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ORIGINAL ARTICLE, u+ o5 O6 y: D; T1 ?
Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on3 R2 C% O8 j! |
in vivo tumor growth- [0 m; T+ @% ~- |) Q) X$ W% T
R Ramasamy1, EW-F Lam2, I Soeiro2, V Tisato1, D Bonnet3 and F Dazzi1
1Stem Cell Biology Section, Kennedy Institute of Rheumatology and Division of Investigative Sciences, Imperial College Faculty of
Medicine, London, UK; 2Cancer Research UK Labs, Department of Cancer Medicine, Imperial College Faculty of Medicine,: P' [9 Q. I# B# K
London, UK and 3Cancer Research-UK, London Research Institute, London, UK& {% V1 M/ X6 f% i, a6 k- q
Mesenchymal stem cells (MSC) have received much attention in% X' V; R+ y8 c- }/ g7 U/ O4 f
the field of hematopoietic stem cell transplantation because not
only do they support hematopoiesis but also exhibit a profound
immunosuppressive activity that can be exploited to prevent
undesired alloreactivity. We have previously shown that their
immunosuppressive activity is mainly exerted at the level of
T-cell proliferation. Here, we show that MSC exhibit a similar
antiproliferative activity on tumor cells of hematopoietic and
non hematopoietic origin. In vitro, MSC produced the transient
arrest of tumor cells in the G1 phase of cell cycle; this was3 P9 I1 t  g8 J$ ~
accompanied by a reduction in the apoptotic rate even when
survival factors were limiting. However, when tumor cells were1 [: N5 m4 g  N+ A) |: d" T
injected into non-obese diabetic–severe combined immunodeficient
mice in conjunction with MSC, their growth was much
faster as compared to the group receiving only tumor cells. To* a& A( G5 k7 T3 D
explain the discrepancy between the in vitro and in vivo
behavior, we suggest that MSC have the ability to form a( E( f: d7 I2 K. M. a
cancer stem cell niche in which tumor cells can preserve the% x; c5 y! n& G  i) ^, q8 w. J- ]
potential to proliferate and sustain the malignant process. We
conclude that the clinical use of MSC in conditions in which a& J* a0 _) s( w  ~6 x1 [
malignant disease is involved should be handled with extreme  Z" E8 T- y9 p' ]& K' P& P; l
caution.
Leukemia (2007) 21, 304–310. doi:10.1038/sj.leu.2404489;. a! l" r. [/ _7 p3 B" n
published online 14 December 2006
Keywords: mesenchymal stem cells; tumors; cell cycle; apoptosis/ A0 s6 D: b, X
Introduction
Mesenchymal stem cells (MSC) constitute a rare non-hematopoietic
population in the adult bone marrow (BM), which can be/ }" f) w  ?5 j9 d3 O: K, x' {( F: _
defined according to its ability to self-renew and differentiate! N) @' P! V' N$ r3 K
into tissues of mesodermal origin (osteocytes, adipocytes,
chondrocytes).1,2 They are progenitors of bone marrow stroma
and thus play a crucial role in supporting hematopoiesis3,4 by0 d( D& X0 C* [6 {& y2 e& r' ~
providing hematopoietic progenitors, the necessary cytokines' w! ^8 ~. A. ~- S1 j) z2 |
and cell contact-mediated signals to self-renew and/or differentiate., p# K4 J( m8 {* \+ _4 h
5 It has also been widely demonstrated that MSC exhibit  J# R* N9 e$ E% \1 X
a potent immunosuppressive activity, which targets virtually all
types of immune cells of both lymphoid and myeloid lineage.$ B! E- X' a7 l2 P1 Q5 Q9 }
There is evidence that such a broad activity results from a8 O7 b* j# J( ?
selective inhibition of cell cycle at early stages of cell3 p+ `8 z' t' X; F' t& [
commitment (G0/G1)6 and whereas cell proliferation is vigorously( u$ p' D& X% v+ Z# v$ ?6 @- G# w
reduced, most of immune effectors functions are4 f) Y% x7 ?4 \' ^' Y
substantially preserved.
Because of these properties, MSC have been tested for# F  l6 F  P; p
therapeutic applications in the field of hemopoietic stem cell
(HSC) transplantation whereby preliminary evidence suggests, g0 N1 o6 I: G& @4 c5 ?( ?3 i5 F
that they improve HSC engraftment7 and suppress graft-versushost
disease after allogeneic HSC transplantation.8,9 Large
unphysiological numbers of MSC are apparently required for
clinical efficacy. As these therapeutic applications often involve
malignant conditions, investigating the effect of MSC on tumor: E5 B# f8 ~5 o5 o1 E- M2 c( I0 C: d
cells is mandatory. Furthermore, such a question becomes
critical in view of the fact that the development and progression  ~* O, h5 u$ q! o
of some tumors depends on the surrounding stroma, which' H, I4 p/ n, O. g, V/ W
consists of cells deriving from bone marrow stromal precursors.
Several studies have outlined a direct effect of stromal fibroblasts8 C- \6 |, F2 [
in cancer initiation and progression, especially in epithelial
tumors.10,11
Although some studies have observed that these cells inhibit
tumor growth in murine12 and rat13,14 models, others have
demonstrated an opposite effect.15,16 Depending on the system: t4 \' x9 D8 E9 t- n
used, MSC have been shown to favor tumor growth either by
promoting their invasive abilities via the activation of matrix
metalloproteinases15 and neoangiogenesis16 or by preventing
tumor cells recognition by the immune system.17 Regardless of6 Q+ p* t6 F$ r/ w) j
the effect on tumor growth and progression, most studies have
documented a selective migration of MSC to the tumor site and% F; q2 p+ d( \2 }
this property has been successfully exploited in animal models
to deliver therapeutic molecules using MSC transduced with
specific genes.18
Here, we show that although human MSC exhibit a potent
antiproliferative activity in vitro on different tumor cell lines, this
effect is transient and when assessed in vivo, it results in9 k( c' \7 ~; q) d* }
facilitation of tumor engraftment and growth. Similarly to what$ I5 [/ o, ]+ [4 B
observed for T cells, MSC induce the downregulation of cyclin$ h- |+ Z) E1 R
D2 and thus halt tumor cells in the G1 phase of the cell cycle.
Such a effect is transient and reduces the proportion of, h  Z/ ]/ y4 }% c
spontaneous apoptosis associated with proliferation. Our findings
suggest that MSC may preserve the self-renewal ability of
cancer cells and a new mechanism by which stromal environment  U& E. c; t: \+ q& R3 }3 N
can influence the course of malignant diseases. The
clinical use of large doses of MSC in the treatment strategies of
malignant conditions might therefore favor the establishment of7 n2 H# j1 e' G# Q2 K2 M" u; E6 j& V
a tumor niche with long-term proliferative potential.9 a, L1 i' z  k/ A" f
Materials and methods* m4 k  z3 {4 F  q" G& g) C
Generation of MSC) ]1 o; P. S. A  H1 @; {& X
Ten to 20 ml of BM suspensions cells were obtained from
normal donors, ranging in age from 20 to 50 years. All samples
were obtained with written, informed consent in accordance
ethical committee requirements. To isolate MSC, ficolled BM
mononuclear cells (Ficoll-Paque, Amersham-Phamarcia, Piscataway,% W' b1 p) H2 d6 U/ Q( S
NJ, USA) were plated in 25 cm2 flasks (Costar, Cambridge,1 v  ]. O. ^: F# r
MA, USA) at a concentration of 1106/ml in
Dulbecco’s modified Eagle’s medium (DMEM), with high
glucose concentration, GLUTAMAX I (Gibco BRL, Gaitherburg,
MD, USA), 10% fetal bovine serum (Stem Cell Technology Inc.,& ]: A; o; h9 U$ D" {
London, UK), 100 U/ml penicillin and 100 mg/ml streptomycin
(Gibco BRL). After 72 h incubation at 371C in a 5% CO2. @) q4 x7 ~) t( h# ?3 e& Z# M4 F
atmosphere, non-adherent cells were removed. When 70–80%
confluent, adherent cells were trypsinized and expanded for 3–5
weeks. Before their use in the experiments, MSC were checked
for positivity of CD105, CD106, CD73, HLA-class I, and the
lack of expression of CD45.
Tumor cell lines! r: v, Z# O* l0 z, d6 [0 s. b
BV173 is derived from a lymphoid blast crisis of chronic
myeloid leukemia (CML);19 K562 is an undifferentiated erythroleukemia
cell line derived from a CML in blast crisis;20
KG1a is an undifferentiated blast cell line from acute( t1 P  E' H3 E: S
myelogenous leukemia;21 the Jurkat cell is a human T-cell
leukemia line22 and COLO 320DM (CC3) is a semi-adherent
colon adenocarcinoma cell line.23 The Epstein–Barr virus -
infected B cell line wS9-B-LCL/B was provided by G Lombardi
(King’s College, London, UK), whereas the small-cell lung5 B$ H' t7 t0 {
cancer cell line UCH10 is a kind gift of P Beverley (Edward
Janner Institute, Berkshire, UK). All cells were grown in. H/ v8 h$ y' |# k
Rosewell’s Park Memorial Institute (RPMI) (Gibco, BRL)
supplemented 10% fetal bovine serum (FBS) (Labtech International,
Sussex, UK) and 1% antibiotic/antimycotic solution6 d) V) g( s, b" |
(Gibco, BRL). Cells were incubated at 371C in 5% CO2
humidified cell culture incubator and fed every 2 days.
Proliferation assays
Cell proliferation assays were performed in round-bottom 96-) y2 @. }, D# p, b
well plates (Costar, Cambridge, MA, USA) in a total volume of
0.2 ml RPMI 1640 supplemented with 10% fetal calf serum6 D/ i! X; ~' B9 J& |
(FCS), GLUTAMAX I (Gibco, BRL, Life Technologies Ltd, UK),
50 U/ml penicillin and 50 mg/ml streptomycin. A total of 0.5 mCi/
well of [3H]-thymidine (ICN, Costa Mesa, CA, USA) was added: C3 M8 Q2 b; h
after 5 days of culture and the cells were harvested 18 h later0 ?. j4 \: Q2 s0 ?+ s6 ?
onto glass fiber filters using an LKB 96 well-harvester (Wallac
Oy, Turku, Finland). [3H]thymidine uptake was measured on an
LKB Betaplate counter (Wallac Oy). The results are expressed as5 t1 z; C( t8 r' }/ d( `
mean count per minute for triplicate cultures (standard errors' c6 Q+ h+ d( M: v/ B  X
were routinely o10%).. C& E7 _1 q4 g: m3 C
Immunophenotype
For surface marker immunophenotyping, cells were incubated- L7 g4 E; i5 g- W# s2 _
with the specific monoclonal antibody for 300 at room
temperature and then analyzed after extensive washing with
phosphate-buffered saline (PBS). Background fluorescence was, s3 ]( L5 q- N, {" U
subtracted after analyzing unstained cells and cells stained with8 _$ p) ?9 O0 \- L
the relevant isotype control.
For cell cycle analysis, bromodeoxyuridine (BrDU; Sigma7 ?( |! B, L/ g
Aldrich, St Louis, MO, USA) was added to cell cultures for 1 h
before cell harvest and fixed in 70% ethanol. Fixed cells were
treated with 0.5% Triton-X-2M HCl (Sigma Aldrich) for 30 min
to denature the DNA and neutralized by sodium tetraborate
(Na2B4O7 10H2O, pH 8.5, Sigma Aldrich). Cells were stained
with 5 ml of anti-BrDU-fluoroscein isothiocyanate antibody; after% S7 u% w( i6 s, A0 n. y6 [
30 min, 1ml of PBS containing 5 mg/ml propidium iodide (PI;* A" c8 x# L3 B8 R( z$ C. ?; s0 t9 P
Sigma, St Louis, USA) was added before flow cytometry analysis/ a5 s+ ^% P+ |% _# b+ r- i0 @
using a fluorescence-activated cell sortiong (FACS) Calibur
cytofluorimeter (Becton Dickinson, San Jose, CA, USA).
Mice  F0 m3 A% j7 ^! W6 C3 v4 ]
Non-obese diabetic–severe combined immunodeficient (NOD/3 ~/ M1 l  f; s# M5 [) b
SCID) mice used in vivo study were obtained from Jackson
Laboratories (Bar Harbor, ME, USA), bred and maintained in a* B! Z! e% `4 D( W: N' I
pathogen-free environment at Cancer Research UK Laboratories.
Mice used were between 6 and 10 weeks of age and all
procedures were carried out in accordance with the Home
Office Animal (Scientific Procedures) Act of 1986. Mice did not  Z1 D! O; s+ ~9 g, R1 z4 J1 j
receive any conditioning before receiving the cells that were
administered subcutaneously in a total volume of 0.2 ml sterile9 O6 ]! L9 a) b" h' k
phosphate-buffered saline (PBS). At autopsy, spleen, liver, BM,
lymph nodes and the tumor (when applicable) were removed
and fixed in 10% neutral buffered formalin solution for3 x8 C2 @9 \' ]+ W& J& O
histologic preparations (BM was decalcified in 10% formalin/. }9 q9 {; D* z* U& r; |
5% formic acid).
Western blotting
Cell suspensions were lysed in Nonidet P-40 lysis buffer (1%
Nonidet P-40, 100mM NaCl, 20mM Tris-HCl pH 7.4, 10mM9 t( c8 ^$ }3 }6 S  \
NaF, 1mM sodium orthovanadate, 30 nM Na-glycerophosphate)/ s; [" C$ p- c$ n6 J* C% \2 M
and protease inhibitors (Roche Applied Science, Basel, Switzerland)# C, `9 o$ D8 N6 e, D/ ]
in ice for 15 min. Protein concentration was determined by
Bio-Rad Dc protein assay (BioRad Lab Ltd, Hertfordshire, UK).( r1 @4 c, @. l( {3 A( c8 P' l* w
Twenty five micro grams of proteins were electrophoretically
separated by 7 and 10% SDS-polyacrylamide gel electrophoresis4 [. |) K3 B+ h2 d
(SDS-PAGE) gels (Invitrogen-Novex, Carlsbad, CA, USA),2 [0 y$ H5 ^4 e
transferred onto Protran Nitrocellulose transfer membranes
(Schleicher and Schnell) and the membranes were incubated
with the following primary antibodies: cdk4, cyclin D2, cyclin
E, cyclin A, p27Kip1 and actin as control (Santa Cruz; X8 N2 W+ K" X3 _
Biotechnology, Inc., Santa Cruz, CA, USA). The immune
complexes were detected using horseradish peroxidase-linked+ J) \/ ]2 e3 Q
anti-mouse or anti-rabbit conjugates as appropriate (DAKO,5 S0 ?( @$ d" w. U( g+ B8 V; R# Y2 g
Glostrup, Denmark) and visualized using enhanced chemiluminescence+ O& x5 K0 R( L- N
detection system (Amersham Biosciences, Amersham,: q- N+ @9 I4 X3 d# e* z
UK).
Results- [4 a# u+ o' L
MSC inhibit the proliferation of malignant cells of4 O/ l) X& H3 M& W
hematopoietic and non-hematopoietic origin2 S* m3 I. a6 R" B9 G5 @* O( K) f
We studied the effect of MSC on the proliferative activity of
malignant cells of different lineages. Tumor cell lines of9 y% C4 G  Q, \/ U1 d9 H8 L
hematopoietic (BV173, K562, Jurkat, KG1a and wS9-B-LCL)) Q# N! I1 L$ Z: Y6 c
and non-hematopoietic (UCH10 and CC3) origin were cultivated,; L! F2 P2 T  ~2 o0 o; c
at different ratios, in the presence of MSC and tested for  ~# k5 J* t: d' Y" F1 j4 Z4 u6 P
their proliferative activity after 3 days of co-culture. MSC# R3 y& Y- T7 ]9 a
exhibited a dose-dependent antiproliferative effect on all cell
lines investigated (Figure 1a and b).
Soluble factors are involved into the anti-proliferative
effect exerted by MSC
It has been shown that soluble factors contribute to the
immunosuppressive effect of human MSC.24,25 To examine8 `5 A% \8 T9 F, f" f3 R
whether the MSC-induced inhibition of tumor cells proliferation
was mediated by soluble factors, MSC were cultured physically
separated from BV173 cells using a transwell system or replaced
by their culture supernatants. An inhibitory effect was detected
MSC influence tumor cell proliferation and apoptosis& M3 }, O' J& ~
R Ramasamy et al
305
Leukemia
tumor proliferation (data not shown), thus reasonably excluding9 l5 ], k# L! j1 O
a role of TGFb in the MSC mediated inhibitory effect.
MSC favor tumor growth in vivo
To investigate the effect of MSC on the in vivo growth of tumor; V$ j0 Y! t" a( `  _+ W7 a6 ?  p  I
cells, we assessed in NOD-SCID mice the kinetic growth of* U& S* C3 E+ ^$ g: |
tumor cells in presence of MSC. Mice received 106 BV173 cells3 F3 K3 o. R: J( G
with or without 0.5106 MSC by subcutaneous injection. After, H. f* B  G4 w+ ]- ~- s8 E3 m
8 weeks, in three different experiments, the 75% of the mice coinjected) E$ i/ h. i0 M* H
with BV173 and MSC developed tumors at the site of
injection, whereas only the 12% of animals receiving BV173
alone showed signs of tumor growth (Figure 2a). The tumor cell3 `: c9 k8 o) w9 L) {
suspensions expressed the phenotype of human B cells and did3 h3 `1 l+ ^. y" T
not contain any detectable levels of MSC as assessed by CD105
staining (Figure 2b). BM from all mice was finally evaluated for
the engraftment of tumor cells and MSC. Of the mice receiving  V9 }( t# t6 X& d# A" P1 ]
MSC, only those which developed the tumor showed a small
proportion of MSC in their BM as identified by the co-expression
of CD105 and human major histocompatibility complex (MHC); X% Z$ D( i! H+ p
class I. No presence of tumor cells (CD19þ/human MHC class
Iþ coexpression) was detected in the BM of any of the animals,
irrespective of whether they had developed the tumor! S7 k2 M0 P; v8 U( M) j, ^' p
(Figure 3a). However, when BM cells were cultivated for 2
weeks, a population with the phenotypic features of BV173 took
over (Figure 3b). At subsequent analysis, these cells exhibiting/ w- ]; X% Z/ m
indefinite self-renewal ability in vitro.2 [- ?% m  `+ U* @& L/ \
MSC transiently arrest tumor cells in the G1 phase of the( L8 M; y# q2 U* f; H+ e7 ~
cell cycle( r( b$ r9 ]( @$ u+ U- f9 g9 K
In order to explain the discrepancy between the in vitro and7 E9 U; h. [6 i' h. k& w' H, v2 _! e
in vivo findings, we characterized the effect of MSC on the cell

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