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



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发表于 2012-5-17 17:15 |只看该作者 |倒序浏览 |打印

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the
tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour2 c6 U/ V6 i# j: v2 ~
pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived humanmesenchymal stem cells,: Z# @/ I+ K+ h. ~
when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their* a( d% _6 X! o+ a4 M) z/ T
metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour& i/ O$ e/ x" n( Z8 {6 \" u: r, K
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+ f6 u- }2 \) K  q5 E
metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine- y# `% h" k5 ^3 a
receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by
eliciting reversible changes in the phenotype of cancer cells.
The origins of the invasive and metastatic phenotypes of carcinoma
cells have been the subjects of intense investigation. Whereas some
current models depict these phenotypes as cell-autonomous alterations( v# m) u' h( r4 o0 q$ G+ P% w
specified by the genomes of cancer cells, alternative views propose6 r  m- S( V+ S$ \8 J5 {* t; \
that metastatic traits are acquired through exposure of epithelial
cancer cells to paracrine signals that they receive from mesenchymal, X, n3 ?8 j2 |
cell types within the tumour-associated stroma. Although several
lines of evidence demonstrate the contributions of stromal cells to+ P0 k3 N  E' Y7 u
primary tumour growth1, direct experimental demonstration of the
influence of these various cells on the metastatic abilities of cancer) M. O- O5 L% A0 O1 h# r  d. g) l
cells has been difficult to obtain. This is due, in part, to the complexity/ I) }! b: S1 k" ~7 z5 _
of the mesenchymal cell types that are recruited into the stroma, and
to the elusive nature of the putative paracrine signals that are
exchanged between the mesenchymal and epithelial compartments
of a tumour. Recent reports proposed that the bone-marrow-derived
mesenchymal stem cell (MSC) is a cell type that is recruited in large
numbers to the stroma of developing tumours2. To characterize better! K) r& k$ R$ L& ], t
the role of this stromal cell in tumorigenesis, we set out to determine* u9 q; ~. M$ h0 H) @  w
whether MSCs could supply contextual signals that serve to/ C. p! Q4 L9 P% V7 _6 K1 ^: S
promote cancer metastasis.
Mesenchymal stem cells are pluripotent progenitor cells that contribute9 ~; Q  U  E  Y' H+ t/ {
to the maintenance and regeneration of a variety of connective
tissues, including bone, adipose, cartilage and muscle3. Although! o2 X1 w0 @# a$ w* v8 y! u' R& u
MSCs reside predominantly in the bone marrow, they are also distributed$ u. n* f  H! N2 r: k, ?
throughout many other tissues, where they are thought to6 \& U2 J% Y: ?. V5 e* H( `. z
serve as local sources of dormant stem cells4,5. The contributions of
MSCs to tissue formation become apparent only in cases of tissue5 b2 A: J) D$ M5 R/ T
remodelling after injury or chronic inflammation. These conditions
are typically accompanied by the release of specific endocrinal signals9 s& |. ^4 a/ L3 v6 g# @
from the injured or inflamed tissue that are then transmitted to the
bone marrow, leading to the mobilization of multi-potent MSCs and& F2 g" i: {, o9 F
their subsequent recruitment to the damage site6. For example, MSCs
have been shown to contribute to the formation of fibrous scars after/ E- t9 N$ ~3 ]4 N: c$ q
injury7." ^& J6 n1 P8 `* {
The formation of breast carcinomas is often accompanied by a
well-orchestrated desmoplastic reaction, which involves the recruitment" B& ]5 J# R& m$ R$ i
of a variety of stromal cells with both pro- and anti-tumorigenic) I) l6 U/ v# w4 l$ J2 `' y8 y6 w
activities1. Such response closely resembles wound healing and scar/ w; U0 \6 l: ~" W
formation, and entails the constant deposition of growth factors,
cytokines and matrix-remodelling proteins that render the tumour
site a ‘wound that never heals’8. This suggests that, similar to sites of
injury, actively growing tumours recruit MSCs through the release of
various endocrine and paracrine signals. Indeed, as we have found,
mouse stroma prepared from developing human MCF7/Ras or
MDA-MB-231 breast cancer xenografts is rich in cells with an ability
to generate fibroblastoid colony-forming units (CFU-F) in vitro
(Supplementary Fig. 1a), a hallmark of MSCs3. The absence of such" Y! }: r" q' Y2 A
colonies from control Matrigel plugs or from neighbouring tissues& m  O/ e& u: w0 D1 g) ^; X& Y
(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+ e5 E; s7 W* r; j2 N5 P( u0 V- ^5 n
ability to attract human MSCs, we established a transwell assay9 \' ^9 i( a+ a; k2 E# h
in which bone-marrow-derived human MSCs were allowed to2 Y( M) s5 I% ?- A$ M& M2 X& A
migrate towards media derived from MCF7/Ras or MDA-MB-231' i  w* l( r4 Z9 }1 ^( m" _8 h  Z* }2 P
cultures. We found that human MSCs migrated much more avidly
(,11-fold more) towards media derived from these cancer cells
than towards control media (Supplementary Fig. 1b). More importantly,
green fluorescent protein (GFP)-labelled human MSCs) e' u0 k4 f# D8 ~0 {8 F9 n
infused into the venous circulation of mice bearing MCF7/Ras! J- i( L- ~$ X8 q2 [2 L
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, }; o; V. p1 y: G$ q# C
and spleen (data not shown). Such findings indicated that MSCs* @% b# Z+ Z; ^( m6 J' t8 j
are specifically recruited by subcutaneous breast xenografts, and corroborated' _/ Q  W5 ?( n4 r
recent studies that described the localization of systemically
infused MSCs to other types of malignancy, such as gliomas9,10,
colon carcinomas11,12, ovarian carcinomas13, Kaposi’s sarcomas14 and. W3 Z- d# M5 e/ o+ `+ C* R
melanomas15.MSCs enhance breast cancer metastasis
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 MSCs) V- _4 E1 M# P5 N* o. @; ~
(hereafter referred to as MSCs) and injected subcutaneously into
immunocompromised mice. The growth kinetics of the MSCcontaining
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.  v+ D! o& I, q# t5 R# |
We found that MSCs accelerated the growth of MCF7/Ras, O$ {( n( C/ y% {
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-
4351MSC and HMLER1MSC tumours displayed a marked
increase in the numbers of micro- and macroscopic lung metastases/ D% s4 Y: u- e' B
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs
of BCC1MSC-bearing mice compared to their BCC-control littermates
revealed two-, three-, four- and sevenfold enhancements in8 I: L! y5 C" w( b; a0 K
the overall numbers of detectable HMLER, MDA-MB-435, MCF7/
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c)." B- Y- V- R5 p1 n1 ^/ l
Furthermore, in contrast to the MDA-MB-231-bearing mice, the
MDA-MB-2311MSC-bearing mice showed metastases to various9 k' `; a5 \3 W+ W/ A2 x0 t- `2 g
other tissues, including the mammary glands (Supplementary2 J5 u; X1 d) W: R
Table 1). Although all four of the tested cell lines exhibited enhanced: o8 z+ ~1 Y3 q7 G8 m
metastatic potential after admixture of MSCs, we chose to focus
further analysis on the MDA-MB-231 tumour model, because it
displayed the greatest relative increase in MSC-induced metastasis
without any concomitant effect on either tumour cell proliferation
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall primary. N' I& O  H6 o: [5 e
tumour growth kinetics.  f' u6 r, _  {; L1 {8 c0 V
We note that admixture of other types of mesenchymal cells—" z6 @9 b* z- N) d1 C' w
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 3
and data not shown)—to MDA-MB-231 cancer cells before injection1 K* q8 s% L0 ?. i! T7 i
into host mice did not result in either enhanced growth kinetics
(Supplementary Fig. 3a, b) or increased numbers of lung metastases+ V# }. h0 ~: J: _4 a% S
(Supplementary Fig. 3c, d). Taken together, these observations indicated, h& F0 ]; e9 z' ^" m! Q
that the metastasis-enhancing powers were a specific property4 A; I8 e. w" Z; @' r" W
of admixed MSCs or derivatives thereof.
Reversible metastasis1 w& N, L7 w, _6 z' e
Implantation of MSCs either contralaterally to MDA-MB-231 cells or2 [) r% S% e2 }- ^
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: ?' g/ ?7 e" p4 K) W& d. B/ M8 o
be ascribed to various effects that MSCs exert on the commingled
carcinoma cells. Thus, the MSCs might favour the outgrowth of rare
variants within the MDA-MB-231 cell populations that exhibit
unusually high metastatic powers. Alternatively, the MSCs might
cause otherwise weakly metastatic MDA-MB-231 cells to acquire
enhanced metastatic abilities. This latter mechanism suggests the
possibility that the acquisition of the metastatic phenotype might! C0 }* I% F1 o2 e' A$ G3 ^% c, v. g# G
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)! k+ q2 _4 q- E' I1 D, n+ e$ n, H
as well as from their derived lung metastases (L-explants),
expanded in vitro, cleared from contaminating stromal components,
and then re-injected into subcutaneous sites in host mice in order
to evaluate their respective metastatic powers (Fig. 2a). Although! G6 J2 {0 \2 Q8 K7 u# d# X
the growth rate of the resulting L-explant primary tumours was
marginally enhanced compared to their T-explant counterparts
(Fig. 2b, c), these L-explant cells were no more metastatic than the
parental T-explant cancer cells (Fig. 2d). This suggested that the
a. \4 ?0 P6 S# }& u
c d
Days after injection
Tumour volume (mm3)1 Q; V- {2 q! h: G* B$ V4 x) Z% I
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* \4 d- ]% ]+ F7 y; X$ k/ W
1 mm 1 mm6 F% g( n/ U! u" o1 I7 _
1 mm 1 mm& ]& V, K/ T' y
300 μm
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC- E* N# S' g* R
MCF7/Ras MCF7/Ras+MSC  B7 h; b6 v9 R' Z/ a# {7 d
100 μm0 F8 ?; N2 W3 D! F3 ]) I
300 μm
100 μm
MDA-MB-231- O6 J! q+ w5 Z: _  M9 x* S+ {
MDA-MB-435
MCF7/Ras' |& |/ H  a. _1 b9 r/ ]
**
Metastasis index (fold)
0
1- s1 H$ b2 [$ `0 [) q
2
3
45 P1 A) k4 q' N1 N, T0 Y8 b& Z+ h1 j
59 i* k; l/ j$ ?
6
7
8, `( J7 ~8 I8 S$ R" j" M
9
*/ F: z8 m) A7 Z
**# r- C1 ^9 ^' w, _' N7 O
**
MSC – + – + – + – +
*3 R. C- Q) p( Q9 i
HMLER9 K: P4 ?, Z$ i3 f% \
700 1,200* g1 o) s- a, p+ h, s  C) o
1,0006 z- v4 G* ?+ K/ O- b
800
600
400
200* m9 N, q" g% C! z! a; ?! r; o/ f) w
0% _7 H/ C& N4 p# P+ x% S
800
700
600
5000 K: P! V; m/ c  |1 ^+ U7 J6 E
4001 y) q0 P1 I( R  K0 K4 h$ e1 Q  W
300
2009 g, w0 A8 q3 b! f
100
0
3,500- H8 F) B6 t- I# S
0- d* v# {1 F7 K2 T) ^' f& u
500! g3 n0 c% M: i7 z0 U0 K' e& B) f2 k
1,000
1,500: G9 i. Q# ?  _& g6 O0 C
2,000& C  u- g& _6 A) R" ^; {
2,500" _! N/ Q: b3 F- j
600 3,000
5008 L: P4 O( K8 v; @4 C9 |
400
3004 p  P! x' L4 M; X4 E9 S
200/ D& H$ P% i5 W$ i: F( Y2 w$ |
1006 e2 u, {. L1 \
0. s9 _9 i3 J. @+ U, Y8 }
0 21 31 38 49 56 63 70 78
MCF7/Ras alone
MCF7/Ras+MSC
MDA-MB-231 alone
MDA-MB-231+MSC
MDA-MB-435 alone; O3 k* ~3 N! `6 e& w
MDA-MB-435+MSC
HMLER alone
HMLER+MSC: Q# B& U4 r$ u3 K0 I. O5 {) W* x
HMLER HMLER+MSC# L8 n  L9 f* S- x" O4 M
MCF7/Ras MCF7/Ras+MSC
MDA-MB-231 MDA-MB-231+MSC4 l5 w' K$ ^2 z5 P7 k/ A
MDA-MB-435 MDA-MB-435+MSC5 ~6 i8 t0 {' l: Z0 q5 [
Figure 1 | MSCs promote breast cancer metastasis. a, Tumour volume/ F+ F; k+ u8 v: D$ A' Q
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
alone, n55–7 mice per group; squares, BCCs plus MSCs, n55–8 mice per
group. b, Representative bright-field/fluorescence images of lungs of mice6 Y6 m1 K: F5 L  p0 G; g
bearing the indicated tumours. Cancer colonies are in green. MCF7/Rasbearing
mice were killed at approximately day 150 to allow these tumours to
grow to comparable sizes to their MCF7/Ras1MSC counterparts. c, The8 j4 W" }6 v" b/ X+ {
lung metastasis indices pooled within each cohort of mice in a are expressed
as fold increase (6s.e.m.) over controls. Data shown are representative of
multiple repeats. Asterisk, P,0.01, double asterisk, P,0.05 using onetailed3 r& M& b0 e5 N, D7 Z
Student’s t-test. d, Representative haematoxylin-and-eosin-stained  \6 q& c) n- x; k
sections of lungs of mice bearing the indicated tumours. Metastases are+ J" \9 X0 w/ d" N- y
delineated by a dashed line.MSC-induced metastatic powers reflected a reversibly induced trait' I/ L7 I* ]9 @. T7 }
of the MDA-MB-231 cells, and that the ability of these cells to metastasize
to the lungs was a consequence of their ‘education’ by MSCs in; [( E2 A% z' l* t
the primary tumour rather than the selection of rare variants of
MDA-MB-231 cells that display elevated metastatic potency in a1 R0 c) r, _+ e9 z# \
stable fashion.
The effects that the MSCs exerted on the BCCs might have9 e% ~8 \4 O- r2 s* m
occurred within the site of primary tumour formation. Alternatively,
the MSCs might have accompanied the metastasizing BCCs: d! M- ~4 [! u% l
to sites of metastasis formation. To distinguish between these two& W& }: t* y) [6 G5 W
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled' f5 q5 t0 Z8 o* O$ `% X
MDA-MB-231 cells and implanted the mixture subcutaneously in
host mice. We found that the tumour-derived lung metastases contained
green-labelled MDA-MB-231 cells but no detectable redlabelled
MSCs (or their derivatives; Supplementary Fig. 4a) when( B2 X% Z* @6 n) c* B! `" j; k
scored 4, 5 or 6 weeks after primary tumour implantation. The5 m7 h0 ?7 P# k* g% M/ o1 U( G
absence of red-labelled MSCs from the lung metastatic sites cannot/ K* m8 V% d/ C7 A5 n  s! P" l7 [
be ascribed to an inhospitable lung parenchyma, as MSCs that lodge/ `# c7 C* F9 g0 V9 j. p6 M
in the lungs of recipient animals after tail-vein infusion survive in that
environment for ,6 weeks after injection (Supplementary Fig. 4b).
Hence, it appeared that the admixed MSCs do not migrate in large
numbers to the sites of metastasis, and that they exerted their prometastatic2 E5 S8 D' Q4 F6 v
effects on BCCs in the context of primary tumours." x9 b( S; m4 U0 ~' J/ a" b( w
CCL5 in MSC-induced metastasis: _# b9 x/ j' |1 S5 M1 [
The aforementioned observations indicate that MSCs supply locally
acting paracrine cues that induce BCCs within primary tumours to. a9 A2 R+ X7 y4 I. @6 d7 k: e
metastasize. To understand this crosstalk better, in vitro co-cultures" V( Z  W1 f: R0 |+ ^) Y3 x  J
of MDA-MB-231 breast cancer cells and MSCs were established and' D% x9 J2 Z# K% D7 O
their conditioned media were screened for the levels of various cytokines,8 ]. k5 n) ]' M/ j
chemokines and growth factors using the Luminex-based Bio-& S! W# i& _& ^8 z# \, S# R9 C
Plex suspension array system (Fig. 3a). In some cases, the resulting
a
b c d+ X/ [4 o2 n* ]
Days after injection7 ]* ~- I( [% i, h0 Y1 f
Tumour volume
(× 100 mm3)
00 ?0 n. C8 x+ r! X: d$ K
54 q3 B2 K9 G4 J; E8 w# I2 p1 |: N
10- }) G% _7 [7 t" ?7 C8 j& B
15
20' `/ }/ h6 u( q, T1 a' P5 u) f
25
14 17 21 24 28 31 34 38 42 45 48 66 71 77# N5 }( g, Y8 P& x0 D6 }4 E" V
Primary tumour explants
Lung explants
MDA-MB-231
MSC
+
Lung explants
Primary tumour! D0 [2 B4 r* G4 L- G
explants+ }  z9 q5 n7 }* k6 x
Antibiotic7 k$ Q, Z+ V; R$ J0 o* \" ]
BCC selection
T-explant7 B# g" T: N! K: x& l$ D8 e) U! m
L-explant) V) n/ c1 G0 p4 B' ^! N9 I5 x" ?4 [
T-explant" l/ ~0 F3 N% m* q% r$ g8 B
L-explant: c. I* }* i2 E* v9 U
Tumour mass (g)5 L. ?1 O0 l" w
3.09 N" t& s: [. {; X  a
2.0
1.06 Y7 I6 a! R" ]0 i5 M
0 0
0.47 K- v& \) B; P5 u" [0 h
0.8$ s' p' j) j  D* V2 A7 n3 i3 z' {
1.25 W& w* W8 Z, l# ]
1.6: f: c, e% I. _1 p( s, d& c, u
2.0
#
Metastasis index (fold)
##
Figure 2 | MSC-induced increase in the metastasis of MDA-MB-231 cells
involves reversible mechanisms. a, BCCs were recovered from lung or1 ?5 J8 Y2 [# n! g* P
primary tumour tissues, cleared of stromal contaminants by culture in
blasticidin-containing media (5 mgml21), and re-injected as primary
subcutaneous tumours in recipient animals. b, Tumour growth& w6 s7 d/ V9 V) p* F" \
(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* z" v# f4 V2 K7 p
four different paired batches of L-explant and T-explant cultures were assayed
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 using2 E4 d* W! |2 G; j% m$ k
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.% x9 J6 T# u6 ]7 X, A* K- [; Y/ F
a
CCL4
bFGF. [- [/ B$ t6 D% {. c
VEGF3 v6 V, d, G& c2 j. |$ i) M
IFN-γ
TNF-α
G-CSF
GM-CSF
CCL3  Y' T! o7 b/ |- }$ x0 n
CCL5
MMP1& \5 Q4 c( C& j* p) Q
MMP33 y; o5 l0 A" B( M
MMP9
MMP13
IL-1α
IL-1β' r9 |4 J; K7 Y( W1 V' s
IL-46 X5 Y1 q: u' B0 R
IL-5
IL-6, T) t0 S1 m1 r- M; |% M
IL-7
IL-8
IL-10
IL-121 t: v/ u9 q/ {: R" w2 V9 E
IL-13
IL-17! r1 ^$ ~7 N4 _! p7 L! }; c
IL-22 r1 E) W- d+ R, G, N
TGF-β
Fold induction. T! S) E; X2 a+ z. Q* ?, n4 w
MSC alone
MDA alone0 v' E! U$ u: H2 O! f. V6 D! [
MDA+MSC (2:1 ratio)
1 * * * * * * * * *
3
5
7& H% i* I7 [& j# c: Z
9
11, T" ]+ a  e/ h$ X8 M
13
603 O  i& ~- g" ?/ O1 x2 L
b c8 m2 Y: f" R% V' @" ~
0.4 μm1 e- X$ }9 }  V' r
2.0
1.8
1.6
1.4
1.2: Y8 [& ?! F0 X) K, g$ v% X* N$ j
1.0
0.8, \! X8 p1 x" y+ M. g
0.6
0.4
0.26 h5 N6 s. {; g
0 0- \* T% o! G# [5 K4 o
5
10) e3 n; z: b0 d
15; A) P8 F/ t2 n1 {- A6 {: {
20/ `: S' a1 B6 w
25
303 k  P! ^8 @1 F' s3 C4 h
351 S4 {) i! I1 i1 G
40  T0 a+ s3 R; V4 ~* @" Z
45
50
d1 d2 d3 d4
MDA alone8 L' z1 y4 F& j" j: k+ @
MSC alone
MDA+MSC
CCL5 levels (pg ml–1)
Co-culture- ?) [% k# G4 H6 `3 ~$ R! c
Fold CCL5 induction" k* u, A9 w7 C# W
MDA alone4 x9 |* c5 d# C2 j2 w# f# w& G
MSC alone: J9 y1 Q: y& x0 v( L' g
MDA+MSC* e$ m0 q1 J& _: t' [  M
d
CCL5 (A.U. × 100)
30; [" Y, v( j/ p3 B9 c2 E
25
20- R; ^5 b$ \8 C' \' ?2 X4 L, c# Q2 X
15+ S& R% B$ Z( m; O: y
10
5" Q- m  G* [& y8 _0 J  C5 P
01 l; o9 p1 j" n8 G+ q. F7 j) I
+# r0 z) o6 i6 ^9 N6 U
MSC.c
+# A4 W" R9 o5 e9 d' }
MSC.1( t+ }4 J9 O' b5 s6 s
+5 z& ^3 h( Y& K& l9 r
MSC.5
MDA. g& h  E& q* Y! I2 B
MSC% C9 E- H- ~* l; H( d
MDA.1
MDA.c
MDA.5
TC-MSC" b- x8 q( Q  K& i
MSC (from MDA tumour)8 @6 i4 _' y; A) g2 _& s" J# s
BCC (from MDA tumour)
Control (MDA/CCL5)
CCL5
GAPDH2 l8 P9 m8 L% S+ O. m, W
e
Figure 3 | The interaction of BCCs with MSCs causes a rise in the levels of/ M  {3 X! m: w% |
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+ G" Z$ s* |3 h: Y* `; S
supernatants were measured by xMAP Bio-Plex cytokine arrays at day 3, and
were normalized to the levels observed in the media of BCCs cultured alone.3 l3 M/ i6 L1 z3 y' G
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.* B. t2 n1 A; G# S- S
Data points representmeans6s.d. of quadruplicates. c,BCCs were separated9 W0 E& p8 y" R! ]
from co-cultured MSCs by a 0.4-mmmembrane. CCL5 levels were probed by2 H7 F* t; @6 S9 k: F
ELISA on the culture supernatants. Data are expressed as fold induction over
levels seen inMDA-MB-231 culture supernatants (mean6s.d. of triplicates).
d, CCL5 ELISA on the supernatants of MSC-siluc (MSC.c), MSC-siCCL5.1, U* |5 ?2 L3 h+ U% W* I
(MSC.1) and MSC-siCCL5.5 (MSC.5) co-cultured with MDA-MB-231-siluc8 G' P  m4 d$ A! ~" m# B+ ?) |* z
(MDA.c), MDA-MB-231-siCCL5.1 (MDA.1), or MDA-MB-231-siCCL5.5, c' @. l7 h$ }
(MDA.5). Data are expressed as means6s.d. of triplicates in arbitrary units4 m4 ]" Z/ a# X7 E2 J5 R
(A.U.). e, RT–PCR analyses of CCL5 in MSCs and BCCs sorted from9 W/ ^% Y* f! B, u  ^
GFP–MSC1MDA-MB-231 tumours (3:1 ratio) 4 weeks after tumour
implantation. Tissue-cultured MSCs (TC-MSC) and MDA-MB-231/CCL5
cells were used as controls. GAPDH was used for equal loading.levels of certain released factors (for example, interferon-c or; [* X+ {4 I" V; d' Q* m
tumour-necrosis factor-a) reflected the additive contributions of$ j( ?3 u1 W; Z: @% }- V
the two cell types when cultured on their own. Notably, the levels
of only one cytokine, CCL5, reflected a synergistic interaction
between the MSCs and BCCs, as it accumulated to levels ,60-fold
higher than those produced by pure BCC cultures (Fig. 3a). This; e% ~/ ?( i2 L& {+ ^# v" i. F
cooperative induction of CCL5 was proportional to the numbers of0 c+ Q4 v5 a4 h( |4 x& Y
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was apparent8 g7 p9 f% L6 I- x$ O2 a1 ?
as early as the third day of co-culture (Fig. 3b). Moreover, this
induction required close physical contact between MSCs and cancer& D) k- `1 ~6 d, u1 S4 q
cells, because it failed to occur when the two cell populations were: k) j0 A3 f% {
separated by a permeable membrane (Fig. 3c).
We undertook to determine the source of the CCL5 produced
under conditions of co-culture. To do so, we stably reduced the% H7 x* n. P' \) v
expression of CCL5 in MDA-MB-231 cells by.80% using short# t8 T8 l+ J0 X. g; \4 q1 X
hairpin (sh)RNA (variant siCCL5.1; Supplementary Fig. 6). Importantly,
however, subsequent co-culture of these MDA-MB-231.1 cells9 K) h9 A. _2 R/ T' t% W
with MSCs continued to allow accumulation of CCL5 in the culture; F4 S* f) \: _( w
supernatants to levels that were comparable to those observed in the
co-cultures of MSCs and control cancer cells (Fig. 3d). This suggested, f& l, \4 Q6 Q' \* v, j" X
that the source of CCL5 was the admixed neighbouring MSCs.
Indeed, inhibition of CCL5 protein expression in MSCs using the" s( v3 `# W7 y% l" [
same shRNA hairpin vector (MSC.1; Fig. 3d) resulted in more than& I" A5 v& p6 I
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 of& a0 m* e  H: H1 {. ~* [: C
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) [# k0 W) T* `4 o' G5 ^
Fig. 5b). Finally, polymerase chain reaction with reverse% W. O# b- s* B# l! `9 n
transcription (RT–PCR) analysis of the RNA prepared from these coculture-/ G$ |/ l4 x8 \  }0 S: e  |7 V" F
derived MSCs (Supplementary Fig. 5c), as well as from the
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after' @' Z" M5 n' U3 b- r2 z
tumour implantation (Fig. 3e), indicated a strong accumulation of
CCL5 messenger RNA, suggesting that an active signal transduction) x; T4 e" \- F) D; u5 B
pathway is triggered in MSCs by the nearby BCCs.* q6 ?' Q2 V# b) `
A series of observations has linked CCL5 signalling and cancer. For$ y. d* o$ b2 U" \% |9 T5 X, o
example, CCL5 levels in the plasma of breast cancer patients have  U9 P/ H1 \" O6 @
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.
However, the precise contributions of CCL5 to cancer development! A' U; m1 H% p9 |
and progression are poorly understood. To investigate further the. I; A' B! o6 L3 |# O
possible causal role of CCL5 in cancer cell metastasis, we overexpressed/ C: d( b5 l( A, F3 F
this chemokine in the MDA-MB-231 BCCs (Supplementary
Fig. 7a) and analysed its effects on cancer cell growth and$ q' w# a* w9 y5 Y  H9 \0 X# f( a
tumorigenesis. The overexpressed CCL5 did not confer any proliferative
advantage on cultured cancer cells when compared with
those lacking such overexpression (Supplementary Fig. 7b), and had
no effect on the ability of BCCs either to grow in an anchorageindependent; E, y/ i' H3 s7 C6 S" ^2 e
fashion in vitro (Supplementary Fig. 7c), or to form
primary subcutaneous tumours in immunocompromised mice (Fig.' J  o" E6 w' Q9 G. F5 J, ]. {
4a). However, these tumours exhibited a ,5-fold enhancement in( x" ]! X# P, }1 n7 j, p. {
their metastatic potential when compared with control tumours
lacking ectopic CCL5 (Fig. 4a). Similarly, overexpression of CCL51 R0 }% ~; l* L, {/ y
in WI-38 fibroblasts sufficed to enable these cells to promote the2 E* v+ S. c5 D& |2 i- T( U9 ~- m* }
metastasis of admixed MDA-MB-231 BCCs (Fig. 4b), indicating that
the actions ofCCL5 are responsible formuch, if not all, of the observed
MSC-induced metastasis by the BCCs.
CCL5 promotes lung colonization; P% _: x- \- p8 h3 D& S& s* e
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,- B+ C0 E8 F9 q/ l9 y
CCL5 expression has been associated with increased tumour neovascularization,  ~8 Y( A, T/ r5 D/ F
suggesting that endothelial cells, which express a variety
of chemokine receptors, may also be attracted by CCL5 to sites of& C9 ^1 q6 U- e0 _
tumour formation, thereby enhancing tumour angiogenesis20. Such  y1 i+ O+ N. f7 D
observations suggest that CCL5 may contribute to breast cancer% t. o. [, ]) q
metastasis through the recruitment of a number of stromal cell types
to sites of primary tumour growth.
However, immunohistochemical analyses indicated that the/ {' N8 E7 ?6 M/ ?$ k0 o
MDA-MB-231 control and CCL5-overexpressing MDA-MB-231$ m" A* y( W7 Q0 X( w1 L; j/ q
(MDA-MB-231/CCL5) tumours exhibited comparable numbers of5 u+ ]8 Y9 R: M/ f
tumour-infiltrating macrophages and had similar vessel densities (as
evident by F4/80 and MECA-32 staining for macrophages and$ P( ~" i3 d) ?( Z0 }9 c: k
endothelial cells, respectively; Supplementary Fig. 8). In addition,) d- E; w- W) W5 R; v
we found that ectopicCCL5 expression did not cause an accumulation8 f% s. H" I5 l8 L, o! a0 H3 x# Z6 p
of other stromal cells, such as SMA-positive cells, in the examined
tumours (Supplementary Fig. 8a). Together, these data indicated that  n: h0 g- r9 S: i, G  N
the observed CCL5-induced metastasis could not be attributed to
significant effects on the numbers of the major constituents of the
stroma or to the vascularity of these tumour xenografts.
Invasion and metastatic dissemination of carcinoma cells are often
facilitated by their transdifferentiation through the process termed- m/ h; ]$ N; J8 s
d f: t2 |+ X4 \* L
Bcl-XL( v! }! n; S+ Z. l& k! ]
Bcl-2. k: P8 D% s# X) A) y5 x+ G
β-Actin& c- S( u& O$ E# p
S473-Akt% A$ k- y  I% L
Motility
0.5%→10%4 H* E& @, L6 l
LY – – + +
g
Extravasated clusters (mean); {+ X8 u0 Y9 r4 p  A
Migration (fold)
*
e( z9 T$ I2 U2 K6 g1 ]- d% V
0
MDA/vector; J. ]/ E  _4 a* E( J: e
MDA/CCL5' y* o- c3 y3 G$ U7 z4 z
***" o: l& b  j* ]1 X
Invasion
10%→10%
Invasion
0.5%→10%
Migration (fold)& l% y; t* t  d  w3 Y+ @, E+ b
**/ g' b, p& a! ]" h8 p
00 i3 l! v+ e6 l/ e8 K4 ]$ e
0.5
1.03 j( ^- m" I7 l5 e
1.5
2.0# o/ m$ y- e6 E# l
2.5
3.0
3.50 Y, `- i+ Q* m$ D2 Z' w: M
*; C( P! T" \7 g! B) {4 H
MDA/vector# P4 o$ v4 Q$ c, Z$ J
MDA/CCL5
a b
*4 T( Q, v# o( {4 I/ |; U; P/ Y
Nodules per lung (mean)( `: ?/ A" v4 y% {% c8 L5 [
c5 \% f/ i; f0 [. w% t4 _& z4 H8 `( u
30) c- I; \. H0 n) J! {
25
20) a! _4 ]" L$ E- I2 U  J
15& X$ b" |1 @% a5 c5 x
10) g& p6 d3 q7 p+ v: Q
5
0
Tumour mass (mg)2 M: F' b0 w! h8 b2 @
302 ?" Q7 D4 g: A
25
20
15
10
5
0 06 S7 i, j- i, ]
50& b6 `+ R4 l. C) s- i' A& l
100. a4 a6 `* ~" m, }+ U; a5 q9 m; w* G
150
200
250; _% t4 L& G  M( m5 }
0- D! \7 c! Q" H- \( [
50
100' ^2 U5 l" ], Z( I6 ]
150" F: _# Q7 o* o$ b; ]$ }5 K
200# t  |6 C9 [' n0 G* M
250
300
3501 O( D0 V, w. u% o
Tumour mass (mg)4 z- M0 e/ @& f8 I; w& a
Ctrl. x0 V# x+ G9 C. L, ^  @
CCL5
Ctrl
CCL5
Ctrl* {% ^, r+ R4 F, b: |
CCL5
Metastasis index (fold)' X( a1 f3 {. {% ]) o! k8 q
Metastasis index (fold)7 g! S6 `  r& f8 x7 g( F4 U1 c+ O% C- I8 y
0
1
2" Y) p; Y) _& S3 J: ]& e# |- o
3
4
5
* 6
0/ L( G3 F  d8 J5 d- c+ y
14 Y, l# ]  i( [* Y* H+ j  W
2
3
41 Z4 L! L2 p0 j. ~
5
6) _: z- ?4 l1 [4 e( u
7
* 86 ]4 {4 E. h, f' i# I4 m1 k* y; d
MDA/vector
MDA/CCL55 h9 Z3 G  Z2 ]5 @7 n5 [+ ]
MDA+WI-38/vector* b- G( M: l4 O" K
MDA+WI-38/CCL5
1
2
3% [1 Y- ^1 X0 j% c9 W
4+ \" N$ x! w0 e( L9 [: ?/ S
*
Figure 4 | CCL5 enhances breast cancer cell migration, invasion and- K( I# o$ K5 X) o, x6 L/ t
metastasis. a, A total of 500,000 MDA-MB-231/vector (ctrl) or MDA-MB-2 B: D5 T4 E" a0 x; o. j
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour+ O, ]" t* c6 N( x9 U
masses (mean6s.e.m., n56 each group) were taken at 10 weeks. Lung) `9 X( g6 K" o$ l, {
metastasis indices are expressed as fold increase (6s.e.m.) over controls. Data
shown are representative of multiple repeats. Asterisk, P,0.01 in one-tailed
Student’s t-test. b, A total of 500,000 MDA-MB-231 cells were admixed to) h( p# G3 C) b; \  M+ \9 i9 U
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 are1 ]) t) x& e/ {" N
expressed as fold increase (6s.e.m.) over controls. Asterisk, P,0.01 in onetailed1 e2 Y& @& S" A0 Q/ V( ^
Student’s t-test. c, A total of 800,000 indicated BCCs were introduced
into the circulation of NOD/SCID hosts. GFP-positive cancer colonies in the+ K/ T; ]9 q/ K6 _
lungs were counted 6.5weeks later. Bars representmeans6s.e.m. (MDA-MB-% H7 |8 P! \9 m
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
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
multiple independent experiments and are expressed asmeans6s.d. Asterisk,7 i' M% N! d6 R' r3 G
P,0.05; double asterisk, P,0.05; triple asterisk, P,0.01 in one-tailed8 K4 d* N+ U4 f& x: H" Y
Student’s t-test. f, One million GFP-labelled BCCs were injected into the tail2 |# q' m' O: U+ j1 Y
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( L) h! L* Z' Z! e6 I
mice; MDA-MB-231/CCL5, n510 mice). Asterisk, P,0.01 in one-tailed0 p; n' k* d- G! b! T
Student’s t-test. g, Transwellmigrationassays on50,000MDA-MB-231 control
orMDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;* o; o% I; O/ O: U! ]7 D/ |6 Z
asterisk, P,0.01 in one-tailed Student’s t-test.the epithelial-to-mesenchymal transition (EMT), in which cells shed' S. b4 Z. i/ S* J0 D, U
their epithelial characteristics and acquire instead a series of mesenchymal
markers that enable their invasiveness and intravasation21.
Despite their lack of E-cadherin and their expression of detectable levels
of mesenchymal markers such as fibronectin (data not shown), the$ m; i! S4 |5 H# i
MDA-MB-231 cells studied here exist in an intermediary phenotypic
state of ‘partial EMT’, as they retain a distinctive epithelialmorphology
in vitro and are still responsive to EMT-inducing stimuli in culture. In+ V& [5 w! ~' B2 l$ }* z
fact, we observed that ectopic CCL5 expression did not cause MDAMB-3 [$ R& N0 i# D, ^7 }' O
231 cells to undergo themorphological changes usually associated
with an EMT(Supplementary Fig. 9a), did not cause rearrangement of
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on  {9 C: m2 E  ]& ]
the expression of mesenchymal markers closely associated with the
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c)4 l$ l" I; Z8 W4 U1 P& \( G/ E
and fibronectin (data not shown). These data suggested thatCCL5 does
not directly promote the EMT programme of MDA-MB-231 cells.
We proceeded to explore an alternative possibility: that CCL53 D" Z* a$ ?+ g* }  m+ \# |* Q( C
expression affected some of the later, critical steps of the invasion–
metastasis cascade, namely the lodging of cancer cells in secondary
organs and the subsequent step of colonization. For that purpose,
MDA-MB-231/CCL5 cells were injected intravenously into host$ W/ ?4 L7 X2 W; q- z
mice, and the lungs of these hosts were examined ,6 weeks later: g7 P) q' \" ]# k6 X4 ?
using fluorescence microscopy. These experiments revealed that
CCL5-overexpressing cells indeed had a significant ,1.8-fold8 q  S# j5 {" _/ T$ v4 _- Y
advantage over their control counterparts in colonizing the lungs
(Fig. 4c), suggesting that CCL5 exposure has effects on later steps
of the invasion–metastasis cascade. We note once again that this# h( P+ O. [+ _! Y6 J/ e7 x. q( t: g
enhanced tissue-colonizing ability was not due to CCL5’s effects on, |1 v# M- k' E- e7 Y- }5 ~
cellular proliferation measured either in vitro (Supplementary Fig.- I2 U  k- \( |" c0 _
7b) or in vivo (Supplementary Fig. 7g, Ki67 staining).
Because improved colonization can be due to enhanced cellular
survival, we tested whether CCL5 protects against apoptosis.0 `  X* W; P* T- ]) I$ t
Notably, we found that MDA-MB-231/CCL5 cells exhibited higher' I. f. q: P8 C! x$ x% l% @; E# z
levels of the Ser 473-phosphorylated, activated form of Akt, but( W4 k7 x0 N1 Q1 T+ n3 g* @
exhibited no difference in the levels of other pro-survival proteins,3 G* `4 [# m8 m* f. v
such as Bcl-XL or Bcl-2 (Fig. 4d), or a reduction in the levels of
pro-apoptotic molecules such as BAX or BAD (data not shown).
Moreover, we found that overexpression of CCL5 had no effect on
the ability of MDA-MB-231 cells to withstand serum deprivation( g, b1 k, Y- C" {, z7 y1 y
(Supplementary Fig. 7b), loss of substrate anchorage (Supplementary
Fig. 7d), or hyperoxia (data not shown). We also observed that
ectopic CCL5 expression did not protect MDA-MB-231 cells from
doxorubicin-induced apoptosis monitored using western blots for* {; X1 m9 M/ I5 C, j% z4 p
cleaved caspase-3 (CC3) and cleaved PARP (as markers of apoptosis;
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling! ]0 p- z- Z' |( ^; D/ Y
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohistochemical" Q9 `+ D4 l+ S% G6 u9 h" C; E( H
analyses on control and CCL5-overexpressing tumours
revealed only minor differences in the levels of apoptotic CC3-
positive cancer cells among the examined groups (Supplementary2 B' R6 j1 y. L. P
Fig. 7g, h). Together, these observations suggested that CCL5 does# E; U* c2 y- r. T
not exert any detectable pro-survival functions in vitro or in vivo, and' Y. A# E  t1 t5 w# [9 i) C. z
that the observed enhancement of lung colonization was not a consequence
of significant anti-apoptotic activities of CCL5.5 X5 A* K7 `; z0 z  S
Akt serves as a key relay switch for upstream signals that promote
both cell survival as well as cellular motility22. Because CCL5-induced# g4 b( P- @+ O7 [2 U$ x# H
Akt phosphorylation did not correlate with enhanced protection) Z* A) @! z. k# w
against apoptosis, we tested whether the CCL5-enhanced lung colonization* q+ |7 E- Z6 H
could be due to an increased ability of MDA-MB-231/
CCL5 cells to invade from the microvasculature into the lung' }- K5 n8 V9 _' D: @
parenchyma through the process of extravasation. Indeed, ectopic* J8 u8 [1 w- G( q7 P! }) I" [
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-' c  \3 ^2 G' |, Q/ J$ e# a
fold in either high or low serum conditions, respectively (Fig. 4e).) y) F4 s. ^' w0 `. j
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein* v; b# s) p" t0 h; x9 j
injection—in order to remove most cells that remained within the0 n" M3 {' m+ I! ]1 R" R
microvasculature of the lungs and thus had not extravasated—we. T. f9 q/ t7 ?6 G! C# D
found twice as many deposits in the MDA-MB-231/CCL5-injected
group than their control-injected littermates (Fig. 4f). This indicates a
clear effect of CCL5 on cancer cell extravasation.8 ]) g, ^. {$ L+ E* @
Finally, we investigated the role of Akt in mediating the actions of+ B: c; M3 W# H+ X
CCL5 on cellular motility by using the phosphatidylinositol-3-OH- A: @9 q. M6 r% Z- a
b silacZ9 ?& o' n# d* S* n+ A+ F# s0 \" q/ b
silacZ
si809
si809
si1867 j+ R, Q# c2 J7 Y
si186
CCR5( p# S, _% _! H! C$ _: F. w# K
β-Actin1 s8 I4 C9 d7 m5 ?' W( ?
a d) V; A+ A% j$ A' \, k
M  T2 n$ H. B$ ?  @' J
+" j) d) K" W7 t
MDA+MSC' d( W7 L7 r0 n" Y8 d( h/ W
+ IgG% }) O6 P1 E: r+ C' j. a+ ^8 u
+Anti-CCL5 Ab$ L% k9 m, V6 o
MDA" K  r5 h% M) l/ v$ \9 S& U* I# Q1 I6 f
+Anti-CCL5 Ab- [& A2 \+ V8 d4 G  Y/ {: b* ~: \
MDA+MSC
MDA  d" Q$ T$ v" A% A  t& G( _
IgG
<
<
<
<
<! z- A% z! w2 @, t8 k
DAPI
CCR5' D% V; Q5 f* X; X. h5 ]
MSCs MDA MDA+MSC: ~1 K+ o, c+ g% l) V8 o9 J
DAPI DAPI  Z: ~- p+ D5 ~  j8 D/ O; G. ~  \
CCR5 CCR5, ]" U4 P+ X7 J  w( t$ q
MSCs MDA MDA+MSC
*! a% y, x3 n4 `$ Q! ]- i# ^
c
Metastasis index (fold)
Metastasis index (fold)
*
Control siCCR5, ?1 V' c% E, S. Q9 p1 g
MSC – + + +
0
1
2$ D" U2 Q( t. j
3+ g7 _9 r9 b; l, _. R- T
42 Q) J2 O& _' {4 C* K8 l/ u$ D
5 e
+ + – –( K. M2 N- ?/ P
Anti-CCL5. ?3 F! }, C  @! Y3 y, d
0! v7 ], V2 r2 U! P5 b$ F- j4 @
1
2' U0 Y! O; C* a' e) v+ ?5 Y& `
3
44 M5 O! D0 U. i, D
5
65 R! n, h3 j. e2 I
– – + +
IgG
MDA
MDA+MSC
<, C- j" `0 f' H6 G: J4 Q, T
Figure 5 | CCL5–CCR5 interaction is essential2 U" A! I" S: T" e- s
for the MSC-induced metastasis.$ T9 D  @, w$ L8 A( Z. [
a, Immunofluorescence analysis of CCR5
distribution in MDA-MB-231 cells cultured with1 J7 \7 W3 V  a$ R
MSCs. DAPI (for nuclei staining) is in blue;
CCR5 detected in green. Arrowheads denote
MSCs. b, Western blot analysis showing CCR5% F9 V9 J1 t% }8 ~1 c' S% w4 {
expression in MDA-MB-231/silacZ, MDA-MB-+ W: k0 ~8 V% o: {" j3 r
231/siCCR5(809) and MDA-MB-231/
siCCR5(186) lysates. b-Actin was used as a
loading control. c, A total of 500,000 cells of the
MDA-MB-231 variants in b were co-mixed with: g  O: Y3 b4 ?
1.53106 MSCs and injected subcutaneously into
nude mice. Mice were killed when tumours- a! Q0 i) X( ~" Y% \
reached 1 cm in diameter and the metastasis/ K* g6 z/ m6 G' D6 T
index was calculated for each cohort (n55 per( t! e" J0 P9 Q# A
group). Results represent means6s.e.m.;
asterisk, P,0.05 using one-tailed Student’s
t-test. d, Anti-CCL5 neutralizing antibody or
control IgG was administered intraperitoneally+ c6 R% K% s4 i+ y3 [9 r: ^
twice weekly in SCID mice bearing MDA-MB-231
(n59) or MDA-MB-2311MSC tumours
(n511). Representative lung pictures of the
indicated cohorts are shown. e, Lung metastasis  o$ x% F4 o1 b% v9 t; q% V* G
indices of mice in d. Data shown are
representative of means6s.e.m. Asterisk,2 i! t: V( m; ^2 k* Y, a- a
P,0.05 in one-tailed Student’s t-test.: u6 v6 V+ D. R7 D
kinase inhibitor LY294002. Drug concentrations that did not inhibit, Y: @! T9 F6 u6 t
the basal motility levels of MDA-MB-231 cells blocked the elevation4 i8 h% Q0 ?: W
of motility induced by ectopic CCL5 expression (Fig. 4g). These' p  S% C2 g+ H9 ~3 g0 u. O
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( [# n- o) z0 @4 \0 _
at sites of dissemination rather than promoting the survival and/or# Q4 W# [. x6 |& f1 s
proliferation of these cells.2 v6 l. F% ]$ E' `, m
Essential role for the CCL5–CCR5 loop
CCL5 acts through three G-protein-coupled receptors, termed: @* v3 i0 W& y. u; C6 l8 x
CCR1, CCR3 and CCR5 (ref. 23). CCR5 has been determined to be& |5 v6 }; J" f
the main receptor for CCL5 in MDA-MB-231 cells, as inhibition of its
surface expression through dominant-negative mutants abrogated
the ability of these cells to respond to CCL5 chemotaxis24. We therefore
focused our efforts on evaluating the importance of the CCL5–
CCR5 interactions in MSC-induced metastasis.
We confirmed that CCR5 is expressed by MDA-MB-231 cells and
not by MSCs (Fig. 5a), supporting the notion that MSC-derived
CCL5 acts primarily in a paracrine fashion on MDA-MB-231 cells1 q: F, t7 w( W% A* u# |
in the BCC and MSC mixed cell populations described above. To- E* c. c$ O: k" K
probe whether the observed MSC-induced metastasis required
CCL5–CCR5 interactions, we inhibited CCR5 expression in MDAMB-
231 cells by more than 85% through shRNA knockdown (ref. 25
and Fig. 5b), and mixed these cells with MSCs before implantation
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,) u+ B/ m  N- M: h1 c3 `
achieved using either of two different shRNA constructs, abrogated
the ability of MSCs to enhance the metastasis of MDA-MB-231 cells  t6 v* T2 G: L. o8 M- a
(Fig. 5c). Furthermore, neutralization of CCL5 protein using intraperitoneal6 C% Y( y# a+ p7 v( o/ D4 H
injections of an anti-human CCL5 monoclonal antibody
also abrogated the MSC-induced metastasis by MDA-MB-231 cells1 v8 F/ n: U* d5 z# k
(Fig. 5d, e). In addition, MSCs in which CCL5 expression was inhibited
by shRNA knockdown failed to promote metastasis of the% f5 R/ Y) b  [: W
admixed MDA-MB-231 cells (data not shown). Taken together, these  m' h8 J& t# {, x
results underscore the critical importance of the CCL5–CCR5 paracrine
interactions in enabling MSCs to induce metastasis of the: p7 V  _  M) `. e1 V2 S( A9 F- n
MDA-MB-231 cells.9 e- [3 X$ q# Q; {% `4 M
Discussion/ E) r. w6 R* X1 b  f
Certain models of metastatic progression propose that cancer cell
invasion and metastasis from the primary tumour site are strongly  U) p* i+ J' {- P' K7 d, Q' v
influenced by contextual signals emanating from the stroma of the. K9 w' n9 u) c" b% x/ S3 j* ^( o
primary tumour. It follows that if carcinoma cells are subsequently( P  _1 j/ S* a0 A) L& _
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 others9 T' h9 R  W+ |- L" q  C+ e" \
on the basis of indirect evidence21. Here, we demonstrate that at least& G0 P- [  b. T0 O5 @' e
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- d6 N2 b' x) `3 O6 U& ~6 v- P
MSCs (or their derivatives) and the BCCs endow the latter with
invasive and metastatic properties.7 q/ g5 L& a0 z4 \) L" V6 k" f
Although the recruitment of labelled MSCs to tumour xenografts- [* T  \5 l3 G5 C
has been established in a variety of experimental models of tumorigenesis,
there is currently no available way to quantify with any accuracy# o: x. x- q5 C. z/ F* }+ b2 f
the number ofMSCs in actual human tumours, in part because no set
of markers has been identified that can uniquely stain these cells without$ _( q1 X/ Y& V8 \' J4 ^4 v
concomitantly staining other mesenchymal types in the tumourassociated9 H5 W* G; Z9 Y* p
stroma6. Our demonstration that the stroma derived from# U; A# ]# v4 h4 W4 E: L* }3 |/ I
tumour xenografts contained appreciable numbers of murine MSCs* |) G5 y- ]5 \# F3 B' N1 J
indicates that significant steady-state levels of these cells aremaintained$ z2 T( _$ q, K$ I: w( }) H) q
in developing tumours. Interestingly, the use of CD10—one of the9 l0 H3 }5 B: |: ?- t6 `3 ^7 K
markers associated withhumanMSCs—to purify cells fromthe stroma
of human primary invasive breast carcinomas yielded a population of* s: a$ e$ O- t  f$ E
cells that expresses a number of other markers collectively used to$ s% }5 I) ~+ h! M
characterize human MSCs (for example, CD44, CD105 and CD106;
Fig. 6a). This suggested that, similar to tumour xenografts, human' `+ z. i- {" \4 ]: C# @. f/ g
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;
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free
stroma of primary invasive ductal carcinomas (Fig. 6d), specifically in
the CD10-positive compartment27 (Fig. 6e). Collectively, these observations
argue strongly for a significant association between stromal0 F' O, l& I8 @( f( C' V' h
CCL5 levels, MSCs and human invasive breast cancers.  _3 V5 h9 n+ b+ k5 T
c3 }: l5 R3 H* n1 s1 l
STT1969B0 v. k8 y+ h# A8 U: _/ n; y: u& S
STT3126
STT3124$ n, H" `6 m+ _4 o$ N: w
STT656B
STT1968B0 w7 U) q# }4 C. N7 z* y+ N
STT3122
STT3053! ?5 b" c( `0 I9 }
STT1986B
STT854
STT3125
STT1975) Z' B) D" V9 c& @3 J
STT1987B! b- ]5 m) l, \4 G+ A+ V& K/ R
STT1079
STT638
STT1774
STT19841 }/ Q# U" V1 E+ n! c, K
STT1737C
STT30688 i1 U. E1 r* c" b- t
STT3120
STT850
STT417B
STT3119
STT1776
STT1777B- S' q( i! X+ c+ O
STT689B
STT19710 ?. `/ r& k- A+ `
STT597
STT626) Z0 H8 y0 m4 w% l
STT154
STT27740 @' x: a' m9 x# D8 e$ j
STT1966
STT2776
STT2775
STT2772
STT1637: b, @0 r1 O9 S. {0 R$ e3 z
STT1220C6 a; H$ z, Q1 z2 ?8 U$ G& ~' Z
STT-094B-10 a; H( a7 N( ~" C# T
STT675
STT2770  A4 U& Q- F5 P5 @& V
STT695B4 ^) K  b% _" |' x& n. s
STT1771
STT1778* B, F7 F) x  D3 M
STT491
STT1823; @) f9 z  T$ w
STT200C) e3 f- G, U1 l, ^# v  L: ?
STT741B
STT335C5 C5 T6 y7 j7 _0 N2 g7 y, J# l$ L
STT709B
STT516D; v. `0 Y6 w5 r. p0 V8 x2 J
STT607B+ g6 r! ^9 v0 J6 T  T
STT680B
STT1148B
STT523B$ o6 Z# H+ G4 {
STT526E* ~* m  S/ d' G( R2 i6 _
STT742F
e
CD13' |, `) B& a6 v$ ^0 @
CD29
CD44
CD49e6 @. u7 U6 @+ d9 Q& l) C, z
CD54- m  s! A! m; c" @/ Y' }, `3 U
CD59$ W9 \* {8 X5 h; Y2 Q8 w4 `8 W, q
CD63" c. q6 Z7 A2 [( t/ q
CD105* g" X7 z1 h: P& U% X3 v3 t
CD106$ M7 h' ?/ p. w$ d% ?/ z
Nestin  M  M" }+ a9 ~5 f
HAS2% J  P( A" L% R2 S  V! {
IGF2  P" V! [, W" W& l5 P* X0 Z
PLAU9 B5 h' _% C& s; ?1 V' ]
TIMP1
CAV1- Z5 T4 i1 G: c. b$ A
IDC-7
T1126037 P4 u5 Y3 G  _/ i$ \
T3923037 O0 v3 I7 {, M* p& _" o
Normal Invasive
CCL5! x5 e5 [5 U( j  F: g0 W
log2 ratios
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.02 y- q3 E' v6 y4 Q
log2 ratios
d
–2
–1) U" j  I/ @$ H4 ]2 J6 t% i
0 1 2
–2.0
–1.5# ?: }. b6 Y5 c) u3 D! _  F
–1.0$ Y1 ?4 l9 x2 W2 C5 H' h, {+ f5 h- p" E
–0.5
0.0( s0 k7 g) D& W8 Y1 F
DTF SFT0 C; v0 m8 d% D) a
DTF# r. g6 _5 Z! Y/ X
SFT
a b) V& B4 ^0 t. _7 O% [
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: \& Y7 `7 A5 {% _3 U6 q' U
tumours27. b, Soft-tissue tumourswere ranked byCCL5 expression26, fromlow, Y1 ?+ y& x3 X/ _8 T" i( }. E
(green) to high (red). Wide blocks indicate expression ratios of tumours
classified as desmoid-type fibromatosis (DTF; yellow outline, n510) or8 Y+ x5 B' \8 ]) n/ U
solitary fibrous tumours (SFT; blue outline, n513); narrow blocks are other7 Y+ {# k' B) w
soft-tissue tumours (n532). c, Box plot showing that CCL5 expression is
higher (P50.004) in SFT than inDTF. The difference in log2 expression ratios2 W' }  B1 W& j* C
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 of7 U: E9 {( _( h# x6 g
CCL5.genelist obtained as in a.Details of thepurificationmethodologies of the: N, l( u) A9 v# S
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-
231 cell interactions, CCL5 seems to have an equally critical involvement
in the functional interaction of MSCs with MDA-MB-435
human BCCs. CCL5 levels accumulate synergistically when the two: H5 q4 `3 U0 Q  f/ d: [8 }: O' I
cell types are co-cultured together (Supplementary Fig. 10a), and
MSCs in which CCL5 expression was compromised by shRNA knockdown
failed to promote metastasis by MDA-MB-435 cells to which* u4 y6 a' V" s9 p
theywere admixed (Supplementary Fig. 10b).With these facts in mind,& H& [, D  y+ k; ^5 c
we point out that CCL5 does not seemto be involved in regulating the
MSC-induced metastasis of MCF7/Ras or HMLER cells, which may& k4 {. F3 n+ q6 j  Q9 I
depend on other paracrine factors such as VEGF and interleukin-8.
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 cells
to the lung that are, on isolation and re-injection into recipient mice,
no more metastatic than their predecessors in the primary tumour
(Fig. 2e). This indicated that acquisition of increased metastatic
powers by these tumour cells was reversible, and suggested that the
maintenance of this phenotype depends on continuing contact with* b: U7 ?/ r6 v0 ~3 {! D9 b
stromal cells. If extended to other tumour types, the present results
hold important implications for the molecular analysis of malignant; t2 b& X, N, i' I+ K2 y/ U
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 to0 |: N( h: u( h6 j0 c* f/ \
contextual signals that tumour cells receive from their stromal microenvironment.
If so, analysis of the gene expression patterns of bulk( ]6 Y  @& M8 w+ Q
primary tumour populations may fail to detect the expression of key
genes mediating invasiveness and metastasis, if only because they are
being transiently expressed in minor subpopulations of cells within1 H; t( |& l9 H
such tumours. Additionally, attempts at determining the metastatic
propensities of tumours may need to be focused on the genes and2 G% u- _- x. p/ S5 _3 Z, @
proteins that confer responsiveness of primary tumour cells to stromal
signals, rather than on the genes and proteins that directly mediate
the cellular phenotypes of invasion and metastasis.
METHODS SUMMARY
Cells labelled with GFP or ds-red, or harbouring various overexpression or' W3 W7 H" w9 j( ]5 A4 t$ B
shRNA constructs, were generated by viral transduction followed by FACS2 F, O& L' w( y/ g# N
enrichment or antibiotic selection. Xenograft experiments were conducted in: l% V# Q  w3 e1 F' y
nude or NOD/SCID mice and metastasis was estimated using fluorescence
microscopy. The levels of cytokines, growth factors and chemokines were
assessed by immunoassays. Migration and invasion assays were conducted using
transwell chambers. Antibody treatment of tumour-bearing mice was conducted
by intraperitoneal injections. See Methods for detailed information regarding
cell culture, viral infections, in vivo colonization and extravasation assays, RT–
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluorescence0 e+ y! T+ o" f0 r6 \7 x! W
determinations, western blotting, and antibodies used.
Full Methods and any associated references are available in the online version of& m/ e% m! d+ W8 r7 f
the paper at www.nature.com/nature.

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