1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Research Materials
3.2. Flow Cytometry
3.3. Fluorescence-Activated Cell Sorting
3.4. CTCs Spiking Experiment
3.5. Confocal Microscopy
3.6. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Giordano, A.; Gao, H.; Anfossi, S.; Cohen, E.; Mego, M.; Lee, B.N.; Tin, S.; De Laurentiis, M.; Parker, C.A.; Alvarez, R.H.; et al. Epithelial-mesenchymal transition and stem cell markers in patients with HER2-positive metastatic breast cancer. Mol. Cancer Ther. 2012, 11, 2526–2534. [Google Scholar] [CrossRef] [PubMed]
- Kaigorodova, E.; Tarabanovskaya, N.; Simolina, E.; Perelmuter, V.; Stakheeva, M.; Cherdyntsevа, N.; Saveleva, O.; Tashireva, L. Circulating tumor cells and bone marrow progenitor cells in the blood of breast cancer patients in the dynamics of neoadjuvant chemotherapy. EJC Suppl. 2015, 13, 22. [Google Scholar] [CrossRef]
- Kaigorodova, E.V. Circulating tumor cells: Clinical significance in breast cancer (Review). Ann. Russ. Acad. Med. Sci. 2017, 72, 450–457. [Google Scholar] [CrossRef]
- Lv, Q.B.; Fu, X.; Jin, H.M.; Xu, H.C.; Huang, Z.Y.; Xu, H.Z.; Chi, Y.L.; Wu, A.M. The relationship between weight change and risk of hip fracture: Meta-analysis of prospective studies. Sci. Rep. 2015, 5, 16030. [Google Scholar] [CrossRef] [PubMed]
- Mego, M.; Mani, S.A.; Lee, B.N.; Li, C.; Evans, K.W.; Cohen, E.N.; Gao, H.; Jackson, S.A.; Giordano, A.; Hortobagyi, G.N.; et al. Expression of epithelial-mesenchymal transition-inducing transcription factors in primary breast cancer: The effect of neoadjuvant therapy. Int. J. Cancer 2012, 130, 808–816. [Google Scholar] [CrossRef] [PubMed]
- Kaigorodova, E.V.; Tarabanovskaya, N.A.; Staheeva, M.N.; Savelieva, O.E.; Tashireva, L.A.; Denisov, E.V.; Perelmuter, V.M. Effect of minor and major surgical injury on the level of different populations of circulating tumor cells in the blood of breast cancer patients. Neoplasma 2017, 64, 437–443. [Google Scholar] [CrossRef] [PubMed]
- Meng, S.; Tripathy, D.; Frenkel, E.P.; Shete, S.; Naftalis, E.Z.; Huth, J.F.; Beitsch, P.D.; Leitch, M.; Hoover, S.; Euhus, D.; et al. Circulating tumor cells in patients with breast cancer dormancy. Clin. Cancer Res. 2004, 10, 8152–8162. [Google Scholar] [CrossRef] [PubMed]
- Kasimir-Bauer, S.; Hoffmann, O.; Wallwiener, D.; Kimmig, R.; Fehm, T. Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells. Breast Cancer Res. 2012, 14, R15. [Google Scholar] [CrossRef] [PubMed]
- Ge, F.; Zhang, H.; Wang, D.D.; Li, L.; Lin, P.P. Enhanced detection and comprehensive in situ phenotypic characterization of circulating and disseminated heteroploid epithelial and glioma tumor cells. Oncotarget 2015, 6, 27049–27064. [Google Scholar] [CrossRef] [PubMed]
- Lin, P.P.; Gires, O.; Wang, D.D.; Li, L.; Wang, H. Comprehensive in situ co-detection of aneuploid circulating endothelial and tumor cells. Sci. Rep. 2017, 7, 9789. [Google Scholar] [CrossRef] [PubMed]
- Spizzo, G.; Fong, D.; Wurm, M.; Ensinger, C.; Obrist, P.; Hofer, C.; Mazzoleni, G.; Gastl, G.; Went, P. EpCAM expression in primary tumour tissues and metastases: An immunohistochemical analysis. J. Clin. Pathol. 2011, 64, 415–420. [Google Scholar] [CrossRef] [PubMed]
- Ladwein, M.; Pape, U.F.; Schmidt, D.S.; Schnölzer, M.; Fiedler, S.; Langbein, L.; Franke, W.W.; Moldenhauer, G.; Zöller, M. The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7. Exp. Cell Res. 2005, 309, 345–357. [Google Scholar] [CrossRef] [PubMed]
- Guillemot, J.C.; Naspetti, M.; Malergue, F.; Montcourrier, P.; Galland, F.; Naquet, P. Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers. Histochem. Cell Biol. 2001, 116, 371–378. [Google Scholar] [CrossRef] [PubMed]
- Maetzel, D.; Denzel, S.; Mack, B.; Canis, M.; Went, P.; Benk, M.; Kie, C.; Papior, P.; Baeuerle, P.A.; Munz, M.; et al. Nuclear signalling by tumour-associated antigen EpCAM. Nat. Cell Biol. 2009, 11, 162–171. [Google Scholar] [CrossRef] [PubMed]
- Osta, W.A.; Chen, Y.; Mikhitarian, K.; Mitas, M.; Salem, M.; Hannun, Y.A.; Cole, D.J.; Gillanders, W.E. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res. 2004, 64, 5818–5824. [Google Scholar] [CrossRef] [PubMed]
- Münz, M.; Kieu, C.; Mack, B.; Schmitt, B.; Zeidler, R.; Gires, O. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 2004, 23, 5748–5758. [Google Scholar] [CrossRef] [PubMed]
- Baeuerle, P.A.; Gires, O. EpCAM (CD326) finding its role in cancer. Br. J. Cancer 2007, 96, 417–423. [Google Scholar] [CrossRef] [PubMed]
- Spizzo, G.; Went, P.; Dirnhofer, S.; Obrist, P.; Moch, H.; Baeuerle, P.A.; Mueller-Holzner, E.; Marth, C.; Gastl, G.; Zeimet, A.G. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol. Oncol. 2006, 103, 483–488. [Google Scholar] [CrossRef] [PubMed]
- Went, P.; Vasei, M.; Bubendorf, L.; Terracciano, L.; Tornillo, L.; Riede, U.; Kononen, J.; Simon, R.; Sauter, G.; Baeuerle, P.A. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br. J. Cancer 2006, 94, 128–135. [Google Scholar] [CrossRef] [PubMed]
- Hyun, K.A.; Koo, G.B.; Han, H.; Sohn, J.; Choi, W.; Kim, S.I.; Jung, H.I.; Kim, Y.S. Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer. Oncotarget 2016, 7, 24677–24687. [Google Scholar] [CrossRef] [PubMed]
- Lin, C.W.; Liao, M.Y.; Lin, W.W.; Wang, Y.P.; Lu, T.Y.; Wu, H.C. Epithelial Cell Adhesion Molecule Regulates Tumor Initiation and Tumorigenesis via Activating Reprogramming Factors and Epithelial-Mesenchymal Transition Gene Expression in Colon Cancer. J. Biol. Chem. 2012, 287, 39449–39459. [Google Scholar] [CrossRef] [PubMed]
- Elshamy, W.M.; Duhé, R.J. Overview: Cellular plasticity, cancer stem cells and metastasis. Cancer Lett. 2013, 341, 2–8. [Google Scholar] [CrossRef] [PubMed]
- Mitra, A.; Mishra, L.; Li, S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget 2015, 6, 10697–10711. [Google Scholar] [CrossRef] [PubMed]
- Wang, K.; Kao, A.P.; Lin, T.C.; Chang, C.C.; Kuo, T.C. Promotion of epithelial-mesenchymal transition and tumor growth by 17beta-estradiol in an ER(+)/HER2(+) cell line derived from human breast epithelial stem cells. Biotechnol. Appl. Biochem. 2012, 59, 262–267. [Google Scholar] [CrossRef] [PubMed]
- Bhat-Nakshatri, P.; Goswami, C.P.; Badve, S.; Sledge, G.W., Jr.; Nakshatri, H. Identification of FDA approved drugs targeting breast cancer stem cells along with biomarkers of sensitivity. Sci. Rep. 2013, 3, 2530. [Google Scholar] [CrossRef] [PubMed]
- Morel, A.P.; Lievre, M.; Thomas, C.; Hinkal, G.; Ansieau, S.; Puisieux, A. Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS ONE 2008, 3, e2888. [Google Scholar] [CrossRef] [PubMed]
- Creighton, C.J.; Li, X.; Landis, M.; Dixon, J.M.; Neumeister, V.M.; Sjolund, A.; Rimm, D.L.; Wong, H.; Rodriguez, A.; Herschkowitz, J.I.; et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc. Natl. Acad. Sci. USA 2009, 106, 13820–13825. [Google Scholar] [CrossRef] [PubMed]
- Mani, S.A.; Guo, W.; Liao, M.J.; Eaton, E.N.; Ayyanan, A.; Zhou, A.Y.; Brooks, M.; Reinhard, F.; Zhang, C.C.; Shipitsin, M.; et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133, 704–715. [Google Scholar] [CrossRef] [PubMed]
- Asiedu, M.K.; Ingle, J.N.; Behrens, M.D.; Radisky, D.C.; Knutson, K.L. TGFbeta/TNF(alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype. Cancer Res. 2011, 71, 4707–4719. [Google Scholar] [CrossRef] [PubMed]
- Thompson, E.W.; Haviv, I. The social aspects of EMT- MET plasticity. Nat. Med. 2011, 17, 1048–1049. [Google Scholar] [CrossRef] [PubMed]
- Shibue, T.; Weinberg, R.A. EMT, CSCs, and drug resistance: The mechanistic link and clinical implications. Nat. Rev. Clin. Oncol. 2017, 14, 611–629. [Google Scholar] [CrossRef] [PubMed]
- Brabletz, T.; Kalluri, R.; Nieto, M.A.; Weinberg, R.A. EMT in cancer. Nat. Rev. Cancer 2018, 18, 128–134. [Google Scholar] [CrossRef] [PubMed]
- Ye, X.; Weinberg, R.A. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol. 2015, 25, 675–686. [Google Scholar] [CrossRef] [PubMed]
- Liu, X.; Fan, D. The epithelial-mesenchymal transition and cancer stem cells: Functional and mechanistic links. Curr. Pharm. Des. 2015, 21, 1279–1291. [Google Scholar] [CrossRef] [PubMed]
- Liu, S.; Cong, Y.; Wang, D.; Sun, Y.; Deng, L.; Liu, Y.; Martin-Trevino, R.; Shang, L.; McDermott, S.P.; Landis, M.D.; et al. Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem. Cell Rep. 2013, 2, 78–91. [Google Scholar] [CrossRef] [PubMed]
- Kaplan, R.N.; Riba, R.D.; Zacharoulis, S.; Bramley, A.H.; Vincent, L.; Costa, C.; MacDonald, D.D.; Jin, D.K.; Shido, K.; Kerns, S.A.; et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 2005, 438, 820–827. [Google Scholar] [CrossRef] [PubMed]
- Perelmuter, V.M.; Manskikh, V.N. The Concept of a Preniche for Localization of Future Metastases. In Tumors of the Central Nervous System; Hayat, M., Ed.; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2013; Chapter 11; Volume 13, pp. 93–106. [Google Scholar] [CrossRef]
Sample Availability: Samples of the compounds are available from the authors. |

N | СTC Total | CTC-1 | CTC-2 | CTC-3 | CTC-4 | CTC-5 | CTC-6 |
---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | |
Breast cancer patients | |||||||
27 | 2.85 (0.51–4.40) | 0.54 (0.00–1.11) p2-1 = 0.00003 * | 0.09 (0.00–0.70) p3-1 = 0.00009 * p3-2 = 0.47 * | 0.00 (0.00–0.43) p4-1 = 0.00002 * p4-2 = 0.0011 * p4-3 = 0.11 * | 0.02 (0.00–0.63) p5-1 = 0.00009 * p5-2 = 0.33 * p5-3 = 0.43 * p5-4 = 0.68 * | 0.18 (0.00–2.41) p6-1 = 0.00003 * p6-2 = 0.14 * p6-3 = 0.67 * p6-4 = 0.011 * p6-5 = 0.23 * | 0.04 (0.00–0.34) p7-1 = 0.00006 * p7-2 = 0.57 * p7-3 = 0.47 * p7-4 = 0.79 * p7-5 = 0.58 * p7-6 = 0.19 * |
Healthy donors | |||||||
7 | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) | 0.00 (0.00–0.00) |
Level after Biopsy | Level after 1 Course of NACT | Level after 2 Course of NACT | Level after 3 Course of NACT | Level before Surgical Treatment |
---|---|---|---|---|
1 | 2 | 3 | 4 | 5 |
СTC total (EpCam+/-CD45-CD44+/-CD24-Ncadherin+/-) | ||||
2.97 (0.93–5.85) | 2.51 (1.85–21.45) p2-1 = 0.32 | 11.04 (3.46–21.96) p3-1 = 0.77 p3-2 = 0.20 | 19.94 (9.28–130.86) p4-1 = 0.068 p4-2 = 0.26 p4-3 = 0.32 | 14.06 (4.51–74.60) p5-1 = 0.017 p5-2 = 0.035 p5-3 = 0.16 p5-4 = 0.77 |
CTC-1 without stemness and EMT properties (EpCam+CD45-CD44-CD24-Ncadherin-) | ||||
0.90 (0.00–1.73) | 1.25 (0.07–5.21) p2-1 = 0.074 | 3.75 (0.29–5.79) p3-1 = 0.027 p3-2 = 0.916 | 0.74 (0.31–4.80) p4-1 = 0.176 p4-2 = 0.735 p4-3 = 0.735 | 3.45 (0.00–7.09) p5-1 = 0.028 p5-2 = 0.600 p5-3 = 0.310 p5-4 = 0.310 |
CTC-2 without stemness and with EMT properties (EpCam+CD45-CD44-CD24-Ncadherin+) | ||||
1.03 (0.45–22.50) | 0.28 (0.17–0.66) p2-1 = 0.345 | 0.87 (0.39–5.99) p3-1 = 0.916 p3-2 = 0.345 | 0.83 (0.29–1.84) p4-1 = 0.463 p4-2 = 0.735 p4-3 = 0.600 | 0.28 (0.00–1.28) p5-1 = 0.865 p5-2 = 0.046 p5-3 = 0.753 p5-4 = 0.753 |
CTC-3 with stemness and without EMT properties (EpCam+CD45-CD44+CD24-Ncadherin-) | ||||
0.02 (0.00–0.22) | 0.12 (0.00–1.09) p2-1 = 0.310 | 0.20 (0.00–1.63) p3-1 = 0.176 p3-2 = 0.079 | 0.50 (0.00–1.79) p4-1 = 0.115 p4-2 = 0.115 p4-3 = 0.916 | 0.00 (0.00–2.57) p5-1 = 0.498 p5-2 = 0.224 p5-3 = 0.892 p5-4 = 0.753 |
CTC-4 with stemness and EMT properties (EpCam+CD45-CD44+CD24-Ncadherin+) | ||||
0.22 (0.00–0.55) | 0.00 (0.00–0.26) p2-1 = 0.715 | 0.05 (0.00–0.22) p3-1 = 0.345 p3-2 = 0.500 | 0.00 (0.00–1.64) p4-1 = 0.144 p4-2 = 0.108 p4-3 = 0.224 | 0.01 (0.00–0.71) p5-1 = 0.043 p5-2 = 0.043 p5-3 = 0.115 p5-4 = 0.892 |
CTC-5 with stemness, without EMT properties and without EpCAM membrane expression (EpCam-CD45-CD44+CD24-Ncadherin-) | ||||
0.00 (0.00–0.49) | 0.46 (0.00–2.61) p2-1 = 0.892 | 1.08 (0.13–5.32) p3-1 = 0.310 p3-2 = 0.310 | 5.62 (2.12–9.57) p4-1 = 0.068 p4-2 = 0.123 p4-3 = 0.025 | 2.39 (0.10–12.80) p5-1 = 0.086 p5-2 = 0.498 p5-3 = 0.128 p5-4 = 0.207 |
CTC-6 with stemness and EMT properties and without EpCAM membrane expression (EpCam-CD45-CD44+CD24-Ncadherin+) | ||||
0.16 (0.04–1.59) | 0.32 (0.06–0.87) p2-1 = 1.00 | 0.21 (0.02–2.35) p3-1 = 0.498 p3-2 = 0.865 | 2.95 (1.25–13.28) p4-1 = 0.027 p4-2 = 0.062 p4-3 = 0.310 | 3.19 (0.47–9.41) p5-1 = 0.027 p5-2 = 0.090 p5-3 = 0.345 p5-4 = 0.779 |
Clinicopathological Parameters | N (%) |
---|---|
Age (year) (Me (Q1–Q3)) | |
49 (43–57) | 27 (100%) |
Molecular type of breast cancer | |
Luminal A | 6/27 (22%) |
Luminal В1 | 12/27 (44%) |
Luminal В2 | 1/27 (4%) |
HER2-positive | 1/27 (4%) |
Triple-negative | 7/27 (26%) |
Tumor size | |
T1 | 6/27 (22%) |
T2 | 18/27 (67%) |
T3 | 1/27 (4%) |
T4 | 2/27 (7%) |
Lymph node status | |
N0 | 14/27 (52%) |
N1 | 8/27 (30%) |
N2 | 3/27 (11%) |
N3 | 2/27 (7%) |
Neoadjuvant chemotherapy (NACT) | |
NO | 13/27 (48%) |
YES | 14/27 (52%) |
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).