Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells
Background: Cell and tissue homeostasis results from the dynamic balance of cell – cell and cell – extracellular component crosstalk that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposition of extracellular matrix...
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Bezdenezhnykh, N. Semesiuk, N. Lykhova, O. Zhylchuk, V. Kudryavets, Y. 2019-01-20T18:07:05Z 2019-01-20T18:07:05Z 2014 Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells / N. Bezdenezhnykh, N. Semesiuk, O. Lykhova, V. Zhylchuk, Y. Kudryavets // Experimental Oncology. — 2014. — Т. 36, № 2. — С. 72-78. — Бібліогр.: 29 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145344 Background: Cell and tissue homeostasis results from the dynamic balance of cell – cell and cell – extracellular component crosstalk that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components. Aim: The aim of the work was to study the crosstalk between tumor cells and stromal cell components using noncontact co-cultivation in vitro system. Materials and Methods: Human and rat breast cancer (BC) cell lines, normal human fibroblasts (NHF) and endothelial cells, and aspirates of bone marrow (BM) of BC patients with different clinical course of the disease (groups “Remission” (BM-R) and “Progression” (BM-P)) were used in noncontact co-cultivation system in vitro. The cell growth, expression of epithelial-mesenchymal transition (EMT) and tumor stem cell markers (E-cadherin, vimentin, CD44), Ki-67, p21 and Slug were investigated using immunocytochemical analysis. Results: Analysis of expression of E- and N-cadherin, vimentin and Slug in BC cells has shown that T-47D and MRS-T5 cells possess mesenchymal phenotype, while MCF-7 and MRS cells possess mostly epithelial phenotype with a part of cells with mesenchymal patterns. Upon noncontact co-cultivation of fibroblasts with Т-47D or MRS-Т5 cells, BC cells acquired higher proliferative activity compared to the control cells (р < 0.05) or MCF-7 and MRS cells co-cultivated with fibroblasts. Upon noncontact co-cultivation of Т-47D cells with normal fibroblasts and BM cells from BC patients from group “Progression” there were observed increased quantity of CD44+ Т-47D cells (by 26%), decreased quantity of Е-cadherin+ Т-47D cells, and appearance of vimentin-positive cells. In co-cultivation variant Т-47D + NHF + BM-R (“Remission“) the quantity of CD44+ Т-47D cells significantly decreased (р < 0.005) and E-cadherin expression remained unaltered compared to control cells. At the same time, in NHF cell population (co-cultivation variant Т-47D + NHF + BM-P) there was detected significant increase of quantity of р21+-cells (р < 0.005), cytoplasmic localization of p21, and nuclear localization of Slug. Expression of vimentin did not alter in any variant of co-cultivation. Conclusion: The new integration cell system for investigation of the mechanisms of interaction between tumor cells and the tumor microenvironment in vitro was developed. The significant changes in proliferative activity of TC dependently on its ЕМT-status were detected after their interaction with fibroblasts and endothelial cells in noncontact co-cultivation system. BM cells of BC patients had different modifying influence on TC dependent on clinical BC course. The activation of ЕМT program was revealed in TC upon noncontact co-cultivation with BM cells of BC patients with progression of the disease. Key Words: breast cancer, epithelial-mesenchymal transition, microenvironment, bone marrow, co-cultivation. This study was supported with the grant NAS of Ukraine for Young Scientists “Development of new integration cell system for investigation of the mechanisms of interaction TC with microenvironment in vitro”, № 2.2.5.383 from 01.07.2013. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| spellingShingle |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells Bezdenezhnykh, N. Semesiuk, N. Lykhova, O. Zhylchuk, V. Kudryavets, Y. Original contributions |
| title_short |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| title_full |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| title_fullStr |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| title_full_unstemmed |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| title_sort |
impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells |
| author |
Bezdenezhnykh, N. Semesiuk, N. Lykhova, O. Zhylchuk, V. Kudryavets, Y. |
| author_facet |
Bezdenezhnykh, N. Semesiuk, N. Lykhova, O. Zhylchuk, V. Kudryavets, Y. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2014 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
Background: Cell and tissue homeostasis results from the dynamic balance of cell – cell and cell – extracellular component crosstalk that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components. Aim: The aim of the work was to study the crosstalk between tumor cells and stromal cell components using noncontact co-cultivation in vitro system. Materials and Methods: Human and rat breast cancer (BC) cell lines, normal human fibroblasts (NHF) and endothelial cells, and aspirates of bone marrow (BM) of BC patients with different clinical course of the disease (groups “Remission” (BM-R) and “Progression” (BM-P)) were used in noncontact co-cultivation system in vitro. The cell growth, expression of epithelial-mesenchymal transition (EMT) and tumor stem cell markers (E-cadherin, vimentin, CD44), Ki-67, p21 and Slug were investigated using immunocytochemical analysis. Results: Analysis of expression of E- and N-cadherin, vimentin and Slug in BC cells has shown that T-47D and MRS-T5 cells possess mesenchymal phenotype, while MCF-7 and MRS cells possess mostly epithelial phenotype with a part of cells with mesenchymal patterns. Upon noncontact co-cultivation of fibroblasts with Т-47D or MRS-Т5 cells, BC cells acquired higher proliferative activity compared to the control cells (р < 0.05) or MCF-7 and MRS cells co-cultivated with fibroblasts. Upon noncontact co-cultivation of Т-47D cells with normal fibroblasts and BM cells from BC patients from group “Progression” there were observed increased quantity of CD44+ Т-47D cells (by 26%), decreased quantity of Е-cadherin+ Т-47D cells, and appearance of vimentin-positive cells. In co-cultivation variant Т-47D + NHF + BM-R (“Remission“) the quantity of CD44+ Т-47D cells significantly decreased (р < 0.005) and E-cadherin expression remained unaltered compared to control cells. At the same time, in NHF cell population (co-cultivation variant Т-47D + NHF + BM-P) there was detected significant increase of quantity of р21+-cells (р < 0.005), cytoplasmic localization of p21, and nuclear localization of Slug. Expression of vimentin did not alter in any variant of co-cultivation. Conclusion: The new integration cell system for investigation of the mechanisms of interaction between tumor cells and the tumor microenvironment in vitro was developed. The significant changes in proliferative activity of TC dependently on its ЕМT-status were detected after their interaction with fibroblasts and endothelial cells in noncontact co-cultivation system. BM cells of BC patients had different modifying influence on TC dependent on clinical BC course. The activation of ЕМT program was revealed in TC upon noncontact co-cultivation with BM cells of BC patients with progression of the disease. Key Words: breast cancer, epithelial-mesenchymal transition, microenvironment, bone marrow, co-cultivation.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/145344 |
| citation_txt |
Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells / N. Bezdenezhnykh, N. Semesiuk, O. Lykhova, V. Zhylchuk, Y. Kudryavets // Experimental Oncology. — 2014. — Т. 36, № 2. — С. 72-78. — Бібліогр.: 29 назв. — англ. |
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2025-11-27T03:15:15Z |
| last_indexed |
2025-11-27T03:15:15Z |
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| fulltext |
72 Experimental Oncology 36, 72–78, 2014 (June)
IMPACT OF STROMAL CELL COMPONENTS OF TUMOR
MICROENVIRONMENT ON EPITHELIAL-MESENCHYMAL
TRANSITION IN BREAST CANCER CELLS
N. Bezdenezhnykh1,*, N. Semesiuk1, O. Lykhova1, V. Zhylchuk2,3, Y. Kudryavets1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
2Rivne Region Oncology Hospital, Rivne 33001, Ukraine
3Danylo Halytsky Lviv National Medical University, Lviv 79010, Ukraine
Background: Cell and tissue homeostasis results from the dynamic balance of cell – cell and cell – extracellular component crosstalk
that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposi-
tion of extracellular matrix (ECM) components. Aim: The aim of the work was to study the crosstalk between tumor cells and stromal
cell components using noncontact co-cultivation in vitro system. Materials and Methods: Human and rat breast cancer (BC) cell lines,
normal human fibroblasts (NHF) and endothelial cells, and aspirates of bone marrow (BM) of BC patients with different clinical course
of the disease (groups “Remission” (BM-R) and “Progression” (BM-P)) were used in noncontact co-cultivation system in vitro.
The cell growth, expression of epithelial-mesenchymal transition (EMT) and tumor stem cell markers (E-cadherin, vimentin, CD44),
Ki-67, p21 and Slug were investigated using immunocytochemical analysis. Results: Analysis of expression of E- and N-cadherin,
vimentin and Slug in BC cells has shown that T-47D and MRS-T5 cells possess mesenchymal phenotype, while MCF-7 and MRS
cells possess mostly epithelial phenotype with a part of cells with mesenchymal patterns. Upon noncontact co-cultivation of fibroblasts
with Т-47D or MRS-Т5 cells, BC cells acquired higher proliferative activity compared to the control cells (р < 0.05) or MCF-7 and
MRS cells co-cultivated with fibroblasts. Upon noncontact co-cultivation of Т-47D cells with normal fibroblasts and BM cells from
BC patients from group “Progression” there were observed increased quantity of CD44+ Т-47D cells (by 26%), decreased quantity
of Е-cadherin+ Т-47D cells, and appearance of vimentin-positive cells. In co-cultivation variant Т-47D + NHF + BM-R (“Remis-
sion“) the quantity of CD44+ Т-47D cells significantly decreased (р < 0.005) and E-cadherin expression remained unaltered compared
to control cells. At the same time, in NHF cell population (co-cultivation variant Т-47D + NHF + BM-P) there was detected sig-
nificant increase of quantity of р21+-cells (р < 0.005), cytoplasmic localization of p21, and nuclear localization of Slug. Expression
of vimentin did not alter in any variant of co-cultivation. Conclusion: The new integration cell system for investigation of the mechanisms
of interaction between tumor cells and the tumor microenvironment in vitro was developed. The significant changes in proliferative
activity of TC dependently on its ЕМT-status were detected after their interaction with fibroblasts and endothelial cells in noncontact
co-cultivation system. BM cells of BC patients had different modifying influence on TC dependent on clinical BC course. The activa-
tion of ЕМT program was revealed in TC upon noncontact co-cultivation with BM cells of BC patients with progression of the disease.
Key Words: breast cancer, epithelial-mesenchymal transition, microenvironment, bone marrow, co-cultivation.
In most cases the cancer-related mortality is caused
by haematogenous spread of cancer cells into distant
organs and their subsequent growth to overt metastases.
After surgical removal of the primary tumor, minimal re-
sidual disease (MRD) is defined as the presence of tumor
cells (TC) that are not detectable by the current routine
diagnostic procedures used for tumor staging in cancer
patients, but become apparent after a period of time [1].
The crosstalk between tumor and cells of its microenviron-
ment crucially determines the fate of tumor progression.
The stroma is a very heterogeneous milieu including vari-
ous cell types and adhesion molecules, both contributing
to the functional activity and structural support of the tumor
microenvironment (TME) [2]. S. Paget laid the foundations
of the TME research area by formulating the seed and
soil theory. The tumor is directed into one or several pos-
sible molecular evolution pathways by signals originating
in native and/or modified microenvironmental factors.
Many of these pathways may lead to metastasis. The TME
has some characteristics: 1) the molecular composi-
tion of the TME is established by TC and by resident/
infil trating non-tumor cells; 2) intercellular interactions
between TC and components of their microenvironment
are crucial in determining cancer progression; 3) tumor —
microenvironment interactions are bidirectional; 4) certain
tumor — microenvironment interactions may initiate tumor
progression ; 5) microenvironmental factors play opposing
roles in tumor progression by either promoting or alterna-
tively antagonizing this process [3].
The TME was recognized as the product of a de-
veloping crosstalk between different cells types: endo-
thelial cells, carcinoma-associated fibroblasts (CAFs),
adipocytes, mesenchymal cells, mesenchymal stem cells
(MSCs; bone marrow derived — (BM-MSCs), or carcino-
ma-associated (CA-MSCs)), and cells from the immune
and inflammatory systems (tumor-associated macro-
phages (TAM), regulatory T cells, etc.). The stromal cells
interact not only with TC but also with each other. TC in-
duce changes in fibroblasts, which contact with them [4],
transforming them into myofibroblasts, which activate
program of epithelial-mesenchymal transition (EMT)
in TC resulting in the domination of mesenchymal features
Submitted: May 8, 2014.
*Correspondence: E-mail: beznalia@mail.ru
Abbreviations used: BC — breast cancer; BM — bone marrow;
ECM — extracellular matrix; EMT — epithelial-mesenchymal transi-
tion; MC — mononuclear cells; MET — mesenchymal-epithelial
transition; MRD — minimal residual disease; TAM — tumor-associ-
ated macrophage; TC — tumor cells; TME — tumor microenviron-
ment; TNF — tumor necrosis factor.
Exp Oncol 2014
36, 2, 72–78
Experimental Oncology 36, 72–78, 2014 (June) 73
and development of highly metastatic phenotype [5]. All
these processes take place with involvement of soluble
(humoral) factors of microenvironment (cytokines, growth
factors, etc.), which, in turn, activate other links of interac-
tion, involved in “malignant transition”, in particular, tran-
scriptional factors. In the result of this, epithelial cells lost
the capacity to form dense intercellular contacts, their
adhesive properties decrease while capacity for inva-
sion and migration develop. However, when such cells
achieve their “destination point”, the inverse process
takes place — mesenchymal-epithelial transition (MET),
during which they again acquire epithelial phenotype with
increased adhesion that gives opportunity to them to fas-
ten onto the substrate and to give growth to se condary
metastatic focus [6]. Thus, a major TAM-derived inflam-
matory cytokine shown to be highly expressed in breast
carcinomas is tumor necrosis factor alpha (TNF-α) which
is a multifactorial cytokine. TNF-α activity varies under
different physiological conditions and in a cell-type-
dependent manner contributes to a sense of ambiguity
regarding its antitumor effects. Indeed, the permanent
expression of TNF-α in breast tumors actually supports
tumor growth and plays a role in the metastatic behavior
of breast carcinomas [7]. Furthermore, in BM of breast
cancer (BC) patients with advanced cancer significantly
higher TNF-α concentration was detected [8]. Therefore,
the further study of intercellular interaction and cell trans-
differentiation may reveal new targets in antimetastatic
therapy.
It is well known that certain organs and tissues are
typical as dominant sites of metastasis for many types
of tumor. For example, bone marrow (BM) and bones
often become such distant sites in BC. Today is well
known the important role of stromal cells, in particular
fibroblasts, as elements of TME, in control of their phe-
notype and biological behavior [9]. However, the role
of BM cells as components of TME is poorly understood.
Therefore, th e determination of influence of BM cells,
especially in comparative aspect with fibroblast elements,
on the phenotypic conversion and proliferative activity
of cells of BC at their co-cultivation, is of special interest.
In routine practice for in vitro research of compounds
of different nature (antitumor drugs, cytotoxic factors)
isolated cell cultures (primary cell cultures and stable
cell lines) are used. Experimental systems in vitro with
use of human cell lines represent important instrument
for estimation of the level and mechanisms of toxicity
of antitumor drugs with the aim of precise definition
of their possible antitumor action in vivo. The main
disadvantage of such in vitro system is an absence
of numerous components of interaction, which exist
in vivo. The principally new approach for determination
of intercellular interaction in vitro has been developed
by A.P. Li and co-authors [10] who created a system
of integrated discrete cultures of normal cells of diffe-
rent histogenesis and BC cells for the study of simulta-
neous combined toxic action of drugs on the TC-targets
and on the normal cells of organism (liver, kidneys,
lungs, vascular endothelium, etc.). The same scheme
with some modification has been developed by us for
testing the toxicity of chemical compounds, in par-
ticular, heavy metal salts, in vitro, so-called multi-organ
system of toxicity (MOST) [11]. For investigation of cel-
lular interaction in progression of disease, we have
developed in this work new cellular system with use
of primary and stable cell lines of different genesis: hu-
man and rat BC TC, normal human fibroblasts (NHF) and
normal rat fibroblasts (NRF), normal porcine endothelial
cells (PAE), cells from BM aspirates from BC patients
with different disease stage.
The aim of the work was to study in vitro the crosstalk
between TC and stromal cell components using noncon-
tact co-cultivation in vitro system.
MATERIALS AND METHODS
Clinical materials. In co-cultivation in vitro system,
mononuclear cells (MC) isolated from BM of BC pa-
tients with II and III stages of the disease (n = 6), were
used. BC patients were cured in Rivne Regional On-
cological Hospital (Rivne, Ukraine). The patients were
informed about the survey and provided written consent
for participation in the research. The study was carried
out with approval of the local Ethics Committee (R.E. Ka-
vetsky Institute of Experimental Pathology, Oncology
and Radiobiology, NAS of Ukraine).
After conducted treatment (radical surgical inter-
vention and adjuvant chemotherapy) BC patients have
been distributed into 2 groups: “Progression” (n = 3) and
“Remission” (n = 3), at the basis of their clinical state,
presence of TC in BM and high level of tumor-associated
cytokines in plasma of BM and peripheral blood [12, 13].
The BM aspirates (3–4 ml) were taken from sternum
with an incision in the skin to avoid contamination with
epithelial cells. BM samples were obtained from BC pa-
tients prior to therapy.
MC were isolated by Ficoll-Hypaque density gradient
LSM 1077 (PAA, Austria) centrifugation at 1,500 rpm for
20 min. MC were washed three times with RPMI-1640 and
were cryopreserved (3–5•106 cells per vial).
Cell lines. In our co-cultivation study the following
cell lines were used: NHF, NRF of Wistar rats, normal
porcine endothelial cells (PAE), human BC cell lines —
T-47D and MCF-7 and their sublines modified by long-
term action of interferon (IFN) (MCF-7 + IFN and T-47D
+ IFN), rat mammary carcinoma — MRS [14, 15], which
are characterized by domination in population of low
tumorigenic cells with epithelial phenotype as well
as highly tumorigenic subline MRS-T5, which is cha-
racterized by the presence of cells of only me senchymal
phenotype. All cell lines have been obtained from Bank
of Cell Lines from Human and Animal Tissues of R.E. Ka-
vetsky Institute of Experimental Pathology, Oncology
and Radiobiology of National Academy of Sciences
of Ukraine (Kyiv, Ukraine).
The modeling system of noncontact cell co-cul-
tivation. The cells of different type which were cultured
separately, serve as control (Fig. 1, a). For co-cultivation
experiment, the wells were combined with each other via
specially made channels in the wall of wells in such a way
74 Experimental Oncology 36, 72–78, 2014 (June)
that interaction of cells through their nutrient medium
could occur (Fig. 1, b).
NHF
TC
BM
cells
NHF
TC
BM
cells
Stromal
cells TC
Stromal
cells TC
a
b
Fig. 1. Scheme of noncontact co-cultivation in vitro: a — control
wells — isolated; b — wells with noncontact co-cultivation
Cells were cultivated in plastic glassware (“ТРР”,
Italy) in DMEM medium (“PAA”, Austria), which contained
2 mM of L-glutamine and NaHCO3 with 10% of FBS
(“PAA”, Austria) in humidified atmosphere at 37 °С and 5%
CO2. Quantity of cells after their co-cultivation has been
determined by staining with crystal violet (“Sigma”, USA)
with further registration of optical density of well content
using multiwell spectrometer (Labsystems Multiskan
PLUS, Finland) [16].
Immunocytochemical analysis. The slides of cyto-
spins were fixed in methanol + acetone (1:1) solution for
2 h at −20 °С, and then incubated with 1% BSA solution for
20 min. For immunocytochemical analysis, the following
monoclonal antibodies have been applied: anti-E-cad-
herin (Thermo Scientific, UK), anti-CD325 ( N-Cadherin)
(BioLegend, USA), anti-vimentin (Diagnostic BioSys-
tems, USA), anti-CD44 (Diagnostic BioSystems, USA),
Slug (GeneTex, USA), р21/waf1 (NeoMarkers, USA),
Ki-67 (Thermo Scientific, UK). Primary antibodies were
used according to the instructions of manufacturers. For
visualization, Poly Vue system (Thermo Scientific, UK) was
used. The slides were also stained with Mayer’s hema-
toxylin (“Sigma”, USA) and examined using microscope
AxioVert (“Сarl Zeiss”, Germany) with ×320 magnification.
Data were analyzed by calculation of positive (+) cells
using classical H-score method:
S = 1В•A + 2В•B + 3В•C,
where S — H-score index, which value lies within
the limits from 0 (no expression) to 300 (intensive ex-
pression in 100% of cells); А — percentage of poorly
stained cells; В — percentage of moderately stained cells;
C — percen tage of strongly stained cells.
Statistics. Statistical processing of obtained re-
sults has been carried out using mathematical program
STATISTIСA 6.0. Significance of differences between
mean values has been conducted with use of Stu-
dent’s t-criterion.
RESULTS AND DISCUSSION
First of all, the expression of ЕМT markers (E- and
N-cadherin, vimentin and Slug) was analyzed in TC which
were cultured separately as a control and later were used
in noncontact co-cultivation system. It was found that
the mesenchymal antigen expression profile was the most
typical for cells of T-47D and MRS-T5 lines. MCF-7 and
MRS cell lines are characterized by the prevalence of cells
with epithelial phenotype with the presence of cells with
mesenchymal features (Table).
Then, TC and stromal elements (fibroblasts, endo-
thelial cells) were co-cultivated in vitro using the system
described above. In the case of co-cultivation of TC and
fibroblasts, the proliferation of fibroblasts was close
to that of control cells but the quantity of TC varied de-
pendently on domination of epithelial or mesenchymal
phenotype of these cells. In particular, the quantity
of TC with domination of mesenchymal features (Т-47D,
MRS-Т5 cells) in noncontact co-cultivation with fibroblasts
was more prominently increased compared to control cells
(р < 0.05), than quantity of TC with prevalence of epithelial
phenotype (MCF-7, MRS) (Fig. 2). In the case of co-culti-
vation of fibroblasts (RNF) with rat TC with me senchymal
or epithelial features (variant of co-cultivation MRS +
RNF + MRS-T5) significant inhibition of MRS-T5 cells
with me senchymal phenotype in presence of epithelial
MRS cells was detected (see Fig. 2). So, the cells with
more differentiated epithelial phenotype may significantly
repress growth potential of cells with mesenchymal phe-
notype [17].
0
20
40
60
80
100
120
140
160
180
200
NHF1 T-47D NHF2 MCF-7
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
0
20
40
60
80
100
120
140
160
NR
F
M
RS NR
F T5
M
RS T5
M
RS NR
F T5
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
a
b
Fig. 2. Quantity of viable NHF and human TC (a) and NRF and rat
TC (b) upon their combined noncontact co-cultivation. OY axis rep-
resents the percent of viable cells compared to respective control
cells cultured in separate wells (taken as 100% of viable cells)
It is well known that tumor and stromal cells in microen-
vironment can interact and crosstalk between them might
be bidirectional. Tumor angiogenesis is a prerequisite for
tumor progression and metastasis. It is a complex process
Table. EMT marker expression in human BC cell lines
Antigens E-cadherin N-cadherin Slug
TC T-47D MCF-7 MRS T5 T-47D MCF-7 MRS T5 T-47D MCF-7 MRS T5E M E M E M
Scores (H-score system) 98±11 126±7 270±21 22±9 39±4 81±11 53±3 72±8 221±28 174±16 136 ±21 54±10 156±26 224±32 269±11
Experimental Oncology 36, 72–78, 2014 (June) 75
that requires cooperative reciprocal interaction of tumor
and endothelial cells and involves soluble and cellular
components. This requires a coordinated expression
of proangiogenic factors and suppression of antiangio-
genic factors, which leads to endothelial cell proliferation,
migration and vessel formation. M. Buess et al. [18] sug-
gest that the interaction of endothelial cells with TC that
express the CD44+/CD24− signature (indicating a low pro-
liferative potential) might explain association of the CD44+/
CD24− signature with highly proliferative tumors that have
an unfavorable prognosis.
The differences in proliferative activities of human
BC cells (control cells and IFN-modified cells) in co-
cultivation with cellular TME components (fibroblasts
and endothelial cells) were identified. For this study,
we used the following design of noncontact cultivation
cell system:
• set 1: 1) MCF-7/MCF-7 (IFN modified) + NHF;
2) MCF-7/MCF-7 (IFN modified) + PAE;
• set 2: 1) T-47D/T-47D (IFN modified) + NHF;
2) T-47D/T-47D (IFN modified) + PAE.
So, we obtained the following results: using
MCF-7 cell line and MCF-7 + IFN subline, we observed
an inhibition the BC proliferation independently on vari-
ant of stromal cells (NHF or PAE). This inhibition was
more significant in the case with MCF-7 + IFN cells
(20–25%) compared with control cells (Fig. 3).
0
20
40
60
80
100
120
140
160
MCF-7 PAE MCF-7 NHF
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
0
20
40
60
80
100
120
140
MCF-7 + IFN PAE MCF-7 + ІFN NHF
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
a
b
Fig. 3. Proliferative activities of MCF-7 (a) and IFN-modified
MCF-7 (b) cells in noncontact co-cultivation system with NHF
and PAE cells. OY axis represents the percent of viable cells
compared to respective control cells cultured in separate wells
(taken as 100% of viable cells)
Interestingly, proliferative activity of NHF and PAE
was changed, too: quantities of stromal elements were
higher (about 10% in case co-cultivation with PAE and
3–20% in case co-cultivation with NHF). To explain this
fact we will investigate the phenotype of these cells.
It is known that tumor can induce phenotypic changes
associated with transcriptional reprogramming of en-
dothelial cells, which can be detected by expression
profiling: production of growth factors by TC that in-
duce endothelial cells to express specific ligands and
their cognate receptors coordinately [19].
In another set of our study, the proliferative activi-
ties of BC and stromal cells were different indicating
a significant interplay of cells. In particular, cultivation
of T-47D cells with NHF or PAE as well as cultivation
of IFN-modified T-47D cells with NHF or PAE resulted
in increased proliferation of both cell types (Fig. 4).
0
50
100
150
200
250
T-47D PAE T-47D NHF
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
0
50
100
150
200
250
T-47D + IFN PAE T-47D + ІFN NHF
Variant of co-cultivation
Vi
ab
le
c
el
ls
, %
a
b
Fig. 4. Proliferative activities of T-47D (a) and IFN-modified
T-47D (b) cells in noncontact co-cultivation system with NHF
and PAE cells. OY axis represents the percent of viable cells
compared to respective control cells cultured in separate wells
(taken as 100% of viable cells)
BM is considered to be depot for TC and is di-
rect participant of MRD [20–22]. So, the influence
of BM components to TC as an additional factor was
studied. Cells from aspirates of BM of BC patients were
used in new integrated system in vitro. The significant
changes of phenotypic features of both tumor and
normal cells in noncontact co-cultivation system with
addition of BM cells were detected (Fig. 5): variant
of co-cultivation Т-47D + NHF + BM (“Progression”)
and Т-47D + NHF + BM (“Remission”). To investigate
changes at the cellular and molecular level we decided
to analyze the expression of some protein markers
associated with EMT.
The investigation of expression of cell cycle regulator
p21 has shown that the quantity of p21-positive T-47D
cells as well as intracellular localization of p21 remained
unchanged in different variants of co-cultivation (see
Fig. 5, a). The subcellular localization of p21 is very
76 Experimental Oncology 36, 72–78, 2014 (June)
important because this protein performs opposite
functions in nucleus and cytoplasm. In particular,
if p21 localizes in nucleus, it acts as regulator of cell
cycle through inactivation of transcriptional factors
Е2F1, c-Myc, STAT3 [23], as well as inhibitor of replica-
tion due to oppression of subunit of DNA-polymerase
of δ-protein PCNА. When p21 localizes in cytoplasm,
it performs formation of stress-fibrin and focal contacts
that assists the migration of cells. Moreover, p21 in cy-
toplasm demonstrates antiapoptotic functions through
decrease of proapoptotic kinases activity (ASK-1, JNK,
p38) and inhibition of proxapase-2.
0
50
100
150
200
250
300
350
cytoplasmic nucleus
Localization of p21
Ex
pr
es
si
on
o
f р
21
, s
co
re
s
Т-47D isolated
Т-47D + NHF + BM (“Progression”)
Т-47D + NHF + BM (“Remission”)
0
50
100
150
200
250
300
350
CD44 Ki-67 Vimentin E-cadherin
Protein markers
Ex
pr
es
si
on
o
f p
ro
te
in
s,
s
co
re
s
*
a
b
** ***
Fig. 5. Expression of р21 (a), and CD44, Ki-67, vimentin,
Е-cadherin (b) in T-47D cells in noncontact co-cultivation sys-
tem in vitro with NHF and BM cells. *p ≤ 0.005; **p ≤ 0.05; ***p;
evaluation by Н-Score, scores
Next, we investigated CD44 expression. CD44 is ad-
hesion molecule, which plays important role in inter-
action “tumor — microenvironment” and is involved
in processes of migration and invasion of TC. More-
over, CD44 is used as marker of progression and
metastasis at different types of cancer, in particular,
BC. TCs with phenotype CD44+CD24− are considered
as cancer stem cells (CSC). In our study, the quantity
of CD44-positive (CD44+) Т-47D cells were increased
by 26% at their co-cultivation with BM cells (variant
Т-47D + NHF + BM (“Progression”)) compared with
control of cells in isolated wells. Interestingly, the quan-
tity of CD44+-cells was significantly decreased in vari-
ant of co-cultivation Т-47D + NHF + BM (“Remission”)
compared to isolated control (р < 0.005) and com-
pared to these in co-cultivation variant with BM cells
of patients from “Progression” group (р < 0.005)
(see Fig. 5, b).
The next step was to study of E-cadherin expres-
sion in T-47D cells in different variants of co-cultivation:
the quantity of E-cadherin+ T-47D cells in co-cultivation
variant Т-47D + NHF + BM (“Progression”) was de-
creased by 1.8 times compared to isolated control and
was not changed compared to these in co-cultivation
variant Т-47D + NHF + BM (“Remission”) (see Fig. 5, b).
E-cadherin is a marker of epithelial phenotype. Loss
of the E-cadherin molecule is thought to enable metas-
tasis by disrupting intercellular contacts — an early step
in metastatic dissemination and prominently associated
with tumor invasiveness and poor prognosis [24].
Vimentin is a protein of cytoskeleton normally ex-
pressed in cells of mesenchymal origin [25]. It is used
widely as a marker of the EMT and can induces changes
in cell shape, motility, and adhesion during of this pro-
cess [26]. Its expression was investigated in T-47D cells
which were included in different variants of co-cultivation.
Thus, this protein was not detected in control T-47D cells
(isolated) as well as in T-47D cells of variant co-cultivation
Т-47D + NHF + BM (“Remission”), but the expression
of vimentin was observed in T-47D cells in co-cultivation
with NHF and BM cells (“Progression”) (see Fig. 5, b).
Such changes of phenotypic profile of T-47D cells
are the results of their co-cultivation with components
of microenvironment (NHF and BM cells of BC patients
with different BC course). These indicate the sig-
nificant influence of BM cells and humoral factors,
which they produce, on the BC cells which depended
on the state of tumor process in BC patients (progres-
sion or remission of the disease). The phenotypic profile
of TC in co-cultivation variant with BM cells of BC pa-
tients of “Remission” group did not differ from control
cells. But the activation of ЕМT program were observed
in TC in co-cultivation variant with BM cells of BC pa-
tients of “Progression” group, which was associated
with obtaining of more aggressive features by cells and
possible increase of their metastatic potential.
The phenotypic profile of NHF after their co-cul-
tivation with T-47D and BM cells of BC patients with
different courses of tumor process was changed too.
The expression of vimentin, p21 and Slug was inves-
tigated. So, changes of expression of vimentin were
not detected in NHF in any variants of co-cultivation.
Changes of expression of transcription factor of me-
senchymal cells Slug were detected. In co-cultivation
NHF with TC and BM cells of BC patients Slug was relo-
calized to the nucleus of NHF (p < 0.002). Interestingly,
the presence of p21 protein only in cytoplasm of NHF
was observed (Fig. 6). The quantity of p21+ NHF cells
was significantly increased upon co-cultivation with
BM cells of BC patients (“Progression” group) com-
pared with control (р < 0.005).
These results in some way explain the well-
known data about differentiation of fibroblasts into
myofibroblasts in co-cultivation with TC [27]. Stro-
mal elements, which include also fibroblasts, may
be inductors of transcription factors in TC that support
the domination of mesenchymal features in cells.
Such modulations of intracellular processes occur
through the secretion by cells of series of factors
(cytokines, factors of growth, etc.). One of such fac-
Experimental Oncology 36, 72–78, 2014 (June) 77
tors may be epi thelial-stromal interaction 1 (EPST1).
EPST1 has been identified as interferon response
gene exactly in co-cultivation of BC cells with stromal
fibroblasts [28, 29]. Its expression was increased
in postsurgical tumor material of BC patients in con-
trast to control mammary gland tissues. The highest
intensity of expression of EPST1 was determined
exactly in regions of tumor, which closely contacted
with stroma [29].
0
50
100
150
200
250
300
350
p2
cytoplasmic
SLUG
cytoplasmic
SLUG
nucleus
Vimentin
Protein markers
Ex
pr
es
si
on
o
f p
ro
te
in
s,
s
co
re
s
NHF isolated
NHF + Т-47D + BM (“Progression”)
NHF + Т-47D + BM (“Remission”)
*
***
****
****
**
Fig. 6. Expression of р21 (a), Slug and vimentin in NHF cells upon
noncontact co-cultivation with TC and BM cells from BC patients.
*p < 0.005; **p < 0.005; ***p < 0.02; ****p < 0.002; evaluation
by Н-Score, scores
In conclusion, the new integration cell system for
investigation of the mechanisms of interaction between
TC and TME in vitro was developed. The significant
changes in proliferative activity of TC dependently
on its ЕМT-status were detected after their interaction
with fibroblasts and endothelial cells in noncontact
co-cultivation system. BM cells of BC patients had dif-
ferent modifying influence on TC dependent on clinical
BC course. The activation of ЕМT program was revealed
in TC upon noncontact co-cultivation with BM cells
of BC patients with progression of the disease.
ACKNOWLEDGEMENTS
This study was supported with the grant NAS
of Ukraine for Young Scientists “Development of new
integration cell system for investigation of the mecha-
nisms of interaction TC with microenvironment in vitro”,
№ 2.2.5.383 from 01.07.2013.
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