干细胞---Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cel - 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 ... 23 下一页

返回列表

查看: 373457\|回复: 227	go 干细胞---Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cel [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 17:12 |只看该作者 |倒序浏览 |打印

ORIGINAL ARTICLE( q1 i! j& M2 t/ n7 N# U
Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on
in vivo tumor growth" B" m% C9 P6 j, l- {: P+ ~8 [  ?' C
R Ramasamy1, EW-F Lam2, I Soeiro2, V Tisato1, D Bonnet3 and F Dazzi14 k$ n8 S) d7 `1 R( Q
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,& a  M8 I: ?, x1 `
London, UK and 3Cancer Research-UK, London Research Institute, London, UK
Mesenchymal stem cells (MSC) have received much attention in' W. o- f% w1 R6 Z0 Z0 K
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, p0 l6 _! N$ ~2 i7 a3 s
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 was
accompanied by a reduction in the apoptotic rate even when( V2 y8 \1 B. [2 V$ G
survival factors were limiting. However, when tumor cells were
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
explain the discrepancy between the in vitro and in vivo# W; S0 g5 P! n  }" L
behavior, we suggest that MSC have the ability to form a
cancer stem cell niche in which tumor cells can preserve the
potential to proliferate and sustain the malignant process. We# t# h% ~$ f- {0 ^9 d
conclude that the clinical use of MSC in conditions in which a
malignant disease is involved should be handled with extreme
caution.
Leukemia (2007) 21, 304–310. doi:10.1038/sj.leu.2404489;
published online 14 December 2006
Keywords: mesenchymal stem cells; tumors; cell cycle; apoptosis
Introduction
Mesenchymal stem cells (MSC) constitute a rare non-hematopoietic: e- v$ M6 V0 f1 D. S/ ~: l
population in the adult bone marrow (BM), which can be: G5 C* W/ s- k; X! P* D
defined according to its ability to self-renew and differentiate
into tissues of mesodermal origin (osteocytes, adipocytes,4 p, ^+ M. u+ ]" l
chondrocytes).1,2 They are progenitors of bone marrow stroma
and thus play a crucial role in supporting hematopoiesis3,4 by
providing hematopoietic progenitors, the necessary cytokines5 Y( A+ v9 `- z0 f& d, W& @" ?
and cell contact-mediated signals to self-renew and/or differentiate.
5 It has also been widely demonstrated that MSC exhibit2 i) b' p  Q- ^8 k- U$ I1 m* j. [" ~3 v! u
a potent immunosuppressive activity, which targets virtually all
types of immune cells of both lymphoid and myeloid lineage.( N" E& T% {4 \' c0 O) |
There is evidence that such a broad activity results from a3 ~: X5 B( D; H- F# j
selective inhibition of cell cycle at early stages of cell: K& T1 ^& H* a. j; D4 Y
commitment (G0/G1)6 and whereas cell proliferation is vigorously8 d" A7 W! R. z2 F" G& R
reduced, most of immune effectors functions are* g6 t( {# h9 S' \- U# p; }
substantially preserved.! W) \# E' \$ `9 H
Because of these properties, MSC have been tested for
therapeutic applications in the field of hemopoietic stem cell
(HSC) transplantation whereby preliminary evidence suggests5 x, G7 K# l; u+ _+ X5 ^! `
that they improve HSC engraftment7 and suppress graft-versushost1 X! C! ^) E) Q6 p* ^4 m( Y
disease after allogeneic HSC transplantation.8,9 Large
unphysiological numbers of MSC are apparently required for
clinical efficacy. As these therapeutic applications often involve8 m3 g5 b+ D6 w# l4 i/ o) _  e* O
malignant conditions, investigating the effect of MSC on tumor# m9 _) O. s# u# F$ {# a
cells is mandatory. Furthermore, such a question becomes, E) Y" S5 ^. i6 J
critical in view of the fact that the development and progression1 n: B6 X4 M- ]2 z
of some tumors depends on the surrounding stroma, which
consists of cells deriving from bone marrow stromal precursors.' h; C5 K5 _) W' |4 Q, Q
Several studies have outlined a direct effect of stromal fibroblasts& p) U5 v& q6 @9 Y2 i; ~; n& N  _" X
in cancer initiation and progression, especially in epithelial
tumors.10,11
Although some studies have observed that these cells inhibit  ~$ X. G& _+ {' z, ~
tumor growth in murine12 and rat13,14 models, others have
demonstrated an opposite effect.15,16 Depending on the system
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$ Z; X. E. D4 P2 ~4 L* r
tumor cells recognition by the immune system.17 Regardless of
the effect on tumor growth and progression, most studies have! {; j- I# d: H! J2 s
documented a selective migration of MSC to the tumor site and
this property has been successfully exploited in animal models
to deliver therapeutic molecules using MSC transduced with8 J8 C8 J7 H' l/ U4 G
specific genes.18) ]( d0 E+ f$ u3 G! Z" J" [3 v
Here, we show that although human MSC exhibit a potent6 w( A4 Y0 a( Z$ z/ p6 D
antiproliferative activity in vitro on different tumor cell lines, this
effect is transient and when assessed in vivo, it results in: p( i5 v4 w. p$ W- R* Y
facilitation of tumor engraftment and growth. Similarly to what
observed for T cells, MSC induce the downregulation of cyclin
D2 and thus halt tumor cells in the G1 phase of the cell cycle.
Such a effect is transient and reduces the proportion of
spontaneous apoptosis associated with proliferation. Our findings' G7 s, X2 s1 d9 b: K  D' D
suggest that MSC may preserve the self-renewal ability of  S# I. w5 R. D9 a: @" T4 s
cancer cells and a new mechanism by which stromal environment9 |/ w! M5 `4 {. {
can influence the course of malignant diseases. The
clinical use of large doses of MSC in the treatment strategies of5 `( S% }5 Y4 X. T" z4 h& }
malignant conditions might therefore favor the establishment of
a tumor niche with long-term proliferative potential.' ?6 e' U1 Q- i
Materials and methods: I  }9 e/ k7 \; {( u4 U
Generation of MSC& K, F$ @4 R0 T3 S5 q: e7 U
Ten to 20 ml of BM suspensions cells were obtained from
normal donors, ranging in age from 20 to 50 years. All samples* b  A+ r- \3 F' q- b$ Y- w- K
were obtained with written, informed consent in accordance$ g' `; G) c' h5 @/ l+ ^1 |
ethical committee requirements. To isolate MSC, ficolled BM/ N# {3 ?) ]8 ~$ A1 x
mononuclear cells (Ficoll-Paque, Amersham-Phamarcia, Piscataway,
NJ, USA) were plated in 25 cm2 flasks (Costar, Cambridge,
MA, USA) at a concentration of 1106/ml in2 R; v8 }" O" t6 e
Dulbecco’s modified Eagle’s medium (DMEM), with high* \% l9 @" }3 P) b0 b
glucose concentration, GLUTAMAX I (Gibco BRL, Gaitherburg,) Z; g- M+ n- L) T
MD, USA), 10% fetal bovine serum (Stem Cell Technology Inc.,
London, UK), 100 U/ml penicillin and 100 mg/ml streptomycin
(Gibco BRL). After 72 h incubation at 371C in a 5% CO21 y6 B6 ^- H: Y& x+ ^
atmosphere, non-adherent cells were removed. When 70–80%9 c# ?0 e) [7 }5 l0 P
confluent, adherent cells were trypsinized and expanded for 3–52 N) r3 n: q7 P
weeks. Before their use in the experiments, MSC were checked5 h9 F& b0 @( V5 m% S
for positivity of CD105, CD106, CD73, HLA-class I, and the
lack of expression of CD45.( ?0 f# ?# N" w0 L6 A) g" M
Tumor cell lines
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 acute9 H3 u6 R- b; P
myelogenous leukemia;21 the Jurkat cell is a human T-cell, M7 v/ e' F7 a- T+ B4 ]
leukemia line22 and COLO 320DM (CC3) is a semi-adherent
colon adenocarcinoma cell line.23 The Epstein–Barr virus -* j- c- q4 `+ C- |, c% M# V) {
infected B cell line wS9-B-LCL/B was provided by G Lombardi7 Y9 M) I+ ^* G4 r
(King’s College, London, UK), whereas the small-cell lung
cancer cell line UCH10 is a kind gift of P Beverley (Edward
Janner Institute, Berkshire, UK). All cells were grown in% G$ a4 E, M2 B& V
Rosewell’s Park Memorial Institute (RPMI) (Gibco, BRL)
supplemented 10% fetal bovine serum (FBS) (Labtech International,
Sussex, UK) and 1% antibiotic/antimycotic solution, y; x% o" `. F: _
(Gibco, BRL). Cells were incubated at 371C in 5% CO2, O6 m2 b- `& _5 H( M3 W
humidified cell culture incubator and fed every 2 days.
Proliferation assays
Cell proliferation assays were performed in round-bottom 96-
well plates (Costar, Cambridge, MA, USA) in a total volume of3 ]. m7 O2 }4 A* i! W
0.2 ml RPMI 1640 supplemented with 10% fetal calf serum
(FCS), GLUTAMAX I (Gibco, BRL, Life Technologies Ltd, UK),& a' x( a, f- k# [$ `
50 U/ml penicillin and 50 mg/ml streptomycin. A total of 0.5 mCi/% ?# K. w0 ~% J  n6 ?, p8 z4 k
well of [3H]-thymidine (ICN, Costa Mesa, CA, USA) was added
after 5 days of culture and the cells were harvested 18 h later  D" n$ R( }! \) ~; p0 N; \
onto glass fiber filters using an LKB 96 well-harvester (Wallac! m4 r0 B0 R7 K( o) W
Oy, Turku, Finland). [3H]thymidine uptake was measured on an
LKB Betaplate counter (Wallac Oy). The results are expressed as
mean count per minute for triplicate cultures (standard errors/ c) C' a$ ^! j( A# [+ L, N7 O
were routinely o10%).
Immunophenotype
For surface marker immunophenotyping, cells were incubated
with the specific monoclonal antibody for 300 at room
temperature and then analyzed after extensive washing with
phosphate-buffered saline (PBS). Background fluorescence was) g* g' S6 A9 v+ ?
subtracted after analyzing unstained cells and cells stained with' G! e, ~" K- ?
the relevant isotype control.
For cell cycle analysis, bromodeoxyuridine (BrDU; Sigma
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, K' U# `8 G3 o# v0 z. X3 ?
(Na2B4O7 10H2O, pH 8.5, Sigma Aldrich). Cells were stained& N% h1 b# ]* q6 D( j- S" I
with 5 ml of anti-BrDU-fluoroscein isothiocyanate antibody; after, Q1 _' l* [! r! X* W2 J4 f& v
30 min, 1ml of PBS containing 5 mg/ml propidium iodide (PI;
Sigma, St Louis, USA) was added before flow cytometry analysis
using a fluorescence-activated cell sortiong (FACS) Calibur
cytofluorimeter (Becton Dickinson, San Jose, CA, USA).* z6 x& a% D- T. l( H7 K  Q
Mice* Q7 f+ w0 u, A. v' l/ r; B5 t# W; f
Non-obese diabetic–severe combined immunodeficient (NOD/  ^, `2 P: i. c7 w2 U* J2 F) n6 Q
SCID) mice used in vivo study were obtained from Jackson# w- n2 }$ H' ^. i: s& E
Laboratories (Bar Harbor, ME, USA), bred and maintained in a/ Z& V' V- l% W8 g: L7 c1 G  [
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
receive any conditioning before receiving the cells that were7 ~( v+ @, W2 i' E/ L* p
administered subcutaneously in a total volume of 0.2 ml sterile, ~1 z, y1 z+ E7 H
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 for
histologic preparations (BM was decalcified in 10% formalin/* }0 ]7 K8 v/ s5 k- `: {+ |
5% formic acid).
Western blotting! [' W. H8 E& c  v3 j: k
Cell suspensions were lysed in Nonidet P-40 lysis buffer (1%
Nonidet P-40, 100mM NaCl, 20mM Tris-HCl pH 7.4, 10mM
NaF, 1mM sodium orthovanadate, 30 nM Na-glycerophosphate)0 k% U4 ~! n* j2 m" g& W, P
and protease inhibitors (Roche Applied Science, Basel, Switzerland)0 j5 h; i6 }- C3 b, ]' c
in ice for 15 min. Protein concentration was determined by0 w# T( o) @, m8 I( J. `) |* r) b
Bio-Rad Dc protein assay (BioRad Lab Ltd, Hertfordshire, UK).4 r2 A% q* I$ ?# I; `
Twenty five micro grams of proteins were electrophoretically8 p# c' _* S/ g% z5 Q' L
separated by 7 and 10% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) gels (Invitrogen-Novex, Carlsbad, CA, USA),
transferred onto Protran Nitrocellulose transfer membranes
(Schleicher and Schnell) and the membranes were incubated; N& O. S" ?4 v9 n" `  q- e. X
with the following primary antibodies: cdk4, cyclin D2, cyclin3 {4 W$ |" ?; g) S% s
E, cyclin A, p27Kip1 and actin as control (Santa Cruz0 X6 O6 @6 Z9 L* A0 d
Biotechnology, Inc., Santa Cruz, CA, USA). The immune( ]2 y2 _/ r  W0 Y2 b1 d/ n
complexes were detected using horseradish peroxidase-linked
anti-mouse or anti-rabbit conjugates as appropriate (DAKO,. ~1 _! A) K8 Q
Glostrup, Denmark) and visualized using enhanced chemiluminescence" v, \! l1 c& [4 t2 y! ?
detection system (Amersham Biosciences, Amersham,
UK).
Results; J: v1 T2 R& j- d
MSC inhibit the proliferation of malignant cells of. Y' h6 P3 h  z. w4 N
hematopoietic and non-hematopoietic origin
We studied the effect of MSC on the proliferative activity of4 S& q* H5 \# z
malignant cells of different lineages. Tumor cell lines of
hematopoietic (BV173, K562, Jurkat, KG1a and wS9-B-LCL)
and non-hematopoietic (UCH10 and CC3) origin were cultivated,
at different ratios, in the presence of MSC and tested for9 ]7 n, E( e4 P% X3 _  F. c. k
their proliferative activity after 3 days of co-culture. MSC
exhibited a dose-dependent antiproliferative effect on all cell( z3 Z! L7 W# }# c  {1 @9 m& s
lines investigated (Figure 1a and b).
Soluble factors are involved into the anti-proliferative- C# a6 I1 u; d8 q5 E4 N) F, y: F
effect exerted by MSC
It has been shown that soluble factors contribute to the( n3 G- p* _8 P& R) Z# W
immunosuppressive effect of human MSC.24,25 To examine
whether the MSC-induced inhibition of tumor cells proliferation1 X( Z3 I7 H; O* m8 o5 L7 `* n; z1 x
was mediated by soluble factors, MSC were cultured physically. Y" u5 I8 b. \9 ~
separated from BV173 cells using a transwell system or replaced6 @7 C5 j- c! F& _, D2 y5 q
by their culture supernatants. An inhibitory effect was detected2 }, M0 G3 g8 H0 Z
MSC influence tumor cell proliferation and apoptosis/ m8 k! o0 r. o6 N* o9 A
R Ramasamy et al' j7 V( r: h; N. G: h3 p9 \9 i
305, Q, E3 }/ V, C( R, M5 I. M
Leukemia+ I& G7 W; r$ d# ~8 A
tumor proliferation (data not shown), thus reasonably excluding" \$ V7 b$ k9 F3 D' M7 L) m
a role of TGFb in the MSC mediated inhibitory effect.
MSC favor tumor growth in vivo7 W5 ]+ b4 z9 y- Y
To investigate the effect of MSC on the in vivo growth of tumor0 E" z' h( t- m
cells, we assessed in NOD-SCID mice the kinetic growth of
tumor cells in presence of MSC. Mice received 106 BV173 cells5 S! \$ H" K. _2 g
with or without 0.5106 MSC by subcutaneous injection. After
8 weeks, in three different experiments, the 75% of the mice coinjected- f! |* q3 E9 r) h
with BV173 and MSC developed tumors at the site of7 n) V: p# m# v$ w- ?! V
injection, whereas only the 12% of animals receiving BV173& P/ N" `& n5 q0 A( H6 Z
alone showed signs of tumor growth (Figure 2a). The tumor cell" C0 T$ W% o1 l3 q
suspensions expressed the phenotype of human B cells and did; ?/ n) N* u" t+ C; H5 _! w5 z
not contain any detectable levels of MSC as assessed by CD105
staining (Figure 2b). BM from all mice was finally evaluated for% X* c4 {1 [9 S1 t
the engraftment of tumor cells and MSC. Of the mice receiving" w# Z5 D6 H- o
MSC, only those which developed the tumor showed a small% f9 P9 q$ ^" v
proportion of MSC in their BM as identified by the co-expression
of CD105 and human major histocompatibility complex (MHC)
class I. No presence of tumor cells (CD19þ/human MHC class+ K3 ]  t2 C. b" q' F, x& k1 e4 {
Iþ coexpression) was detected in the BM of any of the animals,
irrespective of whether they had developed the tumor  a8 j1 j1 N$ F, E! p
(Figure 3a). However, when BM cells were cultivated for 28 W4 ]1 n5 }1 B
weeks, a population with the phenotypic features of BV173 took; M7 V) y, ?/ b
over (Figure 3b). At subsequent analysis, these cells exhibiting
indefinite self-renewal ability in vitro.8 p. J: P  E. v: R2 w8 u- L
MSC transiently arrest tumor cells in the G1 phase of the7 M% R( E  M( I
cell cycle
In order to explain the discrepancy between the in vitro and+ z2 m4 {$ |, t; n+ Q
in vivo findings, we characterized the effect of MSC on the cell

收藏0 分享0 顶0 踩0