STT850! \$ @* d7 E& G4 E
STT417B8 B) E( s" U* t. D2 p8 C, {
STT3119
STT1776
STT1777B, r8 a* g5 G- [  B9 W+ P' Z. y
STT689B
STT1971
STT597# o$ d* s7 @2 H4 A  s# w
STT626
STT154
STT2774
STT1966
STT2776
STT27751 _% P' L/ {) p
STT27720 q  r2 G: ]( c" V+ m: }
STT1637
STT1220C) i6 q5 \9 G1 |7 W0 q& @. {
STT-094B-13 T% x+ f' N0 ^5 y
STT6756 r. z2 g' {7 R0 p, w
STT2770
STT695B  l8 F( Q/ f# O  f2 b8 ~
STT1771
STT1778, n2 V' I2 ^1 L) C
STT491
STT1823
STT200C
STT741B
STT335C
STT709B
STT516D+ [- U, ~' k5 B1 m
STT607B$ K- P5 A' I4 r& B
STT680B0 a! @* G4 y% b* H
STT1148B
STT523B
STT526E
STT742F
e- A, g1 C$ k- |1 d6 C, q1 V( c9 P
CD13
CD29
CD44
CD49e% Y3 o7 Q! ]% h8 `! X4 m! r! j
CD54
CD59
CD63
CD105+ i7 b* L# l! @: c; g  F
CD106% T9 n! p2 n* s8 M
Nestin
HAS2. p4 h  ]1 N% H
IGF2
PLAU
TIMP1
CAV1/ S6 u# H. Y1 K. ]: {# C
IDC-7+ n) P/ x, R9 y/ S3 v; Y. \
T112603
T392303
Normal Invasive& H+ {1 G  A$ J; G3 C! W& G
CCL5) `" e- n* b3 T# C
log2 ratios4 l0 V# l3 _7 a5 ]- x- p# L2 h
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.05 B+ @2 C, Z$ u' R6 q4 k
log2 ratios
d
–2
–1- A8 g  N, c* r/ X, O" |3 p$ V4 S: g
0 1 29 i! k$ m4 _3 `2 C
–2.0
–1.5) Y% ]& Q5 d( s: E- s% m
–1.0
–0.5
0.05 }5 v1 b# H$ P; e
DTF SFT
DTF# ~# a' L2 z7 I6 k: ]% T6 n
SFT9 D! h6 G+ H$ L
a b
F
Figure 6 | Stromal fibroblastic cells of human invasive ductal carcinomas are
rich in MSC markers and overexpress CCL5. a, SAGE TreeView display of
MSC markers expressed in stromal CD10-positive cells from invasive
tumours27. b, Soft-tissue tumourswere ranked byCCL5 expression26, fromlow( h  @, W* z6 I: t* _, I" N5 J+ E
(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. Y: x, j* D6 W+ k0 R# d
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
between SFT and DTF was tested with the Welch’s test. d, CCL5 Affymetrix
gene expression in the stroma of human invasive ductal cancers compared to
that in normal cancer-free breast tissue (indicated as ‘Normal’; see Methods).
e, CCL5 expression is mostly restricted to the CD10-positive fibroblastic cells
derived from invasive ductal cancers. The heatmap shown is a cluster of; U' h6 x5 a/ C6 j: _
CCL5.genelist obtained as in a.Details of thepurificationmethodologies of the
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-; }  ]" h5 n# b$ \0 y
231 cell interactions, CCL5 seems to have an equally critical involvement
in the functional interaction of MSCs with MDA-MB-435; S, p- {* O% l2 n7 w( L0 ?0 W* Q
human BCCs. CCL5 levels accumulate synergistically when the two
cell types are co-cultured together (Supplementary Fig. 10a), and- `4 O! X, ~8 Y
MSCs in which CCL5 expression was compromised by shRNA knockdown
failed to promote metastasis by MDA-MB-435 cells to which- U& R: R# Y" ~6 u, G4 T) j" m) t
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 may0 `7 p! N8 O& Y/ c0 e9 D
depend on other paracrine factors such as VEGF and interleukin-8.& M3 u5 h  z- b3 Z8 e4 a# u# F0 @
Nevertheless, our observations highlight the recently discovered critical
roles of chemokine networks in malignant progression28,29 and suggest
the possible utility of a variety of CCL5 analogues and CCR5 antagonists
currentlyused in anti-HIVtherapy30 in treatingmetastatic disease.
Notably, we have observed that MSCs induce the metastasis of cells7 A( k2 Z% Q* U. f+ r/ s  a# G
to the lung that are, on isolation and re-injection into recipient mice,
no more metastatic than their predecessors in the primary tumour& z& Q% B0 K( D  ^/ z+ i0 H3 x+ i  J
(Fig. 2e). This indicated that acquisition of increased metastatic1 ^; k; v( {/ c- Q
powers by these tumour cells was reversible, and suggested that the3 m% Z! ]3 [. Q8 U0 h& p& h- T
maintenance of this phenotype depends on continuing contact with
stromal cells. If extended to other tumour types, the present results
hold important implications for the molecular analysis of malignant) h0 S) @+ I6 r1 I: Q4 x
progression. They suggest that many of the cellular functions associated/ I6 G: H6 b5 i% s2 Y; Y
with invasion and metastasis are often not expressed constitutively( }+ w8 ~2 X$ W# w  S5 A* Q7 D/ b
by carcinoma cells, but rather only transiently in response to. S- a" J. v  p7 r" K) v/ u
contextual signals that tumour cells receive from their stromal microenvironment.; N" k3 D9 o2 ?6 ?( Q1 c
If so, analysis of the gene expression patterns of bulk3 I# w+ k, z) E8 m9 I
primary tumour populations may fail to detect the expression of key1 O7 A" e) s0 K) |7 x
genes mediating invasiveness and metastasis, if only because they are& r4 {$ W' Y; J7 r& j1 A- @& }$ `
being transiently expressed in minor subpopulations of cells within
such tumours. Additionally, attempts at determining the metastatic
propensities of tumours may need to be focused on the genes and- k6 o# c+ Y/ \$ ~' J
proteins that confer responsiveness of primary tumour cells to stromal5 w3 s+ {) k# V1 {
signals, rather than on the genes and proteins that directly mediate* s8 n6 @; s/ D2 Y
the cellular phenotypes of invasion and metastasis.% t* a' }9 H, X2 G
METHODS SUMMARY
Cells labelled with GFP or ds-red, or harbouring various overexpression or0 k. ]# h+ Y( ~& Y! ]/ ^1 r2 U5 F+ a
shRNA constructs, were generated by viral transduction followed by FACS8 o; }! {% H. I* ^. s* G% e' I  C
enrichment or antibiotic selection. Xenograft experiments were conducted in5 Y2 _( b! p- a4 _1 \
nude or NOD/SCID mice and metastasis was estimated using fluorescence+ b, {1 M& {# k. X
microscopy. The levels of cytokines, growth factors and chemokines were
assessed by immunoassays. Migration and invasion assays were conducted using: F: _7 [4 U' x1 ]6 v
transwell chambers. Antibody treatment of tumour-bearing mice was conducted
by intraperitoneal injections. See Methods for detailed information regarding
cell culture, viral infections, in vivo colonization and extravasation assays, RT–$ Y/ f+ F" B2 _4 H
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluorescence
determinations, western blotting, and antibodies used.' ^  K* ^% l' j* P+ X
Full Methods and any associated references are available in the online version of2 R% j  ^( G, _* E' Y7 n( ]/ c
the paper at www.nature.com/nature.

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