· web viewthe animal experiment aimed to clarify whether or not tgfβ1-smad pathway was involved...
TRANSCRIPT
Mesenchymal Stem Cells Accelerate the Remodeling of Bladder VX2 Tumor
Interstitial Microenvironment by TGFβ1-Smad Pathway
Jun Chen1,2, Qingya Yang2, Yaofeng Zhu1, Zhishun Xu1
1Department of Urology, Qilu Hospital, Shandong University, Jinan 250012, China
2Department of Urology, Qilu Hospital(Qingdao), Shandong University, Qingdao
266035, China. These two authors contribute equally.
Corresponding author: Jun Chen, Department of Urology, Qilu Hospital, Shandong
University, 107 Wenhuaxi Road, Jinan 250012, China; [email protected]
Abstract
1
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
23
Background: Mesenchymal stem cells (MSCs) have been proved to be able to
differentiate into cells that are conducive to tumor growth and invasion. The
mechanism is not clear. This present study was aimed to clarify whether or not
TGFβ1-Smad pathway was involved in this process.
Methods: For the in vitro experiment, five groups of MSCs were cultured to test
whether or not VX2 culture supernatant could induce the differentiation of MSCs into
myofibroblasts. And then transforming growth factor β1(TGFβ1) receptor or Smad2
of MSCs were blocked with RNA interference technique to test whether or not
TGFβ1-Smad pathway was involved in the above differentiation. In the animal
experiment, different kinds of MSCs were co-inoculated with VX2 cells in bladder to
test whether or not the blockage of TGFβ1 receptor or Smad2 of MSCs could affect
the expression of TGFβ1, epidermal growth factor (EGF), fibroblast activation protein
alpha (FAPa), and matrix metalloprotein 9 (MMP9) in five animal groups.
Results: VX2 culture supernatant could up-regulate the expression of α-SMA and
Vimentin in MSCs, which indicated VX2 culture supernatant could induce the
differentiation of MSCs into myofibroblasts. Blockage of TGFβ1 receptor and Smad2
of MSCs could both lead to decreased expression of α-SMA and Vimentin in MSCs.
In the animal experiment, MSCs could favor VX2 bladder tumor growth and up-
regulate the expression of TGFβ1, EGF, FAPa, MMP9 in VX2 tumor tissue. However,
when TGFβ1 receptor or Smad2 of MSCs were blocked, the above effects were
attenuated.
Conclusions: Under the induction of tumor microenvironment, MSCs can
2
4
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
56
differentiate into myofibroblasts and then affect tumor interstitial microenvironment
remodeling. TGFβ1-Smad2 pathway mediates the process.
Keywords: Mesenchymal stem cells; tumor; stroma remodeling
Introduction
Mesenchymal stem cells (MSCs) are widely present in the body, mainly in the bone
marrow. They have been widely used in tissue engineering, cells and gene therapy.
Regarding to how the target organ induces MSCs’ migration, it is generally thought
that the extracellular signals emitted by the injured organ regulate this process, and
various chemokine receptors expressed by MSCs are closely related to the migration
of MSCs[1, 2]. After the migration of MSCs to target organs, their further differentiation
is characterized by environmental dependence. Different microenvironments can
induce MSCs to different cells[3, 4].
As a rapidly growing and metabolically active disease, tumor can also induce MSCs
to differentiate into cells that are conducive to tumor growth and invasion. Current
research results have indeed confirmed this hypothesis[5]. Studeny et al transferred
adenovirus vectors carrying the β-interferon gene into MSCs and found a large
number of MSCs in tumor tissue[6]. These experimental results show that tumor cells
do have the ability to induce the migration of MSCs. Other related studies also found
3
7
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
89
similar results[7, 8]. Furthermore, studies also found that MSCs could promote tumor
growth and metastasis[9-11].
Our previous research results showed that under the induction of tumor
microenvironment, MSCs could differentiate into myofibroblasts, and further
accelerate the development of tumors by promoting tumor interstitial
microenvironment remodeling[10, 12]. The mechanism of tumor-induced differentiation
of MSCs into myofibroblasts remains unclear. In the study of lysophosphatidic acid-
induced differentiation of MSCs into myofibroblasts, Jeon et al found that blocking
the expression of Smad2/3 would significantly reduce the expression of α-SMA, while
blocking the binding of transforming growth factorβ1(TGFβ1) to its receptor could
reduce the α- SMA expression and Smad2 phosphorylation[13]. This result suggests
that TGFβ1-Smad signaling pathway plays an important role in the differentiation of
MSCs into myofibroblasts. In view of the important role of myofibroblasts in the
remodeling of tumor interstitial microenvironment, blocking TGFβ1-Smad signaling
pathway in MSCs may simultaneously affect the remodeling of tumor interstitial
microenvironment and thus further affect the development of tumor. This study
mainly validated the above hypothesis. And the remodeling of tumor interstitial
microenvironment was validated by testing the expression of TGFβ1, epidermal
growth factor (EGF), fibroblast activation protein alpha (FAPa), matrix metalloprotein
9 (MMP9).
Materials and Methods
The design of the study
4
10
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
1112
MSCs have been proved to be able to differentiate into myofibroblasts and then affect
tumor stroma remodeling, but the mechanism is not clear. This present study was
aimed to clarify whether or not TGFβ1-Smad pathway was involved in this process.
In order to prove the hypothesis, we designed an in vitro experiment and an animal
experiment. The in vitro experiment aimed to clarify whether or not TGFβ1-Smad
pathway was involved in the process that VX2 culture supernatant could induce the
differentiation of MSCs to myofibroblasts. The animal experiment aimed to clarify
whether or not TGFβ1-Smad pathway was involved in the process that MSCs could
affect tumor stroma remodeling.
Animals
Fifty male New Zealand white rabbits (3 months old, weight 1.5-2.0kg) were
purchased from Shandong Academy of Agricultural Sciences. The animal protocols
were approved by the Institutional Animal Care and Use Committee of Qilu Hospital,
Shandong University.
The groups of in vitro experiment
There were five groups for the in vitro experiment, named as control A, control B,
control C, test A, test B, respectively. For group control A, F2 passage MSCs were
cultured in DMEM-LG with 10% calf-serum, 30% VX2 culture supernatant. For
group control B, F2 passage MSCs, which had been transfected by blank liposomes,
were cultured in DMEM-LG with 10% calf-serum, 30% VX2 culture supernatant. For
group control C, F2 passage MSCs were cultured in DMEM-LG with 10% calf-
5
13
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
1415
serum. For group test A, F2 passage MSCs, which had been transfected with siRNA
targeting TGFβ1 receptor by liposomes, were cultured in DMEM-LG with 10% calf-
serum, 30% VX2 culture supernatant. For group test B, F2 passage MSCs, which had
been transfected with siRNA targeting Smad2 by liposomes, were cultured in DMEM-
LG with 10% calf-serum, 30% VX2 culture supernatant. The expression of α-SMA
and Vimentin in MSCs was detected by westernblot 14 days later. 10 samples of each
group, 107 MSCs in each sample, were tested.
The groups of animal experiment
Fifty male New Zealand white rabbits were randomly divided into control 1, control
2, control 3, test 1, test 2, ten rabbits in each group. For group control 1, the cell
suspension, containing 106 autologous MSCs and 106 VX2, was injected into the
bladder submucosa. For group control 2, the cell suspension, containing 106
autologous MSCs transfected by blank liposomes and 106 VX2, was injected into the
bladder submucosa. For group control 3, the cell suspension, containing 106 VX2, was
injected into the bladder submucosa. For group test 1, the cell suspension, containing
106 autologous MSCs transfected with siRNA targeting TGFβ1receptor by liposomes
and 106 VX2, was injected into the bladder submucosa. For group test 2, the cell
suspension, containing 106 autologous MSCs transfected with siRNA targeting Smad2
by liposomes and 106 VX2, was injected into the bladder submucosa.
The animals were sacrificed 4 weeks later. The expression of TGFβ1, EGF, FAPa,
MMP9 were detected by westernblot, and the tumor size was recorded.
6
16
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
1718
MSCs isolation, cultivation and identification
The process of MSCs isolation, cultivation and identification was the same as we
previously described[12]. MSCs with CD34(-), CD44(+) and CD45(-) were used for
study.
VX2 tumor cells isolation and cultivation
The VX2 tumor carrier rabbit was presented by Radiology Department of Qilu
Hospital. The process of VX2 tumor cells isolation and cultivation was the same as
we described previously[12].
Tumor inoculation procedure
We established tumor model by means of injecting mixed cell suspensions under the
bladder mucosa. After the exposure of the urinary bladder through the lower
abdominal incision under sterile conditions, we made a small incision (0.8cm) in the
bladder wall, and inoculated the cell suspension into the bladder wall perpendicular to
the incision. Then bladder wall and abdominal wall were stitched in turn[12].(Figure 1)
Cell transfection
According to LipofectamineTM 2000 liposome transfection reagent
instruction( Lipofect2000, Invitrogen) , F2 passage MSCs were digested by
pancreatic enzyme, and then were counted. They were inoculated at 2x105/mL. When
fusion of cell growth was 70%-80%, OD SiRNA diluted by OptiMEM® and
LipofectamineTM 2000 diluted by OptiMEM® Medium were mixed, and incubated
7
19
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
2021
for 20 min; Mixture was added to each hole, and incubated at 37 and 5% CO2,℃
saturated humidity. After 9 hr they were cultured by DMEM containing 10% FBS/F-
12 media, and then were screened and cultured.
Western blotting
Briefly, the protein concentrations of all samples were measured by BCA protein
assay kit (Pierce, USA) after protein extraction. After boiled for 5 min, the protein
samples were fractionated by SDS-PAGE (10–15% polyacrylamide gels) and
transferred to PVDF membrane (Millipore, Bedford, MA, USA). The samples were
blocked with milk powder for 1 hr at room temperature and then incubated with
primary antibodies (Abcam China) as well as calcineurin and NFATc3 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA USA) at 4°C overnight. After washing, the
membranes were incubated with a secondary antibody (Santa Cruz Biotechnology
Inc., Santa Cruz, CA USA) for 1 hr at room temperature. Western blot bands were
quantified using Gel analysis system by measuring the integrated optical density
(IOD). The protein expression intensity was quantified by relative optical density
(ROD). The ROD was defined as protein IOD/actin IOD.
Statistical analysis
All values were expressed as mean ± standard deviation (x̄
±S). SPSS 17.0 was used
to deal with the data, and t test was used to determine statistical differences between
two groups. P<0.05 was considered as significant difference.
8
22
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
2324
Results
The in vitro experiment showed that blocking the expression of TGFβ1 receptor
or Smad2 affected the differentiation of MSCs to myofibroblast
The ROD values of α-SMA and Vimentin were 0.212±0.018 and 0.289±0.036 when
MSCs were cultured with DMEM-LG in group control C. When MSCs were cultured
with DMEM-LG and 30% VX2 culture supernatant in group control A, the ROD
values of α-SMA and Vimentin were both significantly increased (0.641±0.026,
0.476±0.029), which indicated VX2 culture supernatant could induce the
differentiation of MSCs to myofibroblasts. However, compared with group control A,
the ROD values of α-SMA and Vimentin were both significantly decreased when the
expression of TGFβ1 receptor was blocked by siRNA in group test A (0.302±0.021,
0.378±0.040). The ROD values of α-SMA and Vimentin were also both significantly
decreased when the expression of Smad2 was blocked by siRNA in group test B
(0.270±0.021, 0.368±0.048). The results indicated that both TGFβ1 receptor and
Smad2 were involved in the differentiation of MSCs to myofibroblasts.(Figure 2)
TGFβ1 receptor and Smad2 participate in the process that MSCs accelerate VX2
tumor growth
All rabbits were sacrificed 4 weeks after tumor inoculation. The maximum diameter
was recorded. The mean maximum diameter of group control 3 was 2.01±0.28cm.
When VX2 cells and MSCs were co-inoculated in group control 1, the mean
maximum diameter was significantly increased (2.87±0.43cm), which indicated that
9
25
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
2627
MSCs were conducive to VX2 tumor growth. In group test 1, the expression of
TGFβ1 receptor of MSCs was blocked, and these cells were then co-inoculated with
VX2 cells. Compared with group control 1, the mean maximum diameter was
significantly decreased (2.38±0.36cm). The same tendency was also found in group
test 2. After the expression of Smad2 of MSCs was blocked, these MSCs were co-
inoculated with VX2 cells. Compared with group control 1, the mean maximum
diameter was also significantly decreased (2.28±0.32cm). (Figure 3)
TGFβ1 receptor and Smad2 mediated the process that MSCs facilitated the
expression of TGFβ1 and EGF in VX2 tumor tissue
The ROD values of TGFβ1 and EGF were 0.264±0.054 and 0.459±0.090 in group
control 3. When VX2 cells and MSCs were co-inoculated in group control 1, the ROD
values of TGFβ1 and EGF were both significantly increased (0.899±0.124,
1.053±0.107), which indicated that MSCs facilitated the expression of these two
growth factors in VX2 tumor tissue. Compared with group control 1, the ROD values
of TGFβ1 and EGF were both significantly decreased (0.333±0.074, 0.714±0.072)
when the expression of TGFβ1 receptor of MSCs was blocked in group test 1. The
ROD values of TGFβ1 and EGF were also both significantly decreased when the
expression of Smad2 of MSCs was blocked in group test 2 (0.346±0.089,
0.723±0.084). The results indicated that both TGFβ1 receptor and Smad2 were
involved in the process that MSCs facilitated the expression of TGFβ1 and EGF in
VX2 tumor tissue. (Figure 4)
TGFβ1 receptor and Smad2 mediated the process that MSCs facilitated the
10
28
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
2930
expression of FAPa and MMP9 in VX2 tumor tissue
The ROD values of FAPa and MMP9 were 0.072±0.036 and 1.012±0.087 in group
control 3. When VX2 cells and MSCs were co-inoculated in group control 1, the ROD
values of FAPa and MMP9 were both significantly increased (0.222±0.041,
1.147±0.121). When the expression of TGFβ1 receptor of MSCs was blocked in
group test 1, the ROD values of FAPa and MMP9 were both significantly decreased
(0.149±0.046, 1.036±0.108) compared with group control 1. The ROD values of
FAPa and MMP9 were also both significantly decreased when the expression of
Smad2 of MSCs was blocked in group test 2 (0.153±0.056, 1.022±0.120). The results
indicated that both TGFβ1 receptor and Smad2 were involved in the process that
MSCs facilitated the expression of FAPa and MMP9 in VX2 tumor tissue. (Figure 5)
Discussion
Activation of microenvironment is a critical step for tumor growth and
development[14-16]. Activated mesenchymal cells produce a large amount of
extracellular matrix components, growth factors, and matrix remodeling proteins,
thereby forming a microenvironment that is conducive to tumor growth and
proliferation. These mesenchymal cells mainly include fibroblasts, myofibroblasts,
endothelial cells, and some immune cells[17]. Although all of the above cells play a role
in the growth and development of tumors, it is currently believed that myofibroblasts
are especially important for tumor growth, invasion and metastasis[18-20]. In the
11
31
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
3233
activated interstitial microenvironment, myofibroblasts produce some proteases such
as fibroblast activation protein, metalloproteinases, urokinase, plasminogen activator.
In addition, they also synthesize some extracellular matrix components such as
collagen I, collagen III, fibronectin, mucin, polysaccharide proteins. Some reports
suggest that myofibroblasts can secrete certain growth factors to promote the
development of cancer , such as TGFβ1, EGF, platelet-derived growth factor,
fibroblast growth factor, hepatocyte growth factor, keratinocyte growth factor, stem
cell factor[21, 22].
In the tumor stroma, myofibroblasts are mainly derived from fibroblasts. Since many
scholars believe that the fibroblasts in the granulation tissue can be derived from some
progenitor cells in the circulation[23-25], Alexis et al therefore believe that the
circulating cells will also migrate to tumor tissue and differentiate into myofibroblasts
when the tumor grows to a certain size and local fibroblasts are not sufficient [26].
Ishii's findings further indicate that myofibroblasts in tumor stroma are derived from
bone marrow, and that the higher the tumor stage, the greater the number of
myofibroblasts derived from the bone marrow[27]. However, this study only showed
that myofibroblasts in tumor stroma could also be derived from the bone marrow but
did not specify which cells they were derived from. Jeon's findings indicate that
lysophosphatidic acid secreted by tumors can induce the differentiation of MSCs into
myofibroblast-like cells[13]. Mishra found that the expression of α-SMA, a marker of
myofibroblast, was significantly increased in bone marrow mesenchymal cells
12
34
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
3536
induced by tumor-conditioned medium, and the induced MSCs significantly promoted
tumor growth both in vitro and in vivo[28]. Therefore, at least part of myofibroblasts in
tumor tissue should be derived from bone marrow MSCs. In addition to bone marrow,
MSCs are widely present in other parts of the body. Therefore, myofibroblasts in
tumors may also be derived from MSCs in other sites. In addition, Bagley's results
indicate that MSCs are mainly distributed in tumor stroma after entering tumor tissue,
and they mainly differentiate into myofibroblast precursor cells, fibroblasts[29]. In view
of the important role of myofibroblasts in tumor growth, invasion, and metastasis, one
of the mechanisms by which MSCs facilitate tumor growth is to promote tumor
growth by differentiated myofibroblasts.
Smad2/Smad3 is the first signaling molecule in TGFβ1- Smad signaling pathway. As
a direct substrate of the TGFβ superfamily, Smad2/Smad3 plays a key mediating role
in the transmission of TGFβ1 signaling from cytoplasm to nucleus[30, 31]. In the
previous studies, we found that MSCs could differentiate into myofibroblasts under
the induction of tumor microenvironment, and further promote the growth and
development of tumors by promoting remodeling of tumor interstitial
microenvironment[10, 12]. In this study, we also found that MSCs could differentiate
into myofibroblasts under the induction of tumor cell culture supernatant. Blocking
the expression of TGFβ1 receptor or Smad2 in MSCs can significantly down-regulate
the expression of α-SMA and Vimentin. This indicates that TGFβ1-Smad signaling
pathway is involved in the differentiation of MSCs into myofibroblasts. We further
investigated whether TGFβ1-Smad signaling pathway was involved in the process
13
37
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
3839
that MSCs facilitated tumor growth and tumor interstitial microenvironment
remodeling. We found that blocking the expression of TGFβ1 receptor or Smad2 in
MSCs could down-regulate the tumor growth promoting effect of MSCs, and
significantly down-regulate the expression of TGFβ1, EGF, FAPa and MMP9, which
were closely related to the activation and remodeling of tumor interstitial
microenvironment. We speculate that one of the reasons for this result is that blocking
TGFβ1-Smad signaling pathway affects the differentiation of MSCs into
myofibroblasts.
Although most studies indicate that MSCs facilitate tumor growth and metastasis,
some studies indicate that MSCs can inhibit tumor development. However, the vast
majority of these studies are the results of in vitro cell experiments or
immunodeficient mice, and the MSCs used are allogeneic. So one merit of this study
is that the animals used in this study are normal rabbits and MSCs used in every rabbit
are autologous, which avoids the immune interference on the study results.
Conclusions
Under the induction of tumor microenvironment, MSCs can differentiate into
myofibroblasts and then affect tumor interstitial microenvironment remodeling.
TGFβ1-Smad2 pathway mediates the above process.
14
40
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
4142
Abbreviations
MSCs: Mesenchymal stem cells;
TGFβ1: transforming growth factor β1;
EGF: epidermal growth factor;
FAPa: fibroblast activation protein alpha;
MMP9: matrix metalloprotein 9;
PBS: phosphate buffered saline;
DMEM-LG: dulbecco's modified eagle medium-low glucose;
IOD: intergrated optical density;
ROD: relative optical density
Funding: This work was supported by National Natural Science Foundation of China
grant (no.30900549) ( the design of the study and collection, analysis, and
interpretation of data and in writing the manuscript), Natural Science Foundation of
Shandong Province grant (no.ZR2009CQ026) ( the design of the study and collection,
analysis, and interpretation of data and in writing the manuscript), Shandong
provincial key research and development plan (no. 2017GSF218008) ( collection,
analysis, and interpretation of data), and Scientific research start fund of Qilu Hospital
(Qingdao) of Shandong University(no.QDKY2016ZD04) (in writing the manuscript).
Authors' contributions: JC designed and coordinated the study. QY analyzed data
and wrote the paper. YZ did animal and in vitro experiments, flow cytometry work,
15
43
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
4445
ZX contributed experimental design and data analysis. All authors read and approved
the final manuscript.
Acknowledgements: Not applicable
Conflict of interests: None.
Competing interests: The authors have declared that no competing interest exists
References
1 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells
express a restricted set of functionally active chemokine receptors capable of
promoting migration to pancreatic islets. Blood. 2005;106:419-27.
2 Sordi V. Mesenchymal stem cell homing capacity. Transplantation. 2009; 87 (Suppl
9):S42-S45.
3 Sato Y, Araki H, Kato J, et al. Human mesenchymal stem cells xenografted directly
to rat liver are differentiated into human hepatocytes without fusion. Blood. 2005;
106:756-63.
4 Yamaguchi Y, Kubo T, Murakami T, et al. Bone marrow cells differentiate into
wound myofibroblasts and accelerate the healing of wounds with exposed bones when
combined with an occlusive dressing. Br J Dermatol. 2005;152:616-22.
5 Ridge SM, Sullivan FJ, Glynn SA. Mesenchymal stem cells: key players
in cancer progression. Mol Cancer. 2017;16:31.
6 Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells: potential
precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J
Natl Cancer Inst. 2004; 96:1593-603.
16
46
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
4748
7 Coffelt SB, Marini FC, Watson K, et al. The pro-inflammatory peptide LL-37
promotes ovarian tumor progression through recruitment of multipotent mesenchymal
stromal cells. Proc Natl Acad Sci U S A. 2009; 106:3806-11.
8 Spaeth E, Klopp A, Dembinski J, et al. Inflammation and tumor
microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene
Ther. 2008; 15:730-8.
9 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal
stem cells in the treatment of gliomas. Cancer Res. 2005; 65:3307-18.
10 Chen J, Xu ZS, Zhao HF, et al. Distribution and differentiation of marrow
mesenchymal cells in tumor tissue: experimental with rabbits. Zhonghua Yi Xue Za
Zhi. 2007; 87:2361-4.
11 Karnoub AE, Dash AB, Vo AP, et al. Mesenchymal stem cells within tumor stroma
promote breast cancer metastasis. Nature. 2007; 449:557-63.
12 Chen J, Ma L, Zhang N, et al. Mesenchymal Stem Cells Promote Tumor
Progression via Inducing Stroma Remodeling on Rabbit VX2 Bladder Tumor
Model. Int J Biol Sci. 2018; 14:1012-21.
13 Jeon ES, Moon HJ, Lee MJ, et al. Cancer-derived lysophosphatidic acid stimulates
differentiation of human mesenchymal stem cells to myofibroblast-like Cells.
Stem Cells. 2008; 26:789-97.
14 Condon MS. The role of the stromal microenvironment in prostate cancer. Semin
Cancer Biol. 2005;15:132-7.
17
49
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
5051
15 Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation
and progression. Nature. 2004; 432: 332-7.
16 McCuaig R, Wu F, Dunn J, et al. The biological and clinical significance of
stromal-epithelial interactions in breast cancer. Pathology. 2017; 49:133-40
17 Mahale J, Smagurauskaite G, Brown K, et al. The role of stromal fibroblasts in
lung carcinogenesis: A target for chemoprevention? Int J Cancer. 2016;138:30-44.
18 Powell DW, Adegboyega PA, Di Mari JF, et al. Epithelial cells and their
neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer. Am
J Physiol Gastrointest Liver Physiol. 2005; 289:G2-7.
19 Galiè M, Sorrentino C, Montani M, et al. Mammary carcinoma provides highly
tumourigenic and invasive reactive stromal cells. Carcinogenesis. 2005; 26:1868-78.
20 Rodrigues PC, DA Costa Miguel MC, DE Aquino SN, et al. Stromal
myofibroblasts in potentially malignant and malignant lesions of the oral cavity.
Oncol Lett. 2015; 9:667-70.
21 Frazier KS, Grotendorst GR. Expression of connective tissue growth factor
mRNA in the fibrous stroma of mammary tumors. Int J Biochem Cell Biol. 1997;
29:153-61.
22 Shimo T, Nakanishi T, Nishida T, et al. Connective tissue growth factor induces
the proliferation, migration, and tube formation of vascular endothelial cells in vitro,
and angiogenesis in vivo. J Biochem. 1999; 126:137-45.
23 Abe R, Donnelly SC, Peng T, et al. Peripheral blood fibrocytes: differentiation
pathway and migration to wound sites. J Immunol. 2001; 166:7556-62.
18
52
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
5354
24 Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165)
gene transfer augments circulating endothelial progenitor cells in human subjects.
Circ Res. 2000; 86:1198-202.
25 Yang L, Scott PG, Giuffre J, et al.
Peripheral blood fibrocytes from burn patients: identification and quantification of fib
rocytes in adherent cells cultured from peripheral blood mononuclear cells. Lab
Invest. 2002; 82: 1183-92.
26 Alexis D, Christelle G, Giulio G. The stroma reaction myofibroblast: a key player
in the control of tumor cell behavior. Int J Dev Biol. 2004; 48:509-17.
27 Ishii G, Sangai T, Oda T, et al. Bone-marrow-derived myofibroblasts contribute to
the cancer-induced stromal reaction. Biochem Biophys Res Commun. 2003; 309:232-
40.
28 Mishra PJ, Mishra PJ, Humeniuk R, et al. Carcinoma-associated fibroblast-like
differentiation of human mesenchymal stem cells. Cancer Res. 2008; 68:4331-9.
29 Bagley RG, Weber W, Rouleau C, et al. Human mesenchymal stem cells from
bone marrow express tumor endothelial and stromal markers. Int J Oncol. 2009;
34:619-27.
30 Schiffer M, von Gersdorff G, Bitzer M, et al. Smad proteins and transforming
growth factor-beta signaling. Kidney Int Suppl. 2000; 77:S45-S52.
31 ten Dijke P, Hill CS. New insights into TGF-beta-Smad signalling. Trends
Biochem Sci. 2004; 29: 265-73.
19
55
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
5657
20
58
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
5960
Figure 1. Tumor inoculation procedure:A. Exposure the bladder; B. Cystotomy
along the midline; C. 1 ml needle (containing 300 ul of cell suspension) punctures into
the mucosa on the side wall of the bladder; D. After about 1.5cm of immersion in the
submucosa of the bladder, the thumb of the left hand presses against point of the
needle, and 300ul of cell suspension is injected under the mucosa; E. Blisters formed
by cell suspension after injection; F. Close the bladder with absorbable suture.
21
61
428
429
430
431
432
433
434
435
436
437
438
6263
control A control B control C test A test B
α-SMA 42kd
Actin 42kd
control A control B control C test A test B
Vimentin 54 kd
Actin 42kd
Figure 2 The expression of α-SMA and Vimentin in MSCs was detected by westernblot. Control B was used to test whether or not liposome could affect the result. Compared with control A, p>0.05 indicated liposome did not affect the result. The comparison between group control C and group control A indicated that VX2 culture
22
control A control B control C test A test B0
0.10.20.30.40.50.60.7
The ROD values of α-SMA
* compared with control A, t=1.259, p=0.224 * * compared with control A,t=42.316, P <0.001 *** compared with control A,t=31.766, P <0.001 ****compared with control A, t=34.697, P <0.001
ROD
*
***** ****
control A control B control C test A test B0
0.1
0.2
0.3
0.4
0.5
The ROD values of Vimentin
* compared with control A, t=0.385, p=0.705 ** compared with control A, t=12.826, P <0.001 *** compared with control A, t=6.268, p <0.001 ****compared with control A, t=6.058, P <0.001
ROD
*
**
*** ****
64
439440441442443444
445
446
447448449450451452453454455
456
457
458459460461462
6566
supernatant could induce the expression of α-SMA and Vimentin. Blockage of the expression of TGFβ1 receptor (group test A) or Smad2 (group test B) would both lead to decreased expression of α-SMA and Vimentin.
Figure 3 The maximum diameter of VX2 bladder tumor 4 weeks after tumor inoculation. Control 2 was used to test whether or not liposome could affect the result. Compared with control 1, p>0.05 indicated liposome did not affect the result. The comparison between group control 3 and group control 1 indicated that MSCs were conducive to VX2 tumor growth. The expression of TGFβ1 receptor and Smad2 of MSCs were blocked in group test 1 and group test 2, respectively. Compared with group control 1, the mean maximum tumor diameter was significantly decreased in group test 1 and group test 2
23
control 1 control 2 control 3 test 1 test 20
0.5
1
1.5
2
2.5
3
3.5
the mean maximum VX2 tumor diameter 4 weeks after cells inoculation
* compared with control 1, t=0.670, p=0.512 ** compared with control 1, t=5.345, P <0.001 *** compared with control 1, t=2.79, P =0.012
****compared with control 1, t=3.487, P =0.003
Dia
met
er(c
m)
*
***** ****
67
463464465466467468469470471472473474475476477478479480481482483484485486487488489490491
6869
control control 2 control 3 test 1 test 2
TGFβ1 25kd
Actin 42kd
control control 2 control 3 test 1 test 2
EGF 6kd
Actin 42kd
24
control 1 control 2 control 3 test 1 test 20
0.2
0.4
0.6
0.8
1
The ROD values of TGFβ1
* compared with control 1, t=0.334, p=0.743** compared with control 1, t=14.807, P <0.001 *** compared with control 1, t=12.354, P <0.001 ****compared with control 1, t=11.461 P <0.001
ROD
*
** *** ****
control 1 control 2 control 3 test 1 test 20
0.4
0.8
1.2
The ROD values of EGF
* compared with control 1, t=0.367, p=0.718 ** compared with control 1, t=13.413, P <0.001 *** compared with control 1, t=8.297, P <0.001 **** compared with control 1, t=7.660, P <0.001
ROD
*
***** ****
70
492493
494495496497498499
500
501
502503504505506507508509510
511
512
513514515516
7172
Figure 4 The expression of TGFβ1 and EGF in vx2 tumor tissue was detected by westernblot. Control 2 was used to test whether or not liposome could affect the result. Compared with control 1, p>0.05 indicated liposome did not affect the result. The comparison between group control 3 and group control 1 indicated that MSCs could enhance the expression of TGFβ1 and EGF. Blockage of the expression of TGFβ1 receptor (group test 1) or Smad2 (group test 2) would both lead to decreased expression of TGFβ1 and EGF.
control control 2 control 3 test 1 test 2
FAPa 88kd
Actin 42kd
control control 2 control 3 test 1 test 2
MMP9 92kd
Actin 42kd
25
control 1 control 2 control 3 test 1 test 20
0.05
0.1
0.15
0.2
0.25
The ROD values of FAPa
* compared with control 1, t=0.354, p=0.727** compared with control 1, t=8.641, P <0.001 *** compared with control 1, t=3.720, P =0.002 **** compared with control 1, t=3.158, P =0.005
ROD
*
**
*** ****
control 1 control 2 control 3 test 1 test 20.8
1.2
The ROD values of MMP9
* compared with control 1, t=0.284, p=0.780 ** compared with control 1, t=2.858, P =0.01
*** compared with control 1, t=2.159, P =0.045 **** compared with control 1, t=2.315, P =0.033
ROD
* ** *** ****
73
517518519520521522523524525526
527
528529530531532533534535
536
537
538539540541
7475
Figure 5 The expression of FAPa and MMP9 in vx2 tumor tissue was detected by westernblot. Control 2 was used to test whether or not liposome could affect the result. Compared with control 1, p>0.05 indicated liposome did not affect the result. The comparison between group control 3 and group control 1 indicated that MSCs could enhance the expression of FAPa and MMP9. Blockage of the expression of TGFβ1 receptor (group test 1) or Smad2 (group test 2) would both lead to decreased expression of FAPa and MMP9.
26
76
542543544
545546547548
7778