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



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 29 下一页

返回列表

查看: 616081\|回复: 280	go Mesenchymal stem cells within tumour stroma promote breast cancer metastasis [复制链接]

gkshl

新手上路

Rank: 1

积分: 0
威望: 0
包包: 8

楼主

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 tumour
pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived humanmesenchymal stem cells,
when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their' t, g+ k* a; n1 b, M
metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour$ N/ q: k5 @4 a+ K" l  h
xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from1 q* G/ @+ L; @" ^5 b5 c1 [
mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and
metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine
receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by
eliciting reversible changes in the phenotype of cancer cells.
The origins of the invasive and metastatic phenotypes of carcinoma& `- b0 U) p% B0 F+ @$ p6 \
cells have been the subjects of intense investigation. Whereas some# ]4 n7 j9 o: X6 ^9 J- {
current models depict these phenotypes as cell-autonomous alterations
specified by the genomes of cancer cells, alternative views propose0 s/ S. Y; a+ F: k. O7 O  }* B
that metastatic traits are acquired through exposure of epithelial
cancer cells to paracrine signals that they receive from mesenchymal
cell types within the tumour-associated stroma. Although several( w2 f9 ~& S: s
lines of evidence demonstrate the contributions of stromal cells to
primary tumour growth1, direct experimental demonstration of the1 r( \, m$ y* M
influence of these various cells on the metastatic abilities of cancer' O& e. Y; \$ G, h$ ?
cells has been difficult to obtain. This is due, in part, to the complexity
of the mesenchymal cell types that are recruited into the stroma, and
to the elusive nature of the putative paracrine signals that are
exchanged between the mesenchymal and epithelial compartments
of a tumour. Recent reports proposed that the bone-marrow-derived
mesenchymal stem cell (MSC) is a cell type that is recruited in large
numbers to the stroma of developing tumours2. To characterize better3 B4 g1 V0 O, i6 ?) [8 @4 e
the role of this stromal cell in tumorigenesis, we set out to determine
whether MSCs could supply contextual signals that serve to
promote cancer metastasis.; B: M; y' B, m
Mesenchymal stem cells are pluripotent progenitor cells that contribute! T/ R$ x0 B2 D. m! w# D" g5 |2 t+ t5 \
to the maintenance and regeneration of a variety of connective
tissues, including bone, adipose, cartilage and muscle3. Although) {. e: _* J1 Q8 T, t( n3 A( O- l! V
MSCs reside predominantly in the bone marrow, they are also distributed+ I# f0 e& t: M) C
throughout many other tissues, where they are thought to
serve as local sources of dormant stem cells4,5. The contributions of% [3 `! U- d0 {8 Z
MSCs to tissue formation become apparent only in cases of tissue4 n' g- N9 [; o8 M" v
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: n* @' m0 D+ l8 x, K* {$ @
their subsequent recruitment to the damage site6. For example, MSCs8 `1 E0 f5 I5 Y3 A# p* J: h
have been shown to contribute to the formation of fibrous scars after5 ]& k9 D; s  E9 I
injury7.
The formation of breast carcinomas is often accompanied by a% s5 X$ A6 d  Z# o4 z7 D, `
well-orchestrated desmoplastic reaction, which involves the recruitment
of a variety of stromal cells with both pro- and anti-tumorigenic1 Z2 `5 N) `: p6 L; I+ R
activities1. Such response closely resembles wound healing and scar
formation, and entails the constant deposition of growth factors,
cytokines and matrix-remodelling proteins that render the tumour5 J) Q3 q) x4 ^. T! g
site a ‘wound that never heals’8. This suggests that, similar to sites of9 @; x. f. A# P0 w1 _( U4 W* O
injury, actively growing tumours recruit MSCs through the release of
various endocrine and paracrine signals. Indeed, as we have found,% u% _8 f* `4 `( Z# ]# }
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 vitro8 M" D2 W6 V! g
(Supplementary Fig. 1a), a hallmark of MSCs3. The absence of such6 z' N6 x9 ^3 U6 l6 v
colonies from control Matrigel plugs or from neighbouring tissues  l+ W6 M% m5 Q: f) G$ n/ U
(negative control; Supplementary Fig. 1a) suggested that endogenous* w1 z) e2 _% X0 R1 T3 ]
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' M& o7 w, M. j* F6 B+ Q
in which bone-marrow-derived human MSCs were allowed to* K0 q9 w2 S+ M0 k/ g
migrate towards media derived from MCF7/Ras or MDA-MB-231: n3 Z/ R) ?7 h* _, g. p! V
cultures. We found that human MSCs migrated much more avidly
(,11-fold more) towards media derived from these cancer cells. ^' G! q3 {) I4 c2 b
than towards control media (Supplementary Fig. 1b). More importantly,
green fluorescent protein (GFP)-labelled human MSCs
infused into the venous circulation of mice bearing MCF7/Ras
or MDA-MB-231 human breast cancer xenografts localized specifically
to the developing tumours, with no observable accumulation
in other tissues, such as the kidneys (Supplementary Fig. 1c), liver8 S) A! }/ i1 e) L9 k
and spleen (data not shown). Such findings indicated that MSCs# g1 Z/ y3 }: p: f; f0 F$ P' ~
are specifically recruited by subcutaneous breast xenografts, and corroborated3 ?5 D$ b8 `$ n, P
recent studies that described the localization of systemically' l1 R  h* N' e1 Y2 f% ?& b
infused MSCs to other types of malignancy, such as gliomas9,10,! m* [- i" B! N4 v% g2 t
colon carcinomas11,12, ovarian carcinomas13, Kaposi’s sarcomas14 and9 b, a. K; k' @
melanomas15.MSCs enhance breast cancer metastasis
To investigate the functional consequences of the heterotypic interactions0 |5 n- c; B, Z
between MSCs and mammary carcinoma cells, we established
a xenograft model in which GFP-labelled MCF7/Ras, MDA-MB-231,0 M3 Z1 F9 ?2 Z8 F
MDA-MB-435 and HMLER (see Methods) human breast cancer
cells (BCCs) were mixed with bone-marrow-derived human MSCs; E6 I) g- P/ d1 f
(hereafter referred to as MSCs) and injected subcutaneously into7 q9 F, E& K3 l- {' a8 s. X9 R
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.; c% X. }5 `# A, _; {" S7 r
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,9 D) ?1 F+ S  v: Z& \
whereas mice carrying tumours composed only of BCCs: w# A9 l& l% R, {# B: {( b; \
exhibited few microscopic metastases in the lungs (Fig. 1b, d), mice7 }, V  K: A. i- ]7 a" h
bearing the mixed MCF7/Ras1MSC, MDA-MB-2311MSC, MDAMB-5 [3 ]8 D3 K" L6 Z; J  Q6 ]# T, Q
4351MSC and HMLER1MSC tumours displayed a marked
increase in the numbers of micro- and macroscopic lung metastases
(Fig. 1b, d). Normalized counts of the metastatic nodules in the lungs% F3 A% g8 ~! y( d
of BCC1MSC-bearing mice compared to their BCC-control littermates. i# W: _+ J8 ]8 D& b
revealed two-, three-, four- and sevenfold enhancements in% S( n5 q1 T7 f
the overall numbers of detectable HMLER, MDA-MB-435, MCF7/
Ras and MDA-MB-231 metastatic deposits, respectively (Fig. 1c).
Furthermore, in contrast to the MDA-MB-231-bearing mice, the  ^" x' M9 {9 \( A# T
MDA-MB-2311MSC-bearing mice showed metastases to various4 n# r- a4 S. q1 Z& v- J6 r
other tissues, including the mammary glands (Supplementary) `) ~0 Z( [6 o+ f
Table 1). Although all four of the tested cell lines exhibited enhanced+ L( L6 u! v, O
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/ f$ B5 }6 O* z8 V: i
without any concomitant effect on either tumour cell proliferation
(as revealed by Ki67 staining; Supplementary Fig. 2) or overall primary+ I! b  E2 x% X
tumour growth kinetics.
We note that admixture of other types of mesenchymal cells—
specifically WI-38 or BJ human fibroblasts (Supplementary Fig. 3
and data not shown)—to MDA-MB-231 cancer cells before injection
into host mice did not result in either enhanced growth kinetics
(Supplementary Fig. 3a, b) or increased numbers of lung metastases; E7 v; g0 M* _
(Supplementary Fig. 3c, d). Taken together, these observations indicated$ ]" h" o. c* ]7 y! r* U
that the metastasis-enhancing powers were a specific property9 P+ R6 b( Q# G/ l
of admixed MSCs or derivatives thereof.
Reversible metastasis
Implantation of MSCs either contralaterally to MDA-MB-231 cells or; P8 R7 m' P% a+ T( }6 e" ], f
in nearby separate sites of injection did not affect the metastatic
potential of the resulting primary tumours (data not shown), indicating# }; r1 A, h3 ]* z
that MSCs could enhance cancer metastasis only when they
were in close proximity to the engrafted BCCs. This influence might% L6 u, Z) z6 Q8 C, C
be ascribed to various effects that MSCs exert on the commingled: P% ?0 ~/ I2 F
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' y* Z0 q# j7 W( k2 b
enhanced metastatic abilities. This latter mechanism suggests the0 M" s+ l9 v, `) x
possibility that the acquisition of the metastatic phenotype might
be reversible, in that carcinoma cells might revert to a lower metastatic2 ?9 I& ]: v$ h
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)% v( Z, ?6 Y' m& Z" S% ]/ H
as well as from their derived lung metastases (L-explants),5 `+ N& E# O5 a- r% i& s; {8 P
expanded in vitro, cleared from contaminating stromal components,
and then re-injected into subcutaneous sites in host mice in order
to evaluate their respective metastatic powers (Fig. 2a). Although
the growth rate of the resulting L-explant primary tumours was
marginally enhanced compared to their T-explant counterparts1 i9 V; d/ n( n0 A) }
(Fig. 2b, c), these L-explant cells were no more metastatic than the6 Q% E6 \8 T$ T: h% z% y4 t, N8 H
parental T-explant cancer cells (Fig. 2d). This suggested that the
a
c d
Days after injection2 ~* N# T  S. _* o9 t3 _) B
Tumour volume (mm3)
10 17 24 31 38 46 72 80 89 13 19 26 33 40 47 54 61 68 75 81 10 19 24 31 38 46% z  i7 \) V1 }1 C6 ]! u( g( l
b
1 mm 1 mm( k4 V- O7 O9 y4 O" ?2 h8 W
1 mm 1 mm
300 μm( p) @4 l# V( K: {, R) f) v5 \5 ]
HMLER HMLER+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC
MCF7/Ras MCF7/Ras+MSC4 v. @7 J$ R1 b% l: \
100 μm) ]* h" }2 b/ q6 }9 B8 l
300 μm) Z/ Z$ C: Q' C1 H
100 μm
MDA-MB-231
MDA-MB-435% Z6 _$ f2 J# h& i$ p! G
MCF7/Ras
**
Metastasis index (fold)* H/ f; u% C" l+ t, ?* s
0
1
2' [  x" |' L9 V3 Z9 M* x+ B
39 `4 |2 h3 Y" Q! s0 u
4
58 G/ U* |. _1 U9 L6 Y
6
75 H% }7 R7 ?! M
87 c4 I( G6 ]( P, m( @
99 Z; b6 q, C) Z$ s' n
*' H+ `; S# j( d, m) ]6 Q8 b8 I3 H
**
**
MSC – + – + – + – +; t3 X. [  \  X& ]) R, X  ]
*: e% S6 V: J5 x
HMLER
700 1,2009 p( ~  s: ]2 I% S
1,0007 I, A$ }# F5 M& w$ e
800; _. f0 E6 b, b  ?6 q) {! e
600$ L% W: M. e: v5 n2 `, J/ q4 s' G+ _/ c7 Z( Q
400
200' r  ?9 B5 z2 U& W
0
800
7007 d% m; G6 c% s5 O% P" v
600
500
4009 }: ^% |# f9 H0 }
300) l- ]  J6 M( g. C; S
200  S0 W1 ?' F( `  b: H. `
1004 @6 o1 q( F1 V4 m
06 W& I4 R' i; S+ P4 [  O
3,500( A2 O) s9 Z  x+ L  A
0, B6 p6 t$ O" F, y" N/ T( x4 N
5005 h; K2 B  {% H$ R* j# u: N, D8 j$ [6 N
1,000
1,5000 C. R& ]. n8 T' d2 F( j; S
2,0003 N% @" P! h. Y% U8 e% f0 e
2,500
600 3,0000 Y3 S6 H. c/ J4 ^* k( @
500
400; Y8 ?9 ]0 t2 P3 _& R/ A- x. G) r
300
200
100
01 a# S- Z  m+ a0 m: e. I
0 21 31 38 49 56 63 70 78' d2 t0 w2 N# [* j( T
MCF7/Ras alone
MCF7/Ras+MSC
MDA-MB-231 alone3 \$ l  C' f0 A- c0 U7 \4 T! A/ p& w, y
MDA-MB-231+MSC% |5 ^- t, p) V+ F7 R
MDA-MB-435 alone" w# |- w# P* d2 j8 X6 c$ f
MDA-MB-435+MSC: K: G& \) |' s# z+ `; l( n8 |7 ]
HMLER alone
HMLER+MSC) y6 i. L; W5 h: l  [3 J8 Y4 i
HMLER HMLER+MSC
MCF7/Ras MCF7/Ras+MSC
MDA-MB-231 MDA-MB-231+MSC
MDA-MB-435 MDA-MB-435+MSC' e* `; i, J0 }& R& |
Figure 1 | MSCs promote breast cancer metastasis. a, Tumour volume" u6 A0 [( ?% p! D  ?5 X
measurements (mean6s.e.m.) of 500,000 GFP-labelled BCCs injected
subcutaneously into nude mice with or without 1.53106 MSCs.; x- O! o9 g8 z- ?* k
Representative data from multiple experiments are shown. Diamonds, BCCs
alone, n55–7 mice per group; squares, BCCs plus MSCs, n55–8 mice per$ I6 E$ H2 Z+ F
group. b, Representative bright-field/fluorescence images of lungs of mice9 y5 b9 _, n" `" ~' M2 X
bearing the indicated tumours. Cancer colonies are in green. MCF7/Rasbearing( \) e4 }) o) f! z
mice were killed at approximately day 150 to allow these tumours to9 n$ ^0 E9 B0 l7 @
grow to comparable sizes to their MCF7/Ras1MSC counterparts. c, The7 b! e4 e  s# j/ `! a( h
lung metastasis indices pooled within each cohort of mice in a are expressed" Y7 w) O  |  _4 m. F  O# c6 Z
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 are5 U' U$ Y# J& V, U  r8 j" q
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 in5 Y0 q8 s. o& a1 g$ {7 x+ n8 `( ?& a
the primary tumour rather than the selection of rare variants of
MDA-MB-231 cells that display elevated metastatic potency in a" _% N6 i! w; B8 R3 V
stable fashion.
The effects that the MSCs exerted on the BCCs might have
occurred within the site of primary tumour formation. Alternatively,
the MSCs might have accompanied the metastasizing BCCs; z( K/ j( c6 f$ z8 U
to sites of metastasis formation. To distinguish between these two
possibilities, we admixed ds-red-labelled MSCs to GFP-labelled, Q5 a( b( q# p( v# ^9 K
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 redlabelled5 v) g# i$ G# g& w- W
MSCs (or their derivatives; Supplementary Fig. 4a) when
scored 4, 5 or 6 weeks after primary tumour implantation. The
absence of red-labelled MSCs from the lung metastatic sites cannot
be ascribed to an inhospitable lung parenchyma, as MSCs that lodge
in the lungs of recipient animals after tail-vein infusion survive in that
environment for ,6 weeks after injection (Supplementary Fig. 4b).$ D  j6 d6 l! a% }( `# K0 O
Hence, it appeared that the admixed MSCs do not migrate in large& C: J3 F* ?6 [6 [2 y: E0 j" M  m
numbers to the sites of metastasis, and that they exerted their prometastatic
effects on BCCs in the context of primary tumours.! t) O" v- C; |9 R* P# O' }* U
CCL5 in MSC-induced metastasis
The aforementioned observations indicate that MSCs supply locally
acting paracrine cues that induce BCCs within primary tumours to
metastasize. To understand this crosstalk better, in vitro co-cultures
of MDA-MB-231 breast cancer cells and MSCs were established and
their conditioned media were screened for the levels of various cytokines,5 `+ J" t9 ^' x: Y  X: T
chemokines and growth factors using the Luminex-based Bio-, S+ c7 N% E1 E3 }
Plex suspension array system (Fig. 3a). In some cases, the resulting
a; K, E0 V* _& _0 F
b c d9 t& J* V6 q) D8 ]
Days after injection
Tumour volume( f1 T# ]- s6 i1 ]
(× 100 mm3)
0
5( l  h  k2 K4 |1 C( J8 j9 x6 a2 W
10, T, o' y( a/ A4 s: S( k/ S, E
15
206 ^; A6 S0 t6 T( ~& t0 [* j5 G
25
14 17 21 24 28 31 34 38 42 45 48 66 71 77+ d  A# U* z9 @6 u, @+ c2 D
Primary tumour explants1 E0 _% W4 [5 N/ l) W" o5 s
Lung explants
MDA-MB-231
MSC- q; q; f  o' {/ L
+8 \2 n. J" j& l% _8 b) K
Lung explants4 M) V0 Q- C+ I3 W" E3 [. F% }
Primary tumour" ~, q- l- h+ R: D' Q
explants
Antibiotic
BCC selection- P4 B$ }6 E- V! t( |) t* K
T-explant  d7 i# A! O9 c1 }# Y
L-explant1 Q$ {$ G& f. E& n6 {
T-explant! |! }8 h9 l" ~
L-explant! K; A: Z! _, t" \) L! ~$ b
Tumour mass (g)) Q( a; O8 O# s+ m% p
3.0+ G" m2 E2 R! X: t3 W; j
2.0% R. X: u& v, t2 e& G' C
1.0- b& ~$ |8 j; z8 g6 ~* _+ K
0 0
0.4' M% f! |: p# O/ ~. }, _3 u
0.8
1.20 L  x2 f7 I) A# f4 `
1.64 f: T. i+ N9 V, w. L
2.0
#* z, N" S3 H" Y: F9 u3 N6 g
Metastasis index (fold); g- l6 a# `8 f6 b* Y
##' M$ q7 c2 Y, h. U8 ?2 r
Figure 2 | MSC-induced increase in the metastasis of MDA-MB-231 cells3 L$ v: x) |8 I/ N3 z0 h
involves reversible mechanisms. a, BCCs were recovered from lung or. ~# V& [5 m1 s8 U9 z1 J9 N
primary tumour tissues, cleared of stromal contaminants by culture in, x- y$ t2 O- I' q
blasticidin-containing media (5 mgml21), and re-injected as primary3 c+ f* I. W. T% \$ }) D7 b
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.
Data shown are representative ofmultiple independent experiments in which- s0 A( U; Z$ n# B: Z7 }
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. C: A/ D+ E/ ?! l  {
(n510mice). c,Masses (means6s.e.m.) of tumours in b.Hash,P.0.4 using- t" V% v9 Q- ~+ ~
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’s6 Y: B& d/ P' r) n6 x7 i9 A* T
t-test and indicates no statistical significance.. {  u% n5 B; I3 D+ z$ o
a
CCL4, `! R" O. B2 F- }; i4 M
bFGF5 a; U& h1 r5 r: m8 ~6 }
VEGF. Z( q. c# F8 i
IFN-γ
TNF-α
G-CSF& Q5 b  m' @. w. H" N1 y- R
GM-CSF
CCL3
CCL5
MMP1
MMP3
MMP97 l  r6 H. o  D4 D) y
MMP13
IL-1α. V) S. C3 l8 c5 U" w: i2 [( w2 s2 Z
IL-1β
IL-4& Z* j  f9 Y8 t+ c# M6 Z# t
IL-5
IL-6
IL-7
IL-8
IL-10! K6 M, U. G: @( [, n, [
IL-12
IL-13- A4 }8 D4 R1 {9 F, W6 R+ o
IL-178 K. l3 q6 H. g$ ~& m
IL-25 g. k: x+ f( I. g4 M% O+ G- X" g
TGF-β
Fold induction
MSC alone
MDA alone  N" e  f5 `4 E' A
MDA+MSC (2:1 ratio)( g0 }5 ?( t% t0 o: @
1 * * * * * * * * *
3' J  q/ w- g; C8 O1 C
5' L# E. K: e3 W# B5 R
7
9
11; x1 ]0 n3 V) b, L$ x
13
60
b c
0.4 μm" I2 c3 c- d4 a' G# c9 g
2.0
1.8" d5 x: p1 Y1 x) @
1.6
1.4
1.2
1.0
0.8
0.65 K2 |2 z/ ~( y
0.4- Y2 `) @7 Q" ?" \2 j9 W# }0 ~
0.2) @6 b/ l% Y( G+ r; |" _
0 06 w7 b. ^. ?8 X
5  `0 W" Q; K# q. x4 T. @' F
10
158 H9 @: C9 Q# _# m& L
20
25, x: a4 x$ ^/ H# h
30  a0 V! n) [7 \
35& Z+ S; b' }2 [8 L3 \# e
40; A  k/ Y9 x- y2 G6 e
45" p$ F; i4 V# k7 R  P  O2 A% \
507 s5 V9 [! c1 G0 [* t
d1 d2 d3 d4
MDA alone, i. F2 u# j- l5 S5 ]$ W
MSC alone
MDA+MSC, b& p4 r4 ^$ H8 z8 t& T& d) l& \
CCL5 levels (pg ml–1)/ f' L2 y4 g0 s8 A+ J4 ~
Co-culture4 e( @9 H- F2 S- E. N# f# n
Fold CCL5 induction3 e- Y( T3 B! i& E7 u. H) l$ P' i* M7 R
MDA alone
MSC alone; h4 t, f8 n0 p: h3 H; l4 K( b, j& ~4 y
MDA+MSC- G0 x) S  N4 z
d) ~' I& o& L% ~4 X) J+ k6 ]8 }; u+ Y5 |
CCL5 (A.U. × 100)
30: r, r, m2 \0 ?3 K7 s
25
20
15
10
5
03 A# D+ y6 f% {) f8 }( A
+4 @1 ^/ N) [: P7 j0 ]
MSC.c. S# l# h2 G3 r( i' _( |
+! b! t! d6 S# I, r' u2 T1 ]
MSC.15 ]5 ^: r8 N2 ?0 I7 ~0 m
+8 x8 ?0 e* D9 l; R
MSC.5
MDA
MSC+ N1 d- \# J- E+ p/ R% s1 n% z3 Q# H
MDA.1" y8 ~: I1 [- F/ S, u3 W* R0 `
MDA.c% H5 E) v6 z# \6 g
MDA.5
TC-MSC
MSC (from MDA tumour)
BCC (from MDA tumour), M- V* a/ U) Q- M) r
Control (MDA/CCL5)2 T! O# \5 l; V
CCL5
GAPDH
e
Figure 3 | The interaction of BCCs with MSCs causes a rise in the levels of
CCL5. a, MDA-MB-231, MSCs, or MDA-MB-2311MSCs were cultured in
completemedia for 3 days. The levels of various factors in the cell-free culture/ B$ f: M; S" B6 \5 t* d. K
supernatants were measured by xMAP Bio-Plex cytokine arrays at day 3, and" H2 {' }- V4 q& j
were normalized to the levels observed in the media of BCCs cultured alone.
Data are expressed as fold induction6s.d. of triplicates. Asterisk indicates" _  c' d6 D8 r( c6 o3 c7 D" f
undetectable levels. b, CCL5 ELISA on the media of MDA-MB-231,MSCs, or1 D$ t5 v1 V0 J. B" x" }4 s- t
MDA-MB-2311MSCcultures (1:3MDA:MSCs) at the indicated time points.
Data points representmeans6s.d. of quadruplicates. c,BCCs were separated9 v+ s; Q. Q/ l! q$ l
from co-cultured MSCs by a 0.4-mmmembrane. CCL5 levels were probed by
ELISA on the culture supernatants. Data are expressed as fold induction over
levels seen inMDA-MB-231 culture supernatants (mean6s.d. of triplicates).! D" z2 R/ w- H& `% r% v' ]+ |
d, CCL5 ELISA on the supernatants of MSC-siluc (MSC.c), MSC-siCCL5.1+ a! ]1 C7 L$ m# o: g. J
(MSC.1) and MSC-siCCL5.5 (MSC.5) co-cultured with MDA-MB-231-siluc
(MDA.c), MDA-MB-231-siCCL5.1 (MDA.1), or MDA-MB-231-siCCL5.5  j% }' {4 o" A& V3 D
(MDA.5). Data are expressed as means6s.d. of triplicates in arbitrary units2 _8 S( E( s8 u- \" E/ A1 T! t
(A.U.). e, RT–PCR analyses of CCL5 in MSCs and BCCs sorted from' Y$ l: q" `, K+ ^: z1 j
GFP–MSC1MDA-MB-231 tumours (3:1 ratio) 4 weeks after tumour
implantation. Tissue-cultured MSCs (TC-MSC) and MDA-MB-231/CCL5; N9 X  G* T% B
cells were used as controls. GAPDH was used for equal loading.levels of certain released factors (for example, interferon-c or# Q9 i' R( x% y
tumour-necrosis factor-a) reflected the additive contributions of
the two cell types when cultured on their own. Notably, the levels
of only one cytokine, CCL5, reflected a synergistic interaction
between the MSCs and BCCs, as it accumulated to levels ,60-fold4 w& U* P+ h4 u/ L. ?
higher than those produced by pure BCC cultures (Fig. 3a). This
cooperative induction of CCL5 was proportional to the numbers of
MSCs mixed with the BCCs (Supplementary Fig. 5a), and was apparent1 Q  b( K! _3 e4 p  W5 y
as early as the third day of co-culture (Fig. 3b). Moreover, this- B% E, t" ]2 {: R: e4 \& Q
induction required close physical contact between MSCs and cancer
cells, because it failed to occur when the two cell populations were$ Y8 r$ |2 c9 W0 B' E( K
separated by a permeable membrane (Fig. 3c).
We undertook to determine the source of the CCL5 produced
under conditions of co-culture. To do so, we stably reduced the
expression of CCL5 in MDA-MB-231 cells by.80% using short/ G8 ~$ l: G9 P& U
hairpin (sh)RNA (variant siCCL5.1; Supplementary Fig. 6). Importantly,
however, subsequent co-culture of these MDA-MB-231.1 cells
with MSCs continued to allow accumulation of CCL5 in the culture
supernatants to levels that were comparable to those observed in the
co-cultures of MSCs and control cancer cells (Fig. 3d). This suggested
that the source of CCL5 was the admixed neighbouring MSCs.0 ]9 c( k8 ~: `0 q1 h; H
Indeed, inhibition of CCL5 protein expression in MSCs using the* l) m" X% x/ M' I" I
same shRNA hairpin vector (MSC.1; Fig. 3d) resulted in more than1 v6 Y; H1 B1 c) o: \
75% reduction of CCL5 protein levels in the co-cultures, indicating8 Y& F7 G/ L- r* Y* d) e
that the MSCs were the major source of the CCL5 observed on coculture7 f: y* j) \) P
of the two cell types. In support of this conclusion, analysis of
CCL5 levels in the media of MSCs or MDA-MB-231 cells separated8 ^7 H5 A$ m+ l0 S/ k3 X  [
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 (Supplementary6 z$ ^- ~. {9 M. ?# i
Fig. 5b). Finally, polymerase chain reaction with reverse
transcription (RT–PCR) analysis of the RNA prepared from these coculture-- v7 q( y& p/ {3 v* w- @# S7 l  c
derived MSCs (Supplementary Fig. 5c), as well as from the& r& o: s4 Y2 G6 h! a
MSCs isolated from MDA-MB-2311MSC tumours ,4 weeks after
tumour implantation (Fig. 3e), indicated a strong accumulation of; Y7 P. y: N9 G
CCL5 messenger RNA, suggesting that an active signal transduction
pathway is triggered in MSCs by the nearby BCCs.2 @. f" M& a; ]- h
A series of observations has linked CCL5 signalling and cancer. For* P0 D4 g( C) T8 ^6 J
example, CCL5 levels in the plasma of breast cancer patients have
been correlated with the severity of the disease, and localized CCL56 P- h6 W$ k/ k
protein expression was found to be elevated in invasive tumours$ C: V) Q( {2 H2 J- _0 O
when compared to in situ ductal tumours or benign lesions16,17.# p3 x& ~( ~3 ?5 x- n" @
However, the precise contributions of CCL5 to cancer development9 {8 n* Z3 S- j/ p
and progression are poorly understood. To investigate further the0 T! Q' I/ `) E2 u5 U4 _
possible causal role of CCL5 in cancer cell metastasis, we overexpressed, b, A5 R/ J4 x% Y" s; z+ U, \
this chemokine in the MDA-MB-231 BCCs (Supplementary% D' P2 ]# u2 i' t0 G6 u
Fig. 7a) and analysed its effects on cancer cell growth and3 h' Y. @0 K0 j  h. L- @1 X
tumorigenesis. The overexpressed CCL5 did not confer any proliferative
advantage on cultured cancer cells when compared with9 i& J5 F' ]' u4 E5 `! e
those lacking such overexpression (Supplementary Fig. 7b), and had
no effect on the ability of BCCs either to grow in an anchorageindependent9 y6 v/ F! V0 R/ B9 r3 Z
fashion in vitro (Supplementary Fig. 7c), or to form1 G+ o. J, U. ~. B1 _5 g6 C4 S
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) r5 k( [" R; {; O
in WI-38 fibroblasts sufficed to enable these cells to promote the! d9 g$ G$ S& M
metastasis of admixed MDA-MB-231 BCCs (Fig. 4b), indicating that
the actions ofCCL5 are responsible formuch, if not all, of the observed* p4 E6 I* _# C& }) J+ Y
MSC-induced metastasis by the BCCs.
CCL5 promotes lung colonization( v/ e/ L' Y% c& ~' V% o
Previous reports have described an important role for CCL5 as a
chemoattractant for stromal cells, such as macrophages, that express6 I+ k+ X% ?0 E5 s0 e% f
one of the receptors for CCL5, CCR5 (refs 18, 19). Furthermore,& c' z/ k# l, m9 ^
CCL5 expression has been associated with increased tumour neovascularization,* X# W# o2 X$ ]
suggesting that endothelial cells, which express a variety; t: z- v2 ?* [' s/ B
of chemokine receptors, may also be attracted by CCL5 to sites of, C9 F* o  G. {7 p
tumour formation, thereby enhancing tumour angiogenesis20. Such) B1 b4 d) b! o* [9 p% t
observations suggest that CCL5 may contribute to breast cancer
metastasis through the recruitment of a number of stromal cell types
to sites of primary tumour growth.5 d* @5 a& q% X
However, immunohistochemical analyses indicated that the) q6 H. |8 d% u6 e6 ?+ n
MDA-MB-231 control and CCL5-overexpressing MDA-MB-231) s* m  Y& h% u# l. j: m8 l# _
(MDA-MB-231/CCL5) tumours exhibited comparable numbers of
tumour-infiltrating macrophages and had similar vessel densities (as5 k$ f$ }, o2 N
evident by F4/80 and MECA-32 staining for macrophages and
endothelial cells, respectively; Supplementary Fig. 8). In addition,) M1 [2 \4 {+ A, g7 b
we found that ectopicCCL5 expression did not cause an accumulation; a. |4 i! q$ m7 }
of other stromal cells, such as SMA-positive cells, in the examined0 K% D# a' t3 c
tumours (Supplementary Fig. 8a). Together, these data indicated that# [! @; k, y) V8 y$ f( y3 h8 Q( a
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+ O0 n: m5 E% j) I3 D/ o& ]3 w
facilitated by their transdifferentiation through the process termed
d f& u  ?2 R+ L& F4 M" }4 ^
Bcl-XL' p3 g/ F4 F; x2 @5 C! n6 \
Bcl-2
β-Actin
S473-Akt. j5 o% j  k5 X* x0 o
Motility0 N& d6 W" F. d" P! D
0.5%→10%% \) H, q  i( x) m. q
LY – – + +0 @/ ^; M+ C2 e# K
g8 J# {' S) H( \, N: S" B
Extravasated clusters (mean)% p4 A: o. K2 @
Migration (fold)
*
e8 U7 L- j9 C+ @( a) G
05 }' w* `2 O8 u5 I( S) O9 ~+ B
MDA/vector1 Z3 U% U# M7 h4 G1 W  j
MDA/CCL5
***
Invasion
10%→10%
Invasion' ]8 _9 A$ z6 f4 @. j
0.5%→10%0 I4 R: ?# c- T5 ]; @  G
Migration (fold)/ h( K9 E4 n- E. }+ D8 @  g7 X  O
**3 c/ t* h/ p# ?* d) ]% s/ {
0' F+ ]1 W# h' g- S4 W7 v. R( W
0.53 s9 o+ A! X! V; ~
1.0, {9 Z4 Q0 A* r. D* x# D" S
1.5% T, E" u. C* X  W4 b! |( b) [% A
2.0
2.5
3.0, Z4 a- p" ^0 L9 F3 i- R
3.5# y' L2 L, F* `" Q
*" C" l5 |# l; a, T
MDA/vector' R' C3 e4 M# s0 |2 n& R7 E! N1 y
MDA/CCL55 ?% \# Z/ D" Y7 E" l2 p$ E4 l
a b
*" [' `% K# V* G) ~6 M
Nodules per lung (mean), _0 [5 q  X3 D/ @. j7 r
c
30
25
20
150 A: a+ O5 P# D5 E
10
5, [% Z# }6 s" s4 t! ^
0, s# H) t& R) [4 r
Tumour mass (mg)
30
258 z) p0 Q4 J% o
20
15; v2 `) i* D9 n# Y
10
55 ?( y% E: o# c9 H' E
0 0
50' w) d- p0 A, H# c( U# o
100. z. g6 `5 B. g3 i+ _# G/ F
150
200
2503 k! b6 X8 `0 c$ D- j' g4 c
04 x' |- X% }! L- s6 ~- k; Z0 w
50: X7 j8 u* s8 q' Z, Q3 \
100/ n+ m5 s1 q8 O: Z# l. K
150# g$ e3 r0 }  I1 q2 E  g' h
2005 X: ^. X/ T8 M; [! E3 |8 P
250
300
350
Tumour mass (mg)0 b' \# P* c9 d
Ctrl8 {! U' {2 b0 e& W
CCL5& g0 s% S8 N* b% g! p3 H: G4 m, l
Ctrl) ?8 E. W7 V/ B% b
CCL5
Ctrl4 A# g4 j( C5 e6 m1 v
CCL52 E6 I& s7 O7 I0 M9 O* n
Metastasis index (fold)$ w4 ?5 g4 d5 p$ h
Metastasis index (fold)
0
1
2
3* `" }( }0 ]' o  A3 C3 v- K7 Q
4; h% b% |- w9 j
5' v0 q: }5 p/ r9 p5 N
* 6
0
16 ~; o$ c6 z% f6 h
2( Y' P3 y) o/ o% M, ^; g7 o
30 g! {: E1 ~0 L$ L8 e6 h+ q( P/ h4 c
4
5  m( r9 a' F4 n1 h+ s+ ]
6
7
* 8
MDA/vector! M4 G  }3 X0 u' h
MDA/CCL5
MDA+WI-38/vector
MDA+WI-38/CCL5
1
2" u" w! o5 A3 e$ K( e0 T0 }1 {4 I
3
4
*
Figure 4 | CCL5 enhances breast cancer cell migration, invasion and
metastasis. a, A total of 500,000 MDA-MB-231/vector (ctrl) or MDA-MB-3 {/ Z: `! y" `6 G  N
231/CCL5 cells were injected subcutaneously in NOD/SCID mice. Tumour, h( h2 O' X; s9 T* x
masses (mean6s.e.m., n56 each group) were taken at 10 weeks. Lung) n5 [2 C. W  X/ g
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-tailed3 d1 w* Q, L- L1 |) I8 o$ Z' |$ L8 I
Student’s t-test. b, A total of 500,000 MDA-MB-231 cells were admixed to
250,000WI-38 fibroblast controls (WI-38/vector) or WI-38 fibroblasts
overexpressing CCL5 (WI-38/CCL5) and were injected subcutaneously in2 S# p% n: O% ]8 D
NOD/SCID mice. Tumours (n55 per group) were excised and weighed at
12weeks. Masses shown represent mean6s.e.m. Lung metastasis indices are( s, j, z8 s4 S- T: a) Y
expressed as fold increase (6s.e.m.) over controls. Asterisk, P,0.01 in onetailed0 K8 v' m' ^0 {8 b
Student’s t-test. c, A total of 800,000 indicated BCCs were introduced
into the circulation of NOD/SCID hosts. GFP-positive cancer colonies in the
lungs were counted 6.5weeks later. Bars representmeans6s.e.m. (MDA-MB-
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% p& I$ \5 o# f" [0 D
loading control. e, Transwell migration orMatrigel invasion assays on 50,000
MDA-MB-231 control orMDA-MB-231/CCL5 cells.Data are representative of$ S: B: H+ S* U$ O, B
multiple independent experiments and are expressed asmeans6s.d. Asterisk,
P,0.05; double asterisk, P,0.05; triple asterisk, P,0.01 in one-tailed3 a" N7 m# k+ {$ f; j2 ^$ J8 ]" W
Student’s t-test. f, One million GFP-labelled BCCs were injected into the tail
vein of NOD/SCID mice. Lungs were processed 48 h later and examined for8 n$ F* f% t2 i# u8 H
extravasated cells. Bars represent means6s.e.m. (MDA-MB-231 cells, n57
mice; MDA-MB-231/CCL5, n510 mice). Asterisk, P,0.01 in one-tailed
Student’s t-test. g, Transwellmigrationassays on50,000MDA-MB-231 control2 v5 J8 `2 }5 ]$ {/ F* ~* u; G- C/ t
orMDA-MB-231/CCL5 cells plated with or without the phosphatidylinositol-" R6 g3 q% O% c$ a4 z  l- n9 y
3-OH kinase inhibitor LY290042 (0.5 mM); representative experiment shown;0 P. X. {% H5 O, M' w( M% H
asterisk, P,0.01 in one-tailed Student’s t-test.the epithelial-to-mesenchymal transition (EMT), in which cells shed
their epithelial characteristics and acquire instead a series of mesenchymal
markers that enable their invasiveness and intravasation21.
Despite their lack of E-cadherin and their expression of detectable levels
of mesenchymal markers such as fibronectin (data not shown), the3 ~# O  i: ^. `' x2 Z4 K
MDA-MB-231 cells studied here exist in an intermediary phenotypic/ g: K( n$ u4 p) [/ B  M
state of ‘partial EMT’, as they retain a distinctive epithelialmorphology  K( D3 W0 I! Z% E/ c
in vitro and are still responsive to EMT-inducing stimuli in culture. In0 b" w1 G+ @: V$ v2 N
fact, we observed that ectopic CCL5 expression did not cause MDAMB-
231 cells to undergo themorphological changes usually associated
with an EMT(Supplementary Fig. 9a), did not cause rearrangement of) n3 M! V7 P/ S9 D+ I, _
their actin cytoskeleton (Supplementary Fig. 9b), and had no impact on
the expression of mesenchymal markers closely associated with the- r# y4 e" I! b" q$ q" Q
EMT process, namely vimentin, N-cadherin (Supplementary Fig. 9c)
and fibronectin (data not shown). These data suggested thatCCL5 does3 S, O" P, V8 P0 H3 e/ p4 g; W
not directly promote the EMT programme of MDA-MB-231 cells.  e  i3 M4 u, L9 P$ Y
We proceeded to explore an alternative possibility: that CCL5; b  R" @# F# f! {# [4 I
expression affected some of the later, critical steps of the invasion–( H+ \. _9 C6 c+ h& q" g
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 host3 f/ L- L: H: F4 i* j. Z- j
mice, and the lungs of these hosts were examined ,6 weeks later3 d& w+ e2 r& H2 y$ F
using fluorescence microscopy. These experiments revealed that9 p# Q- L9 r. r: e2 \! {* K
CCL5-overexpressing cells indeed had a significant ,1.8-fold
advantage over their control counterparts in colonizing the lungs% F2 F8 ]3 ]0 s  b: g1 k8 n; V% ?* t
(Fig. 4c), suggesting that CCL5 exposure has effects on later steps9 l7 k# z7 x7 {( e- Y
of the invasion–metastasis cascade. We note once again that this
enhanced tissue-colonizing ability was not due to CCL5’s effects on. N8 ]4 a" b0 B: k
cellular proliferation measured either in vitro (Supplementary Fig.! l8 T1 d5 }5 K7 `  P  j+ |
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.. P3 @( ^; p# J  H- l( w
Notably, we found that MDA-MB-231/CCL5 cells exhibited higher: W  u6 l1 Y. E0 V( t! f' r, e5 k
levels of the Ser 473-phosphorylated, activated form of Akt, but1 B! G2 J& a4 N' Z; [+ R
exhibited no difference in the levels of other pro-survival proteins,
such as Bcl-XL or Bcl-2 (Fig. 4d), or a reduction in the levels of
pro-apoptotic molecules such as BAX or BAD (data not shown).
Moreover, we found that overexpression of CCL5 had no effect on5 n4 `2 s/ R1 S  Y2 [
the ability of MDA-MB-231 cells to withstand serum deprivation
(Supplementary Fig. 7b), loss of substrate anchorage (Supplementary7 W" x6 ?& k  M! R
Fig. 7d), or hyperoxia (data not shown). We also observed that
ectopic CCL5 expression did not protect MDA-MB-231 cells from
doxorubicin-induced apoptosis monitored using western blots for
cleaved caspase-3 (CC3) and cleaved PARP (as markers of apoptosis;- o8 _8 o# k0 r. q+ M
Supplementary Fig. 7e), or TdT-mediated dUTP nick end labelling
(TUNEL) assays (Supplementary Fig. 7f). Finally, immunohistochemical
analyses on control and CCL5-overexpressing tumours+ z+ e% j# V; x; R. N
revealed only minor differences in the levels of apoptotic CC3-( f7 A9 n% |9 s* Q! \' b. ?
positive cancer cells among the examined groups (Supplementary. R  a8 ~5 ?* [9 |5 L/ {
Fig. 7g, h). Together, these observations suggested that CCL5 does2 U$ r6 @* y1 L, R6 E' b* [
not exert any detectable pro-survival functions in vitro or in vivo, and6 D! b- l* U3 d2 t9 s
that the observed enhancement of lung colonization was not a consequence
of significant anti-apoptotic activities of CCL5.
Akt serves as a key relay switch for upstream signals that promote
both cell survival as well as cellular motility22. Because CCL5-induced
Akt phosphorylation did not correlate with enhanced protection1 C/ t+ G) r5 g* B5 g4 x# B! L
against apoptosis, we tested whether the CCL5-enhanced lung colonization# B  i* ^- v- t' a: i& k) ^
could be due to an increased ability of MDA-MB-231// c. h7 ^& ~  y, p$ J# X; ]5 p% ]
CCL5 cells to invade from the microvasculature into the lung
parenchyma through the process of extravasation. Indeed, ectopic
expression of CCL5 enhanced the motility of MDA-MB-231 cells
through permeable Boyden chamber membranes by,1.5-fold as well
as the invasion of these cells through Matrigel layers by,1.6 or,2.5-
fold in either high or low serum conditions, respectively (Fig. 4e).8 a3 J' Z2 q5 h; q
Notably, when we flushed the lungs of mice 48 h after BCC tail-vein+ |( C& h' ^- _3 v" j
injection—in order to remove most cells that remained within the) r& o5 S3 R  P* @. {, E. U" Z
microvasculature of the lungs and thus had not extravasated—we! e4 O" g5 r$ u- Y0 m3 o
found twice as many deposits in the MDA-MB-231/CCL5-injected+ `% d" i% f& _5 T" |- s+ n& S
group than their control-injected littermates (Fig. 4f). This indicates a/ V3 O' H! o- g7 ?# o- K1 z# S# I; m
clear effect of CCL5 on cancer cell extravasation.
Finally, we investigated the role of Akt in mediating the actions of/ {. V2 i) @+ t$ L0 B
CCL5 on cellular motility by using the phosphatidylinositol-3-OH& Y! m7 {: N1 s4 \" ?. J
b silacZ+ e" e$ I- p% Z# ]
silacZ
si8096 d- U- f5 Q" |, A; m% K
si809
si186' L" _9 s6 g4 K4 G! b
si186( S) T, c+ E- Z
CCR5* n6 |# |6 W; R/ r
β-Actin
a d
M& C5 j6 t9 b* v# m2 j1 R% R6 N
+
MDA+MSC
+ IgG
+Anti-CCL5 Ab
MDA
+Anti-CCL5 Ab
MDA+MSC
MDA
IgG
<4 h+ c: d" E$ W/ {: P( X; z, h
<
<
<
<
DAPI
CCR5
MSCs MDA MDA+MSC
DAPI DAPI
CCR5 CCR5
MSCs MDA MDA+MSC
*3 ]9 y% b  P) m+ }& r
c
Metastasis index (fold)
Metastasis index (fold)& v- V1 E& D5 U0 }7 v
*
Control siCCR5! l( O! d0 C* [, [- Y
MSC – + + +& H4 _- j+ z: h
0
1
2* I9 M8 g8 {+ d
36 b) n: h  p5 F( v& S
4, t2 r) R6 l. P  A9 ?. Z6 c' W3 @. q
5 e# O) Z8 ^( n7 O9 H: u8 e2 p, G
+ + – –
Anti-CCL5
0
1
2
3- @9 y% q. B" D6 y
4$ a1 H" _' @4 `  W
5
6
– – + +' |6 G6 o5 s2 q3 S' h
IgG( a' t- c; b4 j! R$ b
MDA
MDA+MSC
<  F( {$ m* |/ z
Figure 5 | CCL5–CCR5 interaction is essential
for the MSC-induced metastasis.
a, Immunofluorescence analysis of CCR53 Q; J: {' o6 c
distribution in MDA-MB-231 cells cultured with' [$ c. N: n* n* c/ ]
MSCs. DAPI (for nuclei staining) is in blue;# o' \! x4 G/ ?( h' l
CCR5 detected in green. Arrowheads denote, h3 Q* D% ^( b( T
MSCs. b, Western blot analysis showing CCR5
expression in MDA-MB-231/silacZ, MDA-MB-
231/siCCR5(809) and MDA-MB-231/
siCCR5(186) lysates. b-Actin was used as a4 k7 B1 c6 l- }
loading control. c, A total of 500,000 cells of the7 N$ |7 Y! o5 |2 V
MDA-MB-231 variants in b were co-mixed with: q! i0 W" U( z& e; u
1.53106 MSCs and injected subcutaneously into2 v6 T( g% |  B8 B$ b
nude mice. Mice were killed when tumours$ J  I1 J5 h" G2 |( w. a7 ^! D
reached 1 cm in diameter and the metastasis
index was calculated for each cohort (n55 per
group). Results represent means6s.e.m.;- n& {% i# K0 S/ E  N
asterisk, P,0.05 using one-tailed Student’s: ~/ O$ O. ~9 F: X- U
t-test. d, Anti-CCL5 neutralizing antibody or
control IgG was administered intraperitoneally$ B. L. @9 \" X: ~0 p. [, U" [
twice weekly in SCID mice bearing MDA-MB-2317 ~# ?7 L/ c! w7 s9 g1 ?8 j% c
(n59) or MDA-MB-2311MSC tumours: i' P* \2 t# V6 c1 c
(n511). Representative lung pictures of the
indicated cohorts are shown. e, Lung metastasis
indices of mice in d. Data shown are: S0 t5 f! ]8 k% x3 u: h
representative of means6s.e.m. Asterisk,
P,0.05 in one-tailed Student’s t-test./ g$ [0 Q% `$ W! B+ a
kinase inhibitor LY294002. Drug concentrations that did not inhibit
the basal motility levels of MDA-MB-231 cells blocked the elevation
of motility induced by ectopic CCL5 expression (Fig. 4g). These2 o9 J1 f% W# q" o3 j% K. S
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 cells9 H9 w& Q3 k0 b$ T# l" ]' T
at sites of dissemination rather than promoting the survival and/or
proliferation of these cells.
Essential role for the CCL5–CCR5 loop
CCL5 acts through three G-protein-coupled receptors, termed
CCR1, CCR3 and CCR5 (ref. 23). CCR5 has been determined to be1 N- r- M) k  J* o6 k# Q: `# U
the main receptor for CCL5 in MDA-MB-231 cells, as inhibition of its2 M) A( I$ e, i  O0 K' Y0 I
surface expression through dominant-negative mutants abrogated3 j( G3 b; e3 c! {/ ?# c
the ability of these cells to respond to CCL5 chemotaxis24. We therefore1 F/ w4 A1 C# e: u  ~$ G
focused our efforts on evaluating the importance of the CCL5–
CCR5 interactions in MSC-induced metastasis.
We confirmed that CCR5 is expressed by MDA-MB-231 cells and
not by MSCs (Fig. 5a), supporting the notion that MSC-derived
CCL5 acts primarily in a paracrine fashion on MDA-MB-231 cells
in the BCC and MSC mixed cell populations described above. To
probe whether the observed MSC-induced metastasis required
CCL5–CCR5 interactions, we inhibited CCR5 expression in MDAMB-$ O# |* G* I$ C6 y
231 cells by more than 85% through shRNA knockdown (ref. 25: X8 n0 z. o" `1 G0 |
and Fig. 5b), and mixed these cells with MSCs before implantation' ?' M$ w: z4 l% S* X* D
into host mice. Indeed, inhibition of CCR5 expression in the BCCs,8 D; ~1 z7 V' p( o! a' P
achieved using either of two different shRNA constructs, abrogated! S6 o9 p" m" T
the ability of MSCs to enhance the metastasis of MDA-MB-231 cells
(Fig. 5c). Furthermore, neutralization of CCL5 protein using intraperitoneal
injections of an anti-human CCL5 monoclonal antibody! H; N, c  K7 q5 U
also abrogated the MSC-induced metastasis by MDA-MB-231 cells
(Fig. 5d, e). In addition, MSCs in which CCL5 expression was inhibited+ N' C- |$ F' u" k) B
by shRNA knockdown failed to promote metastasis of the2 O3 n1 U2 Y7 O8 `* n9 W
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.
Discussion; S8 \, j) v, g* z
Certain models of metastatic progression propose that cancer cell
invasion and metastasis from the primary tumour site are strongly
influenced by contextual signals emanating from the stroma of the0 R4 \2 N9 ]  p  G0 ^
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.) I. O9 A9 V$ A' ~
Indeed, such a model has been proposed previously by others
on the basis of indirect evidence21. Here, we demonstrate that at least  q: h1 k* l! n! \
one mesenchymal cell type, the MSC, can expedite tumour metastasis,0 E6 Z/ n  O  m
and suggest that after primary human carcinomas recruit MSC6 f! @( e" x$ t! ?) y7 l
populations into their midst, subsequent interactions between the
MSCs (or their derivatives) and the BCCs endow the latter with
invasive and metastatic properties." m% M& F2 U4 S. c7 l
Although the recruitment of labelled MSCs to tumour xenografts" X% C  R. Y0 z6 u
has been established in a variety of experimental models of tumorigenesis,: m7 T4 q+ h8 @0 H2 Q0 E6 d
there is currently no available way to quantify with any accuracy! q- y" Z% y$ O+ C2 J) l9 F3 {
the number ofMSCs in actual human tumours, in part because no set$ S8 T2 S* ]" H4 z
of markers has been identified that can uniquely stain these cells without
concomitantly staining other mesenchymal types in the tumourassociated
stroma6. Our demonstration that the stroma derived from
tumour xenografts contained appreciable numbers of murine MSCs
indicates that significant steady-state levels of these cells aremaintained% \: D( k5 Y" k: @; x! p
in developing tumours. Interestingly, the use of CD10—one of the  q1 Q2 S$ I6 [) E5 k( k4 D& f
markers associated withhumanMSCs—to purify cells fromthe stroma
of human primary invasive breast carcinomas yielded a population of
cells that expresses a number of other markers collectively used to! w* ]) p9 J) _
characterize human MSCs (for example, CD44, CD105 and CD106;+ \; V& l. ]# I% r/ b
Fig. 6a). This suggested that, similar to tumour xenografts, human1 t" d3 [5 m+ w! d. @
carcinomas also acquire significant numbers of MSCs. Furthermore,
we note that CCL5, which is prominent in the stromal gene expression( a3 ?; r) ~' `* d
signature associated with poor prognosis of breast cancers26 (SFT;) B( A- E; {* B" m& O# i
Fig. 6b, c), is also enriched in the leukocyte- and endothelial cell-free5 Q5 B( z- B3 ^2 U+ ~
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 stromal
CCL5 levels, MSCs and human invasive breast cancers.
c
STT1969B. K1 J( f" d% |7 L2 j& g! Q
STT3126
STT3124
STT656B
STT1968B6 k5 x& x' J( X1 V* s
STT3122
STT3053: p! o  Z9 |0 o- ?2 C- ?' {# ~
STT1986B9 t2 T9 i6 C2 P) S' d' ?* c
STT854
STT31250 D0 z, w3 k- P. F4 g, N; C
STT1975# w* t2 }( Q# o6 ^. [/ l
STT1987B
STT10794 Q/ ~+ ], v& E, e
STT638& l) k/ G  ~6 ]( _6 ^7 `- ]* Y
STT1774
STT1984
STT1737C
STT3068$ P/ `0 A* ^# ^0 M/ J0 t
STT3120; Y! K" @" }2 J8 J$ B6 F3 v
STT850" q' F: I/ g& E5 G
STT417B
STT3119# T+ [5 x5 l" V7 J6 r; @% ^& r1 M- |4 y
STT17761 w# {$ ^5 U2 _( h: F, u  c5 X
STT1777B
STT689B. q% x7 y5 U3 X& U/ r# K: k# m
STT1971
STT597
STT626
STT154
STT2774
STT1966
STT2776# d2 d# N+ b8 s, @1 k
STT2775" f7 j" ~8 L* Z9 V7 ~: V
STT2772
STT1637
STT1220C$ X, d( j8 c7 M) A. o  P
STT-094B-1, k3 w5 \2 N: R# J. w- q9 M1 h) x
STT675
STT2770; d! Y- S: s5 d. q- Q- M( k9 z
STT695B
STT1771
STT1778' x5 e4 V; F2 l% ]5 O# S9 n  N7 P3 |
STT491
STT1823. p' A+ U5 ~, P) a
STT200C
STT741B
STT335C
STT709B$ M' C9 D) q7 l9 K
STT516D2 H& b. X0 B/ M( V& X
STT607B( X, T$ B4 `& S) F7 V3 {5 W. _
STT680B
STT1148B/ g7 ~; K- T9 U' [. i
STT523B
STT526E
STT742F
e
CD13
CD29
CD44
CD49e6 V! Y0 i$ K# C, y! ]* c0 b. Q
CD54( ]' T/ Q$ O$ ^3 w6 S
CD599 b- _! F6 ]$ A. q0 ?
CD63
CD105  i+ ^4 w6 D7 x  ^
CD106
Nestin7 _$ y( W. H& g/ w" S; T
HAS2: ^* S8 j' a; {9 t4 f9 D! L
IGF2$ j# n3 S, p* H. J+ \
PLAU: P" N9 D0 R- d) f
TIMP1; {% W( }$ Z/ M7 @$ }. g
CAV19 f4 a' X' c4 b! z6 n
IDC-7- S8 J# y. G1 b/ a! C! F8 i9 H
T112603( I) H$ F& S7 b
T392303/ s3 L$ S. C. j1 H  l
Normal Invasive
CCL5
log2 ratios. C* H1 X2 n8 l" [9 x" s
–2.0 –1.4 –0.9 –0.3 0.3 0.9 1.4 2.0! E" v3 ^; N4 q6 M* D
log2 ratios5 ]- I9 U3 H& q9 s$ P& T: v& |5 F" A
d
–25 R3 a! W" ]: c  S0 m9 d7 N. M
–1+ e+ P- `$ s' v: c
0 1 2* T( [- N  \; h' s7 F4 c* J
–2.00 n3 T5 t8 ]* s9 B3 i8 e
–1.5; B' R# H9 b1 Z8 D
–1.01 t% ?5 w( G) x$ [3 X/ X
–0.5
0.0
DTF SFT5 {0 \" {7 C; J/ m6 S; D7 N
DTF8 @3 M& l! T0 a: R0 Y
SFT
a b* F5 L% _: A/ P& X- A
F
Figure 6 | Stromal fibroblastic cells of human invasive ductal carcinomas are
rich in MSC markers and overexpress CCL5. a, SAGE TreeView display of' L3 N+ R, C$ H0 |* J- M: }/ j
MSC markers expressed in stromal CD10-positive cells from invasive  w3 ~2 P5 c# p7 |, H& I
tumours27. b, Soft-tissue tumourswere ranked byCCL5 expression26, fromlow: {: e* K+ {3 `8 ]% E* I
(green) to high (red). Wide blocks indicate expression ratios of tumours
classified as desmoid-type fibromatosis (DTF; yellow outline, n510) or  q  B/ G0 M: `- o1 n
solitary fibrous tumours (SFT; blue outline, n513); narrow blocks are other5 S3 y, |4 r( u1 E8 ^. Z
soft-tissue tumours (n532). c, Box plot showing that CCL5 expression is& ^/ h3 A( }. c& p  A% M
higher (P50.004) in SFT than inDTF. The difference in log2 expression ratios" t5 ~$ q5 R" e4 ]5 Q
between SFT and DTF was tested with the Welch’s test. d, CCL5 Affymetrix7 `) P" A/ M6 p% y
gene expression in the stroma of human invasive ductal cancers compared to1 V3 U) B; Z6 I" ?( }# _: I
that in normal cancer-free breast tissue (indicated as ‘Normal’; see Methods).
e, CCL5 expression is mostly restricted to the CD10-positive fibroblastic cells4 I" f1 j. s1 {) H% r  h! @7 u) B
derived from invasive ductal cancers. The heatmap shown is a cluster of% M- N# G% \9 t2 ?/ g* [
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-
231 cell interactions, CCL5 seems to have an equally critical involvement) a! @! p0 W! b5 ^' v. i8 ^( s3 h
in the functional interaction of MSCs with MDA-MB-435
human BCCs. CCL5 levels accumulate synergistically when the two
cell types are co-cultured together (Supplementary Fig. 10a), and
MSCs in which CCL5 expression was compromised by shRNA knockdown
failed to promote metastasis by MDA-MB-435 cells to which
theywere admixed (Supplementary Fig. 10b).With these facts in mind,
we point out that CCL5 does not seemto be involved in regulating the
MSC-induced metastasis of MCF7/Ras or HMLER cells, which may
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! |0 J5 I/ Y: a: Y" [0 Q# W1 Z6 y
the possible utility of a variety of CCL5 analogues and CCR5 antagonists3 o5 s; y5 U  i) T3 B# u2 \( K
currentlyused in anti-HIVtherapy30 in treatingmetastatic disease.( [% S6 |. y" a/ L$ p! q3 f. W
Notably, we have observed that MSCs induce the metastasis of cells
to the lung that are, on isolation and re-injection into recipient mice,% C1 W9 h5 D& J- `/ I
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$ q6 H* C8 s5 m- m$ r
stromal cells. If extended to other tumour types, the present results2 }+ G& X1 T) t. c
hold important implications for the molecular analysis of malignant
progression. They suggest that many of the cellular functions associated1 Q8 T; r3 `/ w& T# v
with invasion and metastasis are often not expressed constitutively2 d/ {0 F8 s) o5 X1 T
by carcinoma cells, but rather only transiently in response to) T0 c7 a9 n3 W, _
contextual signals that tumour cells receive from their stromal microenvironment.
If so, analysis of the gene expression patterns of bulk
primary tumour populations may fail to detect the expression of key! k; N+ a. J. `! S% M& O5 q
genes mediating invasiveness and metastasis, if only because they are- s$ J: O$ ]: P1 K* [% y
being transiently expressed in minor subpopulations of cells within( O* M( R2 S* Z: F' _. _
such tumours. Additionally, attempts at determining the metastatic: K1 U  M' n: D4 S" R
propensities of tumours may need to be focused on the genes and
proteins that confer responsiveness of primary tumour cells to stromal& ?( G: v. f# U5 f8 m
signals, rather than on the genes and proteins that directly mediate# V7 `$ H3 {9 j, Y( F
the cellular phenotypes of invasion and metastasis.' z/ \( R# d: |9 I9 o
METHODS SUMMARY
Cells labelled with GFP or ds-red, or harbouring various overexpression or
shRNA constructs, were generated by viral transduction followed by FACS" x) C5 h2 a6 o1 C9 S3 g
enrichment or antibiotic selection. Xenograft experiments were conducted in
nude or NOD/SCID mice and metastasis was estimated using fluorescence
microscopy. The levels of cytokines, growth factors and chemokines were
assessed by immunoassays. Migration and invasion assays were conducted using
transwell chambers. Antibody treatment of tumour-bearing mice was conducted
by intraperitoneal injections. See Methods for detailed information regarding* x/ H3 D& F, S4 ], o
cell culture, viral infections, in vivo colonization and extravasation assays, RT–
PCR, TUNEL and anoikis assays, immunohistochemical and immunofluorescence
determinations, western blotting, and antibodies used.
Full Methods and any associated references are available in the online version of$ l  r! z9 v; G: Z- |4 m) W2 H5 ?
the paper at www.nature.com/nature.

收藏0 分享0 顶0 踩0