Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer
Aim: To search for additional molecular-biological markers of cancer stem cell (CSC) involved in the development of intra-tumor heterogeneity for the detection of features of the breast cancer (BC) pathogenesis. Materialts and Methods: Expression of estrogen receptors (ER), progesterone receptors (P...
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| Дата: | 2017 |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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| Назва видання: | Experimental Oncology |
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| Цитувати: | Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer / V.F. Chekhun, N.Y. Lukianova, S.V. Chekhun, N.O. Bezdieniezhnykh, T.V. Zadvorniy, T.V. Borikun, L.Z. Polishchuk, O.М. Klyusov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 203-211. — Бібліогр.: 58 назв. — англ. |
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irk-123456789-1385382018-06-20T03:05:21Z Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer Chekhun, V.F. Lukianova, N.Y. Chekhun, S.V. Bezdieniezhnykh, N.O. Zadvorniy, T.V. Borikun, T.V. Polishchuk, L.Z. Klyusov, O.M. Original contributions Aim: To search for additional molecular-biological markers of cancer stem cell (CSC) involved in the development of intra-tumor heterogeneity for the detection of features of the breast cancer (BC) pathogenesis. Materialts and Methods: Expression of estrogen receptors (ER), progesterone receptors (PR), Her2/neu, E- and N-cadherin, CD24, CD44, Bcl-2, Bax, Slug, P-gp, glutathioneS-transferase (GST) and metallothionein in cell lines was determined by the immunocytochemical method. Expression of ER, PR, Her2/neu, CD24 and CD44 in the surgical material of BC patients were determined by the immunohistochemical method. The levels of the miRNA were determined using real-time polymerase chain reaction. Results: Cells of high-grade malignancy (HGM), MDA-MB-231 and MDA-MB-468 are characterized by high expression of stem cell markers compared to the cells of low-grade malignancy (LGM), T47D and MCF-7: CD44 levels in T47D and MCF-7 cells were in range of 72–79 points, which is significantly lower than in HGM cells (p < 0.05). Also, HGM cells with the properties of CSC were characterized by high expression of antiapoptotic proteins, the transcription factor Slug, and low levels of proapoptotic protein Bax (p < 0.05) compared to LGM cells. In cells with CSC characteristics an increased expression of transferrin and its receptor, ferritin, fentorin and hepcidin was revealed indicating activation of the endogenous iron metabolism. The characteristic feature of HGM cells with CSC phenotype were the increased levels of oncogenic miR-221, -155 and -10b by 60%, 92% and 78%, respectively, and decreased levels of oncosuppressive miR-29b, -34a and -200b by 8.4 ± 0.3, 4.6 ± 0.2, and 3.4 ± 0.6 times compared to MCF-7 line cells. It has been established that the development of resistance to cytostatics is accompanied by increased aggressiveness of tumor cells, loss of expression of hormonal receptors and acquiring of stem phenotype. In particular, increased expression of P-gp was observed in BC cells during the development of resistance to doxorubicin, of GST during the development of resistance to cisplatin along with increased CD44 expression (p < 0.05). We have revealed the relation between the presence of cells with the CSC phenotype (CD44⁺CD24⁻/low ) and clinical and pathological characteristics of BC patients, their survival and BC sensitivity to neoadjuvant therapy (p > 0.05). Conclusions: The dependence between the expression of CSC markers and the degree of malignancy of tumor cells, development of resistance to cytostatics in vitro was established as well as the predictive value of the detection of the CSC for the individual prognosis of the BC course and sensitivity of the tumors to the treatment. 2017 Article Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer / V.F. Chekhun, N.Y. Lukianova, S.V. Chekhun, N.O. Bezdieniezhnykh, T.V. Zadvorniy, T.V. Borikun, L.Z. Polishchuk, O.М. Klyusov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 203-211. — Бібліогр.: 58 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138538 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
| topic |
Original contributions Original contributions |
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Original contributions Original contributions Chekhun, V.F. Lukianova, N.Y. Chekhun, S.V. Bezdieniezhnykh, N.O. Zadvorniy, T.V. Borikun, T.V. Polishchuk, L.Z. Klyusov, O.M. Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer Experimental Oncology |
| description |
Aim: To search for additional molecular-biological markers of cancer stem cell (CSC) involved in the development of intra-tumor heterogeneity for the detection of features of the breast cancer (BC) pathogenesis. Materialts and Methods: Expression of estrogen receptors (ER), progesterone receptors (PR), Her2/neu, E- and N-cadherin, CD24, CD44, Bcl-2, Bax, Slug, P-gp, glutathioneS-transferase (GST) and metallothionein in cell lines was determined by the immunocytochemical method. Expression of ER, PR, Her2/neu, CD24 and CD44 in the surgical material of BC patients were determined by the immunohistochemical method. The levels of the miRNA were determined using real-time polymerase chain reaction. Results: Cells of high-grade malignancy (HGM), MDA-MB-231 and MDA-MB-468 are characterized by high expression of stem cell markers compared to the cells of low-grade malignancy (LGM), T47D and MCF-7: CD44 levels in T47D and MCF-7 cells were in range of 72–79 points, which is significantly lower than in HGM cells (p < 0.05). Also, HGM cells with the properties of CSC were characterized by high expression of antiapoptotic proteins, the transcription factor Slug, and low levels of proapoptotic protein Bax (p < 0.05) compared to LGM cells. In cells with CSC characteristics an increased expression of transferrin and its receptor, ferritin, fentorin and hepcidin was revealed indicating activation of the endogenous iron metabolism. The characteristic feature of HGM cells with CSC phenotype were the increased levels of oncogenic miR-221, -155 and -10b by 60%, 92% and 78%, respectively, and decreased levels of oncosuppressive miR-29b, -34a and -200b by 8.4 ± 0.3, 4.6 ± 0.2, and 3.4 ± 0.6 times compared to MCF-7 line cells. It has been established that the development of resistance to cytostatics is accompanied by increased aggressiveness of tumor cells, loss of expression of hormonal receptors and acquiring of stem phenotype. In particular, increased expression of P-gp was observed in BC cells during the development of resistance to doxorubicin, of GST during the development of resistance to cisplatin along with increased CD44 expression (p < 0.05). We have revealed the relation between the presence of cells with the CSC phenotype (CD44⁺CD24⁻/low ) and clinical and pathological characteristics of BC patients, their survival and BC sensitivity to neoadjuvant therapy (p > 0.05). Conclusions: The dependence between the expression of CSC markers and the degree of malignancy of tumor cells, development of resistance to cytostatics in vitro was established as well as the predictive value of the detection of the CSC for the individual prognosis of the BC course and sensitivity of the tumors to the treatment. |
| format |
Article |
| author |
Chekhun, V.F. Lukianova, N.Y. Chekhun, S.V. Bezdieniezhnykh, N.O. Zadvorniy, T.V. Borikun, T.V. Polishchuk, L.Z. Klyusov, O.M. |
| author_facet |
Chekhun, V.F. Lukianova, N.Y. Chekhun, S.V. Bezdieniezhnykh, N.O. Zadvorniy, T.V. Borikun, T.V. Polishchuk, L.Z. Klyusov, O.M. |
| author_sort |
Chekhun, V.F. |
| title |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| title_short |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| title_full |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| title_fullStr |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| title_full_unstemmed |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| title_sort |
association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138538 |
| citation_txt |
Association of cd44⁺cd24⁻/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer / V.F. Chekhun, N.Y. Lukianova, S.V. Chekhun, N.O. Bezdieniezhnykh, T.V. Zadvorniy, T.V. Borikun, L.Z. Polishchuk, O.М. Klyusov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 203-211. — Бібліогр.: 58 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T06:00:24Z |
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| fulltext |
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ���
ASSOCIATION OF CD44+CD24−/low WITH MARKERS
OF AGGRESSIVENESS AND PLASTICITY OF CELL LINES
AND TUMORS OF PATIENTS WITH BREAST CANCER
V.F. Chekhun1, *, N.Y. Lukianova1, S.V. Chekhun1, N.O. Bezdieniezhnykh1, T.V. Zadvorniy1,
T.V. Borikun1, L.Z. Polishchuk1, O.М. Klyusov1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology,
NAS of Ukraine, Kyiv 03022, Ukraine
2Kyiv City Clinical Oncological Center, Kyiv 03022, Ukraine
Aim: To search for additional molecular-biological markers of cancer stem cell (CSC) involved in the development of intra-tumor
heterogeneity for the detection of features of the breast cancer (BC) pathogenesis. Materialts and Methods: Expression of estrogen
receptors (ER), progesterone receptors (PR), Her2/neu, E- and N-cadherin, CD24, CD44, Bcl-2, Bax, Slug, P-gp, glutathione-
S-transferase (GST) and metallothionein in cell lines was determined by the immunocytochemical method. Expression of ER, PR,
Her2/neu, CD24 and CD44 in the surgical material of BC patients were determined by the immunohistochemical method. The
levels of the miRNA were determined using real-time polymerase chain reaction. Results: Cells of high-grade malignancy (HGM),
MDA-MB-231 and MDA-MB-468 are characterized by high expression of stem cell markers compared to the cells of low-grade
malignancy (LGM), T47D and MCF-7: CD44 levels in T47D and MCF-7 cells were in range of 72–79 points, which is signifi-
cantly lower than in HGM cells (p < 0.05). Also, HGM cells with the properties of CSC were characterized by high expression
of antiapoptotic proteins, the transcription factor Slug, and low levels of proapoptotic protein Bax (p < 0.05) compared to LGM
cells. In cells with CSC characteristics an increased expression of transferrin and its receptor, ferritin, fentorin and hepcidin was
revealed indicating activation of the endogenous iron metabolism. The characteristic feature of HGM cells with CSC pheno-
type were the increased levels of oncogenic miR-221, -155 and -10b by 60%, 92% and 78%, respectively, and decreased levels
of oncosuppressive miR-29b, -34a and -200b by 8.4 ± 0.3, 4.6 ± 0.2, and 3.4 ± 0.6 times compared to MCF-7 line cells. It has
been established that the development of resistance to cytostatics is accompanied by increased aggressiveness of tumor cells, loss
of expression of hormonal receptors and acquiring of stem phenotype. In particular, increased expression of P-gp was observed
in BC cells during the development of resistance to doxorubicin, of GST during the development of resistance to cisplatin along
with increased CD44 expression (p < 0.05). We have revealed the relation between the presence of cells with the CSC phenotype
(CD44+CD24-/low) and clinical and pathological characteristics of BC patients, their survival and BC sensitivity to neoadjuvant
therapy (p > 0.05). Conclusions: The dependence between the expression of CSC markers and the degree of malignancy of tumor
cells, development of resistance to cytostatics in vitro was established as well as the predictive value of the detection of the CSC for
the individual prognosis of the BC course and sensitivity of the tumors to the treatment.
Key Words: cancer stem cells, resistance, breast cancer, aggressiveness.
The �eginning of the XXI century is marked �y the
intensification of research on molecular genetic� epi-
genetic and meta�olic factors of tumor cell heteroge-
neity and their plasticity under conditions of cancer
progression and therapy of patients. The post-genome
era gave impetus to the search for a system of new
coordinates for the causes of these processes� the
features of the �iology of the tumor cell and their par-
ticipation in the formation of the relationship �etween
the tumor and the �ody. The importance of individual
differences� which underlie the therapeutic pro�lems
of modern clinical oncology� �ecame evident.
A prominent arsenal of molecular genetic� immu-
nocytochemical and immunohistochemical methods
allowed to identify the key driving force of the initiation
and progression of the malignant process. The theory
of the Virchow — Kennayma �chool� which more than
a hundred years ago envisioned the possi�ility of tumor
development from the remnants of em�ryonic stem
cells tumors received its confirmation [��4]. The ex-
isting concept of cancer stem cells �C�C� appeared
in the mid-����s of the XX century [5]. Recently� the
�iology of C�C is the su�ject of active research� dis-
cussion� and hope. The �asis for such attention was
the accumulated data on their role in the processes
of active proliferation� the creation of protective niche
and the formation of the latent state of the tumor le-
sion [6]. Numerous data are pu�lished on the involve-
ment of C�C in suppressing the immune response [�]�
promoting metastasis [6]� development of resistance
to chemotherapy [8]� and the occurrence of relapses
[�]. Intensive research is �eing carried out on the
network of signaling cascades of C�C and their inter-
cellular interactions that will allow revealing the nature
of the malignant process and determine the current
strategy of diagnosis and therapy [��]. The phenotype
of C�C is extremely diverse and can vary �oth within
the intra-tumoral and inter-tumoral lesions� therefore�
the definition of a network of markers and factors that
Submitted: August 4, 2017.
*Correspondence: E-mail: chekhun@onconet.kiev.ua
Abbreviations used: BC — breast cancer; CP — cisplatin;
CSC — cancer stem cell; Dox — doxorubicin; EMT — epithelial-
mesenchymal transition; ER — estrogen receptors; �ST — gluta-�ST — gluta- — gluta-
thione-S-transferase; H�M — high-grade malignancy; L�M — low-
grade malignancy; MT — metallothionein; PCR — polymerase chain
reaction; P-gp — P-glycoprotein; PR — progesterone receptors.
Exp Oncol ����
��� �� �������
��4 Experimental Oncology ��� �������� ���� ��eptem�er�
contri�ute to the formation of personified the mani-
festation of the signs of stem tumor phenotype is the
main and actual pro�lem of experimental and clinical
oncology [�� �����].
For many decades� the formation of the main pro-
perties of malignant tumors was thought to �e associ-
ated with the accumulation of genetic changes and
disorders in the network of their signaling cascades�
which promote the activation of cell proliferation� inva-
sion and metastasis [�4� �5]. The results were concen-
trated around two major theories of the development
of malignant neoplasms. Thus� according to the clonal
�stochastic� theory� tumor cells can transform any so-
matic cells� and tumor progression occurs as a result
of the appearance of clones that �enefit from survival
due to oncogenic mutations and/or epigenetic modifi-
cations. However� the existing concept of multiple muta-
tions in somatic cells� which deepen the differentiation
and loss of their normal phenotype� has �ecome too
simplistic model of carcinogenesis [�� �6� ��].
According to the hierarchical theory� the tumor
develops from the stem cells� which in the tumor mi-
croenvironment lose vertical system of legitimate self-
control and transformed into a limited su�population�
which �ecame a central and inexhausti�le element
of tumor development and metastatic potential. Biology
of C�C is a key factor in the development� plasticity� and
progression of the disease. �upporters of hierarchical
model argue that only �.�������% of these cells can
provide awakening of the tumor from latent state and
restore the progression of the disease [��]. It is �elieved
that such cells are capa�le of asymmetric division�
which allows continuous replenishment of the C�C and
the generation of a pool of daughter cells that form the
tumor mass [�].
The dynamic varia�ility of the microenvironment
of the C�Cs and the un�alanced network of signaling
cascades contri�ute to the formation of numerous su�-
clones within which each cell differs from one another
�y the structure of the genome� the nature of transcripts�
proteomic elements� etc. [�4� �8���]. �u�clones with
numerous molecular defects in the conditions of the
intra-tumoral environment �ecome the source of the
heterogeneous pool of cells that can selectively support
oncogenesis� have multiple targets� and exhi�it varia�le
sensitivity to the action of cytostatics.
A large num�er of experimental studies and reviews
is devoted to the identification of surface antigens of C�C
[����5]. However� it has �een proven that most markers
are not sta�le and depend on the individual characteris-
tics� as well as vary at different stages of the tumor pro-
cess. Therefore� our research is aimed at finding markers
and factors for the inter- and intra-tumor features of C�C
that are involved in the formation of a heterogeneous
cell pool and contri�ute to the pathogenesis of �reast
cancer �BC�. Attention was focused on the search for
molecular-�iological indexes that could characterize
additional features of the C�C phenotype and to reveal
the mechanisms of their participation in the varia�ility
and plasticity of malignant cells.
MATERIALS AND METHODS
Cell lines and drug treatment. The studies were
performed in vitro on 6 human BC cell lines: T4�D —
metastatic �reast ductal carcinoma; MDA-MB-��� and
MDA-MB-468 — metastatic �reast adenocarcinoma;
MCF-� — invasive �reast ductal carcinoma� MCF-�/CP�
MCF-�/Dox — its variants� resistant to cisplatin �CP�
or doxoru�icin �Dox�� respectively.
T4�D cells were cultured in RPMI-�64� me-
dium ��igma� U�A� supplemented with �ovine in-
sulin ��.� U/ml� and ��% fetal �ovine serum �FB��.
MCF-� cells were grown in DMEM ��igma� U�A� supple-
mented with recom�inant human insulin ��.�� mg/ml�
and ��% FB�. MDA-MB-��� and MDA-MB-468 cells
were cultured in Lei�ovitz’s L-�5 medium ��igma�
U�A� supplemented with ��% FB�. All cultures were
grown on glass cover slips in humidified atmosphere
with 5% CO� at �� °C. The cell lines were o�tained
from the Bank of Cell Lines from Human and Animal
Tissues of the R.E. Kavetsky Institute of Experimental
Pathology� Oncology and Radio�iology �IEPOR� of the
National Academy of �ciences �NA�� of Ukraine.
The peculiarities of phenotype were evaluated
taking into account receptor status� proliferation ac-
tivity and invasive properties [�6]. MDA-MB-��� and
MDA-MB-468 cells were considered highly malignant
�a�sence of steroid hormone receptors� high invasive
potential and low adhesive properties�. T4�D and
MCF-� cells were considered low malignant �high ex-
pression of estrogen and progesterone receptors� low
invasive activity�.
The resistant variants MCF-�/Dox and MCF-�/CP were
originated �y growing parental MCF-� cells with rising
concentrations of CP �from �.�� to 6 μg/ml� or Dox
�from �.� to �� μg/ml�� respectively. CP and Dox were
added twice a week after reseeding. Every � months�
cell survival rate was analyzed �y MTT assay. IC5� val-
ues for MCF-� and MCF-�/CP cells were �.�5 and
� μg/ml of CP� respectively� and for MCF-� and MCF-�/
Dox cells — �.5 and 8 μg/ml of Dox� respectively.
Therefore� MCF-�/CP were 4 times as much resistant
to the cytotoxic effect of CP and MCF-�/Dox cells were
�6 times as much resistant to the cytotoxic effect of Dox
as parental MCF-� cells.
Immunocytochemical assay. The cells were
fixed on cover slips �in triplicate for each sample�
in ice-cold methanol: acetone ��:�� at −�� °C for
��� min and incu�ated with �% �ovine serum al�umin
solution for �� min. For immunocytochemical assay�
primary anti-CD�4 �clone �N�B� �NeoMarkers� U�A�;
anti-Вcl-� �clone ��4�� anti-Вax �Polyclonal Ra��it
Anti-Human Anti�ody� �DakoСytomation� Denmark�;
anti-CD44/HCAM �clone �56��C���� anti-N-Cadherin
�clone CD��5� �Diagnostic Bio�ystems� U�A�; anti-
E-cadherin �clone NCH-�8�� anti-P-glycoprotein
�clone C����� anti-�lug �clone �H5� �Thermo�cien-
tific� U�A�; anti-transferrin receptor � �clone B��6���
�Bioworld Technology� U�A�� anti-transferrin �clone
a�8�4���� anti-ferritin light chain �clone a�6������
anti-hepcidin �clone a����6��� anti-metallothionein
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ��5
�clone a�����8�� anti-glutathione-�-transferase
�clone a��8�8��� anti-ferroportin �clone a��8�66;
A�cam� �A�cam� U�A�; anti-ferritin heavy chain �clone
GTX6����� �Gene Tex� Bioworld Technology� U�A�.
UltraVision LP Detection �ystem �La� Vision� Thermo
�cientific� U�A� and DAB Quanto �Thermo �cientific�
were used according to the instructions of the manu-
facturers. When the immunocytochemical reaction was
completed� the cells were stained with hematoxylin
�y Mayer and placed in Faramount Aqueous Mounting
Medium �DakoCytomation� Denmark�. Results were
analyzed �y light microscopy �× ����� oil immersion�
with the use of classical H-�core method:
S = 1 • N1+ + 2 • N2+ + 3 • N3+,
where � — “H-�core” index� N�+� N�+ and N�+ —
num�er of cells with low� medium or high marker ex-
pression [��]. The level of studied markers expression
was assigned as follows: low — from � to ��� H-�core
points� medium — from ��� to ��� H-�core points�
and high — from ��� to ��� H-�core points.
Total RNA isolation. Total RNA extraction was
performed� using Nucleo�pin® miRNA �MACHEREY-
NAGEL Gm�H & Co. KG� Germany�. Concentration
of RNA was measured� using NanoDrop ����c �pec-
trophotometer �Thermo �cientific� U�A�. The purity
of isolated RNA was controlled� analyzing the ratio
of OD at �6�/�8� nm. RNA was dissolved in TE �uffer
and stored at −�� С.
�ingle-stranded cDNA was synthesized from
��� ng of total RNA� using TaqMan® MicroRNA Kit for
reverse transcription.
Real-time quantitative reverse transcription
polymerase chain reaction (qRT-PCR). Preparation
of reverse transcription reaction mix was performed
according to manufacturer’s protocol. Reverse tran-
scription was performed at a “Tertsik” thermal cycler
�DNA Tehnologіya� Russian Federation�. qRT-PCR was
performed on Applied Biosystems ����HT Fast Real-
Time PCR �ystem using TaqMan® MicroRNA primers
and manufacturer’s protocol.
�mall nucleolar RNA RNU48 was used as an endo-
genous control for normalization of miRNA expression.
Relative expression of the studied miRNAs was identi-
fied �y comparative Ct method [�8]. Experiments were
performed in triplicates for each line� and PCR was
performed three times for each sample. Expression
differences �etween the studied miRNA levels relative
to control were calculated �y the formula:
Fold change = 2−ΔΔCt [�8]�
where ΔCt �target — control� is equal to the dif-
ference �etween threshold cycles for miRNA �target�
and the threshold cycle for RNU48 �control� �ΔCt
�target — control� = Ct target − Ct control�. ΔΔCt = ΔCt
�experiment� − ΔCt �control�.
Characteristics of the clinical material. The
ex vivo study is �ased on the examination of clinical and
morphological parameters of ��� patients with BC. Tu-
mor samples were stored in the clinical data�ase of the
Department of Monitoring Tumor Process and Therapy
Design at the R.E. Kavetsky IEPOR of the NA� of Ukraine.
A retrospective analysis of the features of C�C
markers expression and the study of the dependence
of their expression on the clinical morphological pa-
rameters of BC and survival rates of patients were
carried out on the material of �4� BC patients with
I�II stages� who received special treatment in Kyiv City
Clinical Cancer Center during ���5����� �Ta�le ��.
Histological type of tumors was verified on histologi-
cal sections of tumors’ paraffin �locks �staining with he-
matoxylin and eosin� according to the WHO International
Histological Classification ����6�. Depending on clinical
indications� patients underwent organ-saving surger-
ies or radical Madden mastectomies� and adjuvant
polychemotherapy �CAF or AC schemes with �� day
interval� the num�er of courses varied from 4 to 6�� ac-
cording to the approved in Ukraine �tandards of Treat-
ment of Breast Cancer Patients. Postoperative radiation
therapy was performed on the postoperative scar� axil-
lary� parasternal and supraclavicular regions; the single
focal dose was � Gy� and the total focal dose — 4� Gy.
General clinical description of �4� BC patients
with stage I�II is presented in Ta�le �. The num�er
of BC patients with stage I was �6.6%� with stage II —
��.4%. Patients’ age varied from �4 to �� years� mean
age was 5�.� ± 4.4 years. According to the results
of complex examination of patients �X-ray� ultrasound�
etc.�� metastases in regional lymph nodes �N���� were
detected in ��.4% cases� distant metastases were not
detected. The morphological study has determined
infiltrating ductal BC �6�.�%� more often� than lo�ular
BC ���.8%�. More often moderate differentiation of BC
�4�.�%� as compared with high and low differentiation
��8.� and ��.�%� correspondingly� was o�served.
Table 1. Clinical characteristics of patients with BC of stage I–II
Index Number of patients
n %
Total number of patients 143 100
Age of patients (years)
Average 52.1 ± 4.4
Range 34–70
Menstrual function
Preserved 51 35.7
Menopause 92 64.3
BC stage by TNM
I 38 26.6
II 105 73.4
Metastases in regional lymph nodes (Category N)
N0 101 70.6
N1–N3 42 29.4
Histopathology of BC
Infiltrative ductal carcinoma 99 69.2
Infiltrative lobular cancer 44 30.8
Differentiation grade of BC
�1 (high) 40 28.0
�2 (moderate) 70 49.0
�3 (low) 33 23.0
Molecular subtype of BC
Luminal А 56 39.2
Luminal B 34 23.8
Her2/neu-positive 31 21.7
Basal 22 15.3
Analysis of the results of immunohistochemi-
cal study of estrogen receptors �ER�� progesterone
receptors �PR� and Her�/neu expression evidenced
luminal A su�type in ��.�% cases� luminal B su�type —
in ��.8%� Her�/neu-positive su�type — in ��.�% and
�asal su�type — in �5.�% cases �see Ta�le ��.
��6 Experimental Oncology ��� �������� ���� ��eptem�er�
The study of the association of expression
of C�C markers and the effectiveness of neoadju-
vant chemotherapy was performed on the material
of �48 BC patients with ІІ�ІІІ stages� who received
special treatment in Kyiv City Clinical Cancer Center
during ��������5. Tumor stage was determined ac-
cording to the TNM classification �6th edition� �����.
The histological type of the resected tumors was
verified upon morphological study �hematoxylin and
eosin staining� according to the International Histo-
logical Classification of the World Health Organiza-
tion ����6�. All patients were treated with NACT. The
course included ��6 cycles of chemotherapy �y FAC�
AC scheme with �� day-intervals. NACT efficacy
was evaluated every � cycles �y mammography
according to RECI�T criteria [��� ��]. Depending
on the degree of clinical effect of NACT �according
to RECI�T criteria� all patients were distri�u ted into
� groups. The �st group included �� BC patients who
have demonstrated a positive response to the NACT:
complete regression was o�served in �� patients�
partial regression — in 68 patients. �nd group was
formed of 65 women with BC resistant to the treat-
ment� including 56 patients with sta�ilization of tumor
growth and �� patients with BC progression in the set-
ting of NACT. All patients were informed and agreed
to the use of �iopsy material for research purposes.
The clinical characteristics of �48 patients with
BC of stage II�III are shown in Ta�le �. According
to the clinical data� the age of patients ranged from
�8 to �� years� mean age was 5�.� ± 6.4 years. The
majority of patients �56.�%� were at menopause�
the menstrual function was preserved in 44.�%
of patients. The num�er of patients with BC of stage
II was 6� �45.�%�� of stage III — 8� patients �54.�%�.
Upon comprehensive examination �X-ray� ultrasound�
la�oratory� conducted �efore treatment� metastases
�N���� in regional lymph nodes were found in ��� pa-
tients ��6.�%�.
Table 2. Clinical characteristics of patients with BC of stage II–III
Index Number of patients
n %
Total number of patients 148 100
Age of patients (years)
Average 51.2 ± 6.4
Range 28–72
Menstrual function
Preserved 65 44.0
Menopause 83 56.0
BC stage by TNM
II 67 45.3
III 81 54.7
Metastases in regional lymph nodes (category N)
N0 35 23.7
N1–N3 113 76.3
Histopathology of BC
Infiltrative ductal carcinoma 111 75.0
Infiltrative lobular cancer 37 25.0
Differentiation grade of BC
�1 (high) 37 25.0
�2 (moderate) 72 48.6
�3 (low) 39 26.4
Molecular subtype of BC
Luminal А 68 45.9
Luminal B 32 21.6
Her2/neu-positive 14 9.5
Basal 34 23.0
The distri�ution of patients �y histological type
of BC showed that most patients had infiltrative duc-
tal carcinoma ��5.�%� of moderate differentiation
�48.6%�. The greatest incidence was registered for
luminal A su�type — 45.�%. Incidence of luminal B�
Her�/neu-positive and �asal su�types of BC was ��.6;
�.5 and ��.�% respectively.
Immunohistochemical assay. Expression of ER�
PR� Her�/neu� proliferative activity marker �Ki-6���
СD�4 and CD44 in tumor cells were studied on paraffin
sections �4�5 microns� of �iopsy and operation ma-
terial. As the primary anti�odies used were the same
as in the immunocytochemical study. To visualize the
reaction� EnVision �ystem kit �Dako L�AB� system�
Denmark� was used according to the manufac-
turer’s recommendations. The sections were stained
with Mayer’s hematoxylin. The expression of molecular
markers was evaluated �y a semiquantitative method.
Analysis of the results was performed using optical
microscopy ����� oil immersion ���� using the clas-
sical method of H-�core [��].
Statistical analysis. �TATI�TIСA 6.� computer
program ��tat�oft Inc.� U�A� was used for statistical
processing of the o�tained results. Differences �e-
tween the average values were compared with the use
of �tudent’s t-test; correlation analysis was performed
using Pearson correlation coefficient. Differences were
considered as significant with the pro�a�ility not less
than �5% �р < �.�5�.
RESULTS AND DISCUSSION
Hypotheses of tumorigenesis and heterogene-
ity of BC with the participation of the C�C have
great �iological and clinical significance and are
actively discussed in the modern scientific litera-
ture. In particular� in ����� for the first time� surface
antigens of CD44+CD�4� on tumor-forming cells
of BC were detected [��]. Their population is less
than �.���% of the tumor mass [��]. High levels
of expression of the epithelial specific antigen �E�A+��
CD44 marker �CD44+� and the a�sence or low ex-
pression of CD�4 �CD�4�/low� were detected on the
surface of these cells. The surface cellular protein
CD44 is a receptor of hyaluronan� as well as some
other ligands� which include osteopontin� collagen
I and IV types� metalloproteinases of the extracellular
matrix [��]. The interaction of CD44 with its ligand
�hyaluronan� leads to activation of a num�er of intra-
cellular signaling pathways that promote cell survival.
�u�sequently� numerous studies confirmed the high
tumorigenic activity of CD44+CD�4�/low cells isolated
from a variety of �iological material �primary cultures
and sta�le cell lines in vitro� �iopsy� operation mate-
rial� primary and serial xenografts of tumor tissue
of the mammary gland� [�4� �5]. According to data
from own studies concerning the markers of C�C�
the expression of CD�4 was noted only in three lines
of BC cells of low and high-grade malignancy: T4�D
�55.� ± �.� points�� MDA-MB-��� ��4.� ± �.6 points��
and MDA-MB-468 ��5�.� ± 4.� points� �Fig. ��.
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ���
0
50
100
150
200
250
300
350
T4
7D
M
CF
-7
M
DA
-М
В-
23
1
M
DA
-М
В-
46
8
M
CF
-7
/С
Р
M
CF
-7
/D
ox
Low High
Malignancy level CD24
CD44
Po
in
ts
, H
-S
co
re
Fig. 1. Expression of C�C markers in human BC cells of varying
degrees of malignancy and sensitivity to cytostatics �H-score�
points�
Instead� the expression of CD44 was esta�lished
in all BC lines� �ut its degree was different. On aver-
age� it was in the range of ����� points in the lines
of low malignant cells� which is significantly less than
in high malignant cells� which also showed the vari-
a�ility of the expression of this marker on average from
�6� to ��8 points. The highest level of CD44 expres-
sion was o�served in malignant MDA-MB-��� cells
���8.� ± �.� points� compared to other lines with ag-
gressive phenotype �p < �.�5�. As can �e seen from the
data presented in Fig. �� the development of the phe-
notype of drug resistance to CP and Dox in MCF-� cells
is accompanied �y an increase in the num�er of cells
with the C�C phenotype. This is evidenced �y a de-
crease in the num�er of cells that are positive for ex-
pression of CD44 ��.4 and �.6 times� in MCF-�/CP and
MCF-�/Dox su�lines� respectively. Consequently�
among all the examined cells� low levels of stemness
were determined in the low-malignant MCF-� line� while
the high stemness level� according to the findings�
was o�served in a population of MDA-MB-468 highly
malignant cells and cells resistant to cytostatics. It has
�een shown that the level of C�C markers expression
directly correlates with the proliferative activity of low-
and highly malignant BC cells �r = �.54�.
A little later C. Ginestier et al. [�6] found that more
significant �iochemical marker of C�C can �e a level
of activity of aldehyde dehydrogenase � �ALDH���
which provides cell resistance to cytotoxic drugs. The
high expression of ALDH� correlates with the aggres-
siveness of the BC course and resistance to chemo-
therapy [��� ��� �8]. In general� it is known that in the
cells with the C�C phenotype high activity enzymes
of phase I and II of xeno�iotics meta�olism is o�served.
In particular� high expression of ATP-�inding trans-
porters of xeno�iotics� including ABCG�� ensures the
withdrawal of many drugs �taxanes� topoisomerase
inhi�itors� antimeta�olites� from cells� while high activ-
ity of various enzymes inactivating anticancer drugs�
including ALDH� NAT� glutathione-�-transferase �G�T��
provides detoxification of cyclophosphamide� Dox�
paclitaxel� etc. [���4�].
According to our data� an increase of the stemness
during the development of drug resistance is accom-
panied �y changes in the expression of P-glycoprotein
�P-gp�� G�T and metallothioneins �MT�. As can �e seen
from the data shown in Fig. �� upon the development
of resistance to Dox expression of proteins of I phase
xeno�iotics meta�olism �ABC-transporter — P-gp�
increased �y �6.5 times� and upon the development
of resistance to CP expression of proteins of I phase
xeno�iotics meta�olism� including G�T� increased
�y �� times. Formation of phenotype of resistance
to CP and Dox is also associated with a reduction
in the num�er of cells positive for expression of MT
��y 8�.4 and 84.8%� respectively�.
Po
in
ts
, H
-S
co
re
0
50
100
150
200
250
300
P-gp GST MT
MCF-7 MCF-7/CP MCF-7/Dox
* *
*
Fig. 2. Expression of proteins of xeno�iotic meta�olism in the
BC cell lines of varying degrees of sensitivity to cytostatics �H-
score� points�. *p < �.�5 compared with MCF-� cell line
A network of signaling cascades� which constrains
the C�C from the active phase of differentiation�
is widely studied. In particular� inhi�ition of one of the
elements of Wnt� kinase cascade associated with
mem�rane glycoprotein of BC cell line results in loss
of stem properties [4�]. �ignificant role in formation
of pathways regulating cell proliferation� differentia-
tion� apoptosis and epithelial-mesenchymal transition
�EMT� �elongs to a conservative intracellular cascade
Notch� which is activated �y the interaction of the trans-
mem�rane ligand from Jagged family �Jagged � and ��
and Delta family with receptors Notch � and 4 [4�].
The family of Notch transmem�rane proteins includes
extracellular sequences — the domains EGF and D�L�
which take an active part in lateral inhi�ition and em-
�ryogenesis [44]. It is known that the first step in the
activation of EMT is the rupture of various �onds �e-
tween epithelial cells� which helps the latter to acquire
the mesenchymal features and increases their migra-
tion and metastatic activity [45�4�]. Transmem�rane
structures involved in cell adhesion — cadherins — are
mem�ers of the family of calcium-dependent adhesion
molecules� which are involved in intercellular contacts
such as zona adherence. The cytoplasmic domain
of E-cadherin is a�le to �ind to cytosolic protein via
catenins� forming the cadherin-catenin complexes that
�ind E-cadherin to other mem�rane proteins [5�� 5�].
The complex of CD44�E-cadherin-catenin is involved
in the complex signaling process in the cell �Wnt/
catenin signaling pathway�� in intracellular integration�
differentiation� inflammation� morphogenesis in normal
state and pathology [48]. It is known that the transcrip-
tion factor �lug is capa�le to suppress the expression
of E-cadherin� �y �inding to its promoter� promoting
an increase in the migration properties of cells. Another
molecule of adhesion associated with an increased level
of invasion of tumor cells is N-cadherin [5�� 5�]. There-
��8 Experimental Oncology ��� �������� ���� ��eptem�er�
fore� the studied �y us proteins of the family of cadherins
and the �lug transcription factor� which may �e associ-
ated with the Notch signaling� allow us to expand our un-
derstanding of the network of cascades involved in the
regulation of C�C. In particular� the highest expression
rates for E-cadherin were o�served in low-grade ma-
lignant BC lines T4�D and MCF-� ��5�.� ± 5.� points
and �68.� ± 4.6 points� p > �.�5� respectively� �Fig. ��.
Po
in
ts
, H
-S
co
re
*
*
*
0
50
100
150
200
250
300
350
T47D MCF-7 MDA-MB-231 MDA-MB-468
Low High
E-cadherin N-cadherin Slug
Malignancy level
Fig. 3. Indexes of expression of markers of intercellular adhe-
sion and transcription factor in BC cell lines of different degrees
of malignancy �H-score� points�. *p < �.�5 compared with cells
of low degree of malignancy
At the same time� in cells of high-grade malignancy
significant varia�ility of expression of this molecule
of intercellular adhesion was revealed. Thus� in the
MDA-MB-468 line it was �5�.� ± �.5 points� while in the
MDA-MB-��� line with a high level of stemness� it was
less than 5�.� ± �.� points. According to the data o�-
tained �Fig. ��� high-grade malignant lines also differed
in the elevated levels of expression of the �lug transcrip-
tion factor. The highest expression rates for this molecu-
lar marker were o�served in the MDA-MB-��� line with
a high level of stemness ���6.� ± 8.4 points�. It should
�e noted that most cell lines of varying degrees of ma-
lignancy are characterized �y low levels of N-cadherin
expression� which is likely to indicate that this intercel-
lular adhesion molecule is not involved in the formation
of a stem phenotype. Thus� the data o�tained �y us show
the key role of the molecules of the cadherin complex
and the transcription factor �lug in the activation of stem
cells. The loss of epithelial properties �y the cells in the
process of dedifferentiation upon EMT can �e one of the
ways for the acquisition of the C�C phenotype �y tumor
cells. Preventing the loss of epithelial dedifferentiation�
in particular the level of E-cadherin� may �e one of the
priority treatment approaches for patients with BC.
However� the main pro�lem of modern clinical oncolo-
gy remains the formation of resistance to medical therapy�
and every step in understanding the mechanisms of this
phenomenon can �ring us closer to overcoming one of the
most complex medical and �iological pro�lems of our time
[�� 54�56]. The study of the plasticity of C�Cs associ-
ated with EMT and the change in the apoptotic program
of cells and the factors of their epigenetic modeling was
the su�ject of our further research� since C�Cs� through
conservative resources� survive on the �ackground
of chemotherapy and restore a heterogeneous popula-
tion of tumor cells. First� this happens at the expense
of C�Cs� which divide slowly in the G� phase of the cell
cycle [��� 4�� 5�]. �econdly� �ut equally important� the
resistance to apoptosis may �e manifested �y changing
the level of expression of pro- and anti-apoptotic proteins
[58]. This is confirmed �y data from our studies� accord-
ing to which high-grade malignant BC cell lines with high
levels of stemness are characterized �y an increase in the
expression of the anti-apoptotic protein Bcl-� and a de-
crease in the level of proapoptotic protein Bax �y more
than �.86 and �.4 times� respectively� in comparison with
MCF-� cells �Fig. 4�.
Po
in
ts
, H
-S
co
re *
*
*
*
0
50
100
150
200
250
300
T47D MCF-7 MDA-MB-231 MDA-MB-468
Malignancy level
Low High
Bcl-2 Bax
Fig. 4. Indexes of expression of proteins-regulators of apoptosis
in BC cells of low- and high-grade malignancy �H-score� points�.
*p < �.�5 compared with cells of low degree of malignancy
Nontraditional �ut important component of the
stem cell phenotype may �e markers of iron-containing
proteins and miRNAs. Thus� the most pronounced dif-
ference in the expression of iron-containing proteins�
according to our data� is o�served in the BC cell lines
of a high degree of malignancy� which are characterized
�y the expression of C�C markers. In particular� in the
T4�D and MCF-� cells� the level of expression of the main
proteins of iron meta�olism �transferrin and its receptor�
ferritin� ferroportin� and hepcidin� was average and did
not exceed �4� points �Fig. 5�. Instead� in cells of high
degree of malignancy� positive for the expression of C�C
markers� MDA-MB-��� line� the expression of these
proteins was higher and amounted to �����4� points.
TFR
TF
FH
FL
FPN
Heps
High malignancy level
Low malignancy level0
50
100
150
200
250
300
Fig. 5. Indexes of expression of proteins of iron meta�olism
in low- and high-grade malignant BC cells �H-score� points�.
TFR — transferrin receptor; TF — transferrin; FH — ferritin
heavy chains; FL — ferritin light chains; FPN — ferroportin;
Heps — hepcidin
�ome differences we have also o�served in miRNA
expression levels depending on the level of human
BC cells stemness of varying degrees of malignancy.
It is shown that the characteristic feature of high-grade
Experimental Oncology ��� �������� ���� ��eptem�er���� �������� ���� ��eptem�er� ��eptem�er� ���
malignant cells with stemness features is increased
expression of oncogenic miRNAs and reduced ex-
pression of antioncogenic miRNA responsi�le for
the passage of the cell cycle� invasive and adhesive
properties� proliferative activity and apoptosis �Fig. 6�.
Thus� in the MDA-MB-��� cell line expression levels
of miR-���� -�55 and -��� were �y 6�%� ��% and
�8% higher� and the levels of miR-���� and -����
-�4a were 8; 4; 4.6 and �.4 times lower compared
with MCF-� cells.
0.76 0.33
1.1
12.02
2.75
1.55
miR-221
miR-10b
miR-155
miR-29b
miR-34a
miR-200b
2.67
2.985
34.985
0.44
0.35
0.55
Fig. 6. Indexes of expression of oncogenic and antioncogenic
miRNAs in low- and high-grade malignant BC cells� positive for
expression of C�C markers �H-score� points�
Thus� the results we received in the in vitro system
indicate that there are significant correlations �etween
the level of aggressiveness of the BC cells and the level
of their stem phenotype.
At the next stage� we analyzed the peculiarities
of the expression of C�C markers in tumors of patients
with BC and their significance in the prognosis of the
disease. We have revealed the dependence of the
frequency of tumors with the C�C markers was deter-
mined on the molecular su�type: the higher num�er
of tumors with C�C markers was o�served in patients
with BC of �asal su�type in comparison with luminal
one �r = �.54� p < �.�5�. We have found the existence
of an associative relation �etween a higher frequency
of tumors with C�C markers in tumors with low dif-
ferentiation grade compared with high differentiation
grade �p < �.�5�� as well as the close correlation �e-
tween the frequency of such cells in the primary tumor
and the metastases of BC in regional lymph nodes
�r = �.6�� p < �.�5� �Fig. ��.
І ІІ Positive Negative G1 G2 G3
Stage Metastases in regional
lymph nodes
Grade of differentiation
Nu
m
be
r o
f C
SC
-p
os
iti
ve
tu
m
or
s,
%
0
10
20
30
40
50
60
70
80
Fig. 7. Distri�ution of tumors with C�C phenotype depending
on clinical and pathological features of BC
We have also esta�lished certain patterns in the
study of the dependence of the expression of C�C
markers in tumor cells from the sensitivity of BC pa-
tients to neoadjuvant chemotherapy. The highest per-
centage of cases positive for expression of C�C mark-
ers was registered in a group of patients with tumors
resistant to neoadjuvant chemotherapy �y the FAC
and AC schemes �sta�ilization or progression of the
tumor process according to RECI�T criteria�. In most
tumors �8� and ��%� of patients with luminal A and
luminal B su�types that showed a positive response
to treatment� no expression of C�C was revealed. The
highest rates of C�C markers expression in tumor cells
of 88% and ��% of patients were identified in the �asal
and Her�/neu-positive BC groups that were resistant
to neoadjuvant chemotherapy �Fig. 8�.
0
10
20
30
40
50
60
70
80
90
100
Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant
Luminal A Luminal B Hеr2/neu -positive Basal
Nu
m
be
r o
f t
um
or
s,
%
CSC positive CSC negative
Fig. 8. Distri�ution of tumors with C�C phenotype depending
on the sensitivity of patients with BC to neoadjuvant chemo-
therapy.
In analyzing the survival rates of patients with BC�
it was found that survival of patients with �asal BC was
significantly higher in the a�sence of CD44+CD�4-/low
cells in tumors and� accordingly� less — in the pres-
ence of such cells �Log-rank test� p < �.�5�. In patients
with luminal A and luminal B su�types no significant
changes in the survival rate of patients� depend-
ing on the presence or a�sence of CD44+CD�4-/low
cells �Log-rank test� p > �.�5� in tumor cells was re-
vealed. To determine the value of expression indexes
of CD44+/СD�4-/low C�C markers in tumor cells in pa-
tients with BC and to determine their use in clinical
practice as a prognostic criterion� a Cox regression
analysis was performed. The relia�le dependence
of the expression of tumor stem cell markers for �asal
�triple receptor-negative� molecular BC su�type was
esta�lished� indicating the possi�ility of using such
molecular markers of tumor stem cells as CD44+ and
CD�4-/low as markers for predictive estimation of indi-
vidual prognosis of patients with �asal BC.
Thus� ex vivo on clinical material we have confirmed
the participation of C�C in the formation of aggressive-
ness of the BC course and its sensitivity to neoadjuvant
chemotherapy.
Consequently� our analysis of the literature and
the results of our studies indicate that there is a wide
regulatory network and markers of activity of C�C indi-
cating their active role in the pathogenesis of BC. The
deepening of knowledge a�out molecular� genetic and
epigenetic links in the formation of the C�C phenotype
can �e useful in the search for new methods that should
�e directed to the deviations and elimination of C�C
��� Experimental Oncology ��� �������� ���� ��eptem�er�
through the influence on their mem�rane markers�
�lockade of the corresponding signaling molecules�
modification of epigenetic molecules and regulation
of levels of growth factors of the microenvironment.
An alternative and promising approach to influence
C�C is immunotherapy. The use of antitumor vaccines
will promote the activation of T cell immunity and allow
recognition of the antigenic components of the stem
phenotype in a heterogeneous tumor cell pool.
�ources of Funding:
• NA� of Ukraine �cientific Project �����������
No �.�.5.4�� “Molecular-�iological factors of can-
cer cells heterogeneity and varia�ility of clinical
course of hormone sensitive tumors”;
• NA� of Ukraine �cientific Project �����������
No �.�.5.��5/� “Investigation of cancer-associat-
ed miRNAs as extratumor predictive �reast cancer
markers”.
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