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DOI: 10.21294/1814-4861-2018-17-4-75-80 УДК: 618.19-006.6-091.8
Для цитирования: Савельева О.Е., Таширева Л.А., Булдаков М.А., Мухамеджанов Р.Х., Кайгородова Е.В., Денисов Е.В., Завьялова М.В., Перельмутер В.М. Экспрессия CXCR4 в различных популяциях циркулирующих и одиночных опухолевых клеток рака молочной железы. Сибирский онкологический журнал. 2018; 17 (4): 75-80. - doi: 10.21294/1814-4861-2018-17-4-75-80.
For citation: Savelieva O.E., Tashireva L.A., Buldakov M.A., MukhamedzhanovR.H., Kaigorodova E.V., Deni-sov E.V., Zavyalova M.V., Perelmuter V.M. CXCR4 expression in different subsets of CTCS and single (detached) breast cancer cells. Siberian Journal of Oncology. 2018; 17 (4): 75-80. - doi: 10.21294/1814-4861-2018-17-4-75-80.
ЭКСПРЕССИЯ CXCR4 В РАЗЛИЧНЫХ ПОПУЛЯЦИЯХ ЦИРКУЛИРУЮЩИХ И ОДИНОЧНЫХ ОПУХОЛЕВЫХ КЛЕТОК
РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ
О.Е. Савельева12, Л.А. Таширева1, М.А. Булдаков12, Р.Х. Мухамеджанов3, Е.В. Кайгородова1, 3, Е.В. Денисов12, М.В. Завьялова123, В.М. Перельмутер1
Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук, г Томск, Россия1
Россия, 634009, г. Томск, пер. Кооперативный, 5. E-mail: olga.sav.1980@gmail.com1 Национальный исследовательский Томский государственный университет, г. Томск, Россия2 Россия, 634050, г Томск, пр. Ленина, 362
Сибирский государственный медицинский университет, г Томск, Россия3 Россия, 634050, г Томск, Московский тракт, 23
Аннотация
Целью исследования явилось определение экспрессии CXCR4 в различных популяциях циркулирующих (ЦОК) и одиночных (дискретных) опухолевых клеток рака молочной железы. Материал и методы. В исследование были включены 35 пациенток с инвазивной карциномой неспецифического типа молочной железы (T1-4N0-2M0) в возрасте от 29 до 69 лет. Экспрессию CXCR4 в различных популяциях ЦОК оценивали методом проточной цитометрии. Для оценки экспрессии CXCR4 в аналогичных популяциях одиночных (дискретных) опухолевых клеток в первичной опухоли использовали метод конфокальной микроскопии. Результаты. Нами было установлено, что CXCR4 экспрессировался ЦОК без признаков стволовости и эпителиально-мезенхимального перехода (ЭМП), ЦОК с признаками ЭМП, но без маркеров стволовости, а также ЦОК с признаками стволовости, но без признаков ЭМП. У всех пациенток в крови ЦОК с признаками стволовости и ЭМП не экспрессировался CXCR4. В первичной опухоли молочной железы CXCR4 обнаруживался как на одиночных (дискретных) опухолевых клетках без признаков стволовости с признаками ЭМП, так и на клетках с маркерами стволовости и ЭМП. У всех пациенток в образцах первичной опухоли отсутствовали стволовые и нестволовые клетки без признаков ЭМП. Заключение. Таким образом, CXCR4 экспрессируются на различных популяциях ЦОК. Экспрессия CXCR4 не зависит от наличия или отсутствия признаков стволовости и/или ЭМП в опухолевых клетках. Также мы показали, что некоторые популяции одиночных (дискретных) опухолевых клеток в первичной опухоли характеризуются способностью презентировать на своей мембране CXCR4 и могут являться источником соответствующих популяций ЦОК.
Ключевые слова: ОХОК4, циркулирующие опухолевые клетки, стволовые опухолевые клетки, одиночные (дискретные) опухолевые клетки, эпителиально-мезенхимальный переход, рак молочной железы.
CXCR4 EXPRESSION IN DIFFERENT SUBSETS OF CTCs AND SINGLE (DETACHED) BREAST CANCER CELLS
O.E. Savelieva12, L.A. Tashireva1, M.A. Buldakov12, R.H. Mukhamedzhanov3, E.V. Kaigorodova13, E.V. Denisov12, M.V. Zavyalova123, V.M. Perelmuter1
Савельева Ольга Евгеньевна, olga.sav.1980@gmail.com СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(4): 75-80
Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences,Tomsk, Russia1
5, Kooperativny Str., 634009-Tomsk, Russia. E-mail: olga.sav.1980@gmail.com1
Tomsk State University, Tomsk, Russia2
36, Lenin Ave., 634050-Tomsk, Russia2
Siberian State Medical University, Tomsk, Russia3
2, Moskovsky trakt, 634050-Tomsk, Russia3
Abstract
The aim of this study was to assess CXCR4 expression in different subsets of CTCs and single (detached) breast cancer cells. Materials and methods. Thirty five patients with invasive breast carcinoma of no special type (IC NST) (T1-4N0-2M0), between 29 and 69 years of age were included in this study. Different subsets of CTCs with CXCR4 expression were evaluated by flow cytometry. A confocal microscopy was used to assess CXCR4 expression in different subsets of single (detached) cancer cells in breast tissue. Results. The CXCR4 was expressed in CTCs without stem-like and EMT phenotype, in CTCs with EMT but not stem markers and in stem-like CTCs without EMT features. In all blood samples, the CXCR4 expression in CTCs with stem-like and EMT phenotype was absent. In breast tumor the CXCR4 was expressed in the non stemlike single (detached) breast cancer cells with EMT features, in the single (detached) breast cancer cells with stem and EMT features. In all tumor samples the stem-like or non stem-like single (detached) breast cancer cells without EMT features were absent. Conclusions. Different subsets of the CTCs exhibited CXCR4. The CXCR4 expression did not depend on the presence or absence of stem or/and EMT features in tumor cells. We showed that some subsets of single (detached) breast cancer cells in the primary tumor were characterized by the ability to express CXCR4 and may be a source of the respective CTC subsets.
Keywords: CXCR4, circulating tumor cells, cancer stem-like cells, single (detached) cancer cells, epithelial-mesenchymal transition, breast cancer.
Introduction
Circulating tumor cells (CTCs) mediate tumor dissemination and play the key role in the metastatic cascade [1, 2]. The prognostic value of CTCs in breast cancer has been previously shown. Thus, presence of CTCs in the peripheral blood is associated with the poor prognosis in breast cancer [3, 4]. However, little is known about exact mechanism of migration and trafficking of CTCs in the peripheral blood circulation [2]. Metastasis of cancer cells to distant locations needs intravasation from the primary tumor sites into blood vessels, survival in the circulation, migration to secondary organs, adhesion, and proliferation of cancer cells in targeting organs and tissues [5]. Chemokines might affect the selection of target organs for metastases formation. For example, the CXCR4/ CXCL12 axis makes breast cancer cells move out of the circulation and traffic into organs with high amounts of chemokines, and thus forming metastases [6, 7]. In our previous study, we showed that CTCs were represented by heterogeneous population. Some cancer cells had stem-like or/and EMT (epithelialmesenchymal transition) phenotype, other cancer cells did not have EMT and stemness features [8]. We suggested that CTCs did not always precede metastasis, because they could not have the necessary features, including their CXCR4 expression. So, in present study we investigated the CXCR4 expression in different subsets of CTCs and single (detached) breast cancer cells in primary tumor.
Material and Methods
Patients (n=35) with invasive breast carcinoma of no special type (IC NST) (T1-4N0-2M0) within the range
of 29 - 69 years of age (mean age: 49.06 ± 9.78) were treated at the Cancer Research Institute, Tomsk NRMC (Tomsk, Russia) between 2011 and 2017 included (Table 1). IC NST was defined according to the World Health Organization's recommendations [9]. The venous blood samples were collected in EDTA-treated tubes before surgical intervention and were used for flow cytometry. The formalin-fixed, paraffin-embedded (FFPE) primary tumor samples were used for immunofluorescence analysis. This study was approved by the institutional review board, all patients signed an informed consent for voluntary participation.
Flow Cytometry
Different subsets of CTCs were evaluated by flow cytometry (BD FACSCanto II (BD, USA)) using anti-CD45 (clone HI30, PE/Cy7) (BD Pharmingen, USA), anti-CD44 (clone G44-26, APC-H7) (BD Pharmingen, USA), anti-CD24 (clone ML5, PerCP-Cy5.5) (BD Pharmingen, USA), anti-CK7 (clone CAM5.2, AF647) (BD Pharmingen, USA), anti-N-cadherin (CD325) (clone 8C11, PE) (BD Pharmingen, USA) and anti-CXCR4 (clone 44717, AF488) (R&D Systems, USA) antibodies. Then, erythrocytes were lysed in BD FACS lysing solution.
Confocal microscopy
The FFPE tumor sections were incubated with the primary antibody cocktail against CK7 (polyclonal, 1:50, guinea pig, Acris Antibodies, Germany), CD133 (clone 3F10, 1:800, mouse, MyBioSource, USA), N-cadherin (CD325) (polyclonal, 1:400, rabbit, Abcam, UK), CXCR4 (polyclonal, 1:50, goat, Thermo, USA) in 1% BSA. As secondary antibodies were used anti-guinea pig IgG H&L (CF405) (Biotium, USA),
Table 1
The clinicopathological parameters of the patients with breast cancer
Clinicopathological parameters
N (%)
49 (29-69)
Luminal A Luminal B HER2-positive Triple-negative
T1 T2 T3 T4
N0 N1 N2 N3
NO YES
Age (year) (Me (Q1-Q3)) Molecular type of breast cancer
Tumor size
Lymph node status
Neoadjuvant chemotherapy (NACT)
35 (100%)
12/35 (34%) 13/35 (37%) 3/35 (9%) 7/35 (20%)
8/35 (22.8%) 25/35 (71.4%) 1/35 (2.9%) 1/35 (2.9%)
21/35 (60.0%) 9/35 (25.7%) 1/35 (2.9%) 1/35 (2.9%)
24/35 (69%) 11/35 (31%)
Table 2
The CXCR4 expression in different subsets of circulating or single (detached) cancer cells
The breast cancer cell subsets
Frequency of cell detection
Cell count
The circulating tumor cells in patient's blood
CK7+CD45-CD44-CD24+/-N-cadherin-CXCR4+ 31.8 % (7/22)
CK7+CD45-CD44-CD24+/-N-cadherin+CXCR4+
CK7+CD45-CD44+CD24-N-cadherin-CXCR4+
68.2 % (15/22)
27.3 % (6/22)
CK7+CD45-CD44+CD24-N-cadherin+CXCR4+ 0.0 %(0/22)
The single (detached) cells in tumor
0.0 % (0/25)
CK7+N-cadherin-CDl33-CXCR4+ CK7+N-cadherin+CDl33-CXCR4+ CK7+N-cadherin-CDl33+CXCR4+ CK7+N-cadherin+CDl33+CXCR4+
44.0 % (11/25) 0.0 % (0/25) 28.0 % (7/25)
0.00
(0.00-0.95) cell/ml 1.96
(0.13-3.59) cell/ml 0.00
(0.00-0.13) cell/ml
0.00 (0.00-8.00)%
0.00 (0.00-1.05)%
anti-mouse IgG H&L (AF488) (Abcam, UK), anti-rabbit IgG H&L (Cy3) (Abcam, UK), anti-goat IgG H&L (AF647) (Abcam, UK). Nuclei were stained with DRAQ5. The single (detached) breast cancer cells were visualized using the LSM 780 NLO confocal microscope (Carl Zeiss, Germany) with a 63 plan apochromat objective, numerical aperture (z1), and immersion oil.
Results
As CXCR4 promotes breast cancer metastasis to distant organs where its ligand, SDF-1, is generated in large quantity [10, 11], we examined the expression of these molecules in different subsets of CTCs in the blood of breast cancer patients (Figure 1). The
CXCR4 expression in CTCs without stem-like and EMT phenotype (CK7+CD45-CD44-CD24+/-N-cadherin-CXCR4+) was present in 31.8% (7/22) patients (0.00(0.00-0.95) cell/ml). In 68.2% (15/22) patients, CTCs with CXCR4 exhibited EMT but not stem markers (CK7+CD45-CD44-CD24+/-N-cadherin+CXCR4+) (1.96(0.13-3.59) cell/ml). The stem-like CTCs expressed CXCR4 in 27.3% (6/22) patients. All this cells (0.00(0.00-0.13) cell/ml) didn't have EMT features (CK7+CD45-CD44+CD24-N-cadherin-CXCR4+). In all patient blood samples (22/22) the CXCR4 expression in CTCs with stem-like and EMT phenotype (CK7+CD45-CD44+CD24-N-cadherin+CXCR4+) was absent (Table 2).
Figure 1. Flow cytometry analysis of the circulating tumor cells with CXCR4 expression
Ms.
\a
1- &
L * *S
DAPI
Cytokeratin 7
CD133
N-cadherin
CXCR4
Figure 2. The single (detached) breast cancer cells with CXCR4 expression. The white cursor points to the non stem single (detached) breast cancer cell with EMT features and CXCR4 expression
The non stem-like single (detached) breast cancer cells with EMT features (CK7+CD133-N-cadherin+CXCR4+) expressed CXCR4 in 44.0% (11/25) tumor samples (Figure 2). Our results suggested that only 0.00(0.00-8.00)% of single (detached) CK7+CD133-N-cadherin+ cells exhibited CXCR4 in primary tumor. The single (detached)
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Received 23.07.18 Accepted 10.08.18
Funding
The study was supported by the Russian Science Foundation (grant №16-15-10221). Conflict of interest
The authors declare that they have no conflict of interest.
ABOUT THE AUTHORS
Olga E. Savelieva, MD, DSc, Leading Researcher, Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences; Tomsk State University (Tomsk, Russia). E-mail: olga.sav.1980@ gmail.com. Researcher ID (WOS): A-6690-2014. Author ID (Scopus): 14065805800. ORCID: 0000-0002-0301-8455. Liubov A. Tashireva, PhD, Senior Researcher, Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences (Tomsk, Russia). E-mail: lkleptsova@mail.ru. Researcher ID (WOS): C-8222-2012. Author ID (Scopus): 55234960400. ORCID: 0000-0003-2061-8417.
Mikhail A. Buldakov, PhD, Senior Researcher, Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences; Tomsk State University (Tomsk, Russia). E-mail: buldakov@oncology. tomsk.ru. Researcher ID (WOS): C-7922-2012. Author ID (Scopus): 55932165800. ORCID: 0000-0001-8276-110X. Rustam H. Mukhamedzhanov, Postgraduate, Department of Pathological Anatomy, Siberian State Medical University (Tomsk, Russia). E-mail: dx.rusya@gmail.com.
Evgenia V. Kaigorodova, MD, DSc, Leading Researcher, Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences; Siberian State Medical University (Tomsk, Russia). E-mail: zlobinae@mail.ru. Researcher ID (WOS): A-5400-2014. Author ID (Scopus): 24778286000. ORCID: 0000-0003-4378-6915. Evgeny V. Denisov, PhD, Senior Researcher, Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences; Tomsk State University (Tomsk, Russia). E-mail: d_evgeniy@ oncology.tomsk.ru. Researcher ID (WOS): C-8662-2012. Author ID (Scopus): 26653961800. ORCID: 0000-0003-2923-9755. Marina V. Zavyalova, MD, DSc, Professor, Head of Department of Pathological Anatomy, Siberian State Medical University; Leading Researcher, Department of General and Molecular Pathology, Tomsk National Research Medical Center, Russian Academy of Sciences; Tomsk State University (Tomsk, Russia). E-mail: zavyalovamv@mail.ru. Researcher ID (WOS): C-8580-2012. Author ID (Scopus): 36711031100. ORCID: 0000-0001-9429-9813.
Vladimir M. Perelmuter, MD, DSc, Professor, Head of Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences (Tomsk, Russia). E-mail: pvm@ngs.ru. Author ID (Scopus): 8091317300. ORCID: 0000-0002-7633-9620.
СВЕДЕНИЯ ОБ АВТОРАХ
Савельева Ольга Евгеньевна, доктор медицинских наук, ведущий научный сотрудник отделения общей и молекулярной патологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук; Национальный исследовательский Томский государственный университет (г. Томск, Россия). E-mail: olga.sav.1980@gmail.com. SPIN-код: 9633-9449. Researcher ID (WOS): A-6690-2014. Author ID (Scopus): 14065805800. ORCID: 0000-0002-0301-8455.
Таширева Любовь Александровна, кандидат медицинских наук, старший научный сотрудник отделения общей и молекулярной патологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук (г. Томск, Россия). E-mail: lkleptsova@mail.ru. SPIN-код: 4371-5340. Researcher ID (WOS): C-8222-2012. Author ID (Scopus): 55234960400. ORCID: 0000-0003-2061-8417.
Булдаков Михаил Александрович, кандидат биологических наук, старший научный сотрудник лаборатории молекулярной онкологии и иммунологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук; Национальный исследовательский Томский государственный университет (г. Томск, Россия). E-mail: buldakov@oncology.tomsk.ru. SPIN-код: 7558-4726. Researcher ID (WOS): C-7922-2012. Author ID (Scopus): 55932165800. ORCID: 0000-0001-8276-110X.
Мухамеджанов Рустам Хусанович, аспирант кафедры патологической анатомии, Сибирский государственный медицинский университет (г. Томск, Россия). E-mail: dx.rusya@gmail.com.
Кайгородова Евгения Викторовна, доктор медицинских наук, ведущий научный сотрудник отделения общей и молекулярной патологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук; Сибирский государственный медицинский университет (г. Томск, Россия). E-mail: zlobinae@mail. га. SPIN-код: 8286-3757. Researcher ID (WOS): A-5400-2014. Author ID (Scopus): 24778286000. ORCID: 0000-0003-4378-6915. Денисов Евгений Владимирович, кандидат биологических наук, старший научный сотрудник лаборатории молекулярной онкологии и иммунологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук; Национальный исследовательский Томский государственный университет (г. Томск, Россия). E-mail: d_evgeniy@oncology.tomsk.ru. SPIN-код: 9498-5797. Researcher ID (WOS): C-8662-2012. Author ID (Scopus): 26653961800. ORCID: 0000-0003-2923-9755.
Завьялова Марина Викторовна, доктор медицинских наук, профессор, заведующая кафедрой патологической анатомии, Сибирский государственный медицинский университет; ведущий научный сотрудник отделения общей и молекулярной патологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук; Национальный исследовательский Томский государственный университет (г. Томск, Россия). E-mail: zavyalovamv@mail.ru. SPIN-код: 1229-0323. Researcher ID (WOS): C-8580-2012. Author ID (Scopus): 36711031100. ORCID: 0000-0001-9429-9813.
Перельмутер Владимир Михайлович, доктор медицинских наук, профессор, заведующий отделением общей и молекулярной патологии, Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук (г. Томск, Россия). E-mail: pvm@ngs.ru. SPIN-код: 6252-5319. Author ID (Scopus): 8091317300. ORCID: 0000-0002-7633-9620.
Финансирование
Исследование было поддержано Российским научным фондом (грант №16-15-10221). Конфликт интересов
Авторы объявляют, что у них нет конфликта интересов.
Поступила 23.07.18 Принята в печать 10.08.18
