Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis
Abstract
BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed.
METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness.
RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p<0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression.
CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination.
KEYWORDS: breast neoplasms, cell line, dedifferentiation, immunohistochemistry, neoplastic stem cells
Full Text:
PDFReferences
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394–424, CrossRef.
Prager GW, Braga S, Bystricky B, Qvortrup C, Criscitiello C, Esin E, et al. Global cancer control: Responding to the growing burden, rising costs and inequalities in access. ESMO Open. 2018; 3: e000285, CrossRef.
Gerber B, Freund M, Reimer T. Recurrent breast cancer: treatment strategies for maintaining and prolonging good quality of life. Dtsch Arztebl Int. 2010; 107: 85–91, CrossRef.
Dragu DL, Necula LG, Bleotu C, Diaconu CC, Chivu-Economescu M. Therapies targeting cancer stem cells: Current trends and future challenges. World J Stem Cells. 2015; 7: 1185–201, CrossRef.
Dales JP, Garcia S, Meunier-Carpentier S, Andrac-Meyer L, Haddad O, Lavaut MN, et al. Overexpression of hypoxia-inducible factor HIF-1α predicts early relapse in breast cancer: Retrospective study in a series of 745 patients. Int J Cancer. 2005; 116: 734–9, CrossRef.
Bomken S, Fišer K, Heidenreich O, Vormoor J. Understanding the cancer stem cell. Br J Cancer. 2010; 103: 439–45, CrossRef.
Maugeri-Saccà M, Bartucci M, De Maria R. DNA damage repair pathways in cancer stem cells. Mol Cancer Ther. 2012; 11: 1627–36, CrossRef.
Chen R, Nishimura MC, Bumbaca SM, Kharbanda S, Forrest WF, Kasman IM, et al. A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell. 2010; 17: 362–75, CrossRef.
Abdullah LN, Chow EK-H. Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med. 2013; 2: 3, CrossRef.
Lou H, Dean M. Targeted therapy for cancer stem cells: the patched pathway and ABC transporters. Oncogene. 2007; 26: 1357–60, CrossRef.
Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997; 3: 730–7, CrossRef.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003; 100: 3983–8, CrossRef.
Hombach-Klonisch S, Panigrahi S, Rashedi I, Seifert A, Alberti E, Pocar P, et al. Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications. J Mol Med. 2008; 86: 1301–14, CrossRef.
Schatton T, Frank NY, Frank MH. Identification and targeting of cancer stem cells. BioEssays. 2009; 31: 1038–49, CrossRef.
Ricardo S, Vieira AF, Gerhard R, Leitao D, Pinto R, Cameselle-Teijeiro JF, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011; 64: 937–46, CrossRef.
Poss KD. Advances in understanding tissue regenerative capacity and mechanisms in animals. Nat Rev Gene. 2010; 11: 710–22, CrossRef.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126: 663–76, CrossRef.
Yamanaka S. Patient-specific pluripotent stem cells become even more accessible. Cell Stem Cell. 2010; 7: 1–2, CrossRef.
Khavari DA, Sen GL, Rinn JL. DNA methylation and epigenetic control of cellular differentiation. Cell Cycle. 2010; 9: 3880–3, CrossRef.
Bhartiya D, Boheler KR, Rameshwar P. Multipotent to pluripotent properties of adult stem cells. Stem Cells Int. 2013; 2013: 813780, CrossRef.
Gómez-López S, Lerner RG, Petritsch C. Asymmetric cell division of stem and progenitor cells during homeostasis and cancer. Cell Mol Life Sci. 2014; 71: 575–97, CrossRef.
Santoro A, Vlachou T, Carminati M, Pelicci PG, Mapelli M. Molecular mechanisms of asymmetric divisions in mammary stem cells. EMBO Rep. 2016; 17: 1700–20, CrossRef.
Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961; 25: 585–621, CrossRef.
Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast Cancer Res. 2011; 13: 215, CrossRef.
Wang J, Liu X, Jiang Z, Li L, Cui Z, Gao Y, et al. A novel method to limit breast cancer stem cells in states of quiescence, proliferation or differentiation: Use of gel stress in combination with stem cell growth factors. Oncol Lett. 2016; 12: 1355–60, CrossRef.
Pasternak T, Tietz O, Rapp K, Begheldo M, Nitschke R, Ruperti B, et al. Protocol: An improved and universal procedure for wholemount immunolocalization in plants. Plant Methods. 2015; 11: 50, CrossRef.
Zhao J, Lin Z, Yao H, Wan Y. Analysis of the relationship between expressions of TF and MMP-9 and prognosis of breast cancer patients. Chinese J Clin Oncol. 2008; 5: 141–5, CrossRef.
Ha W, Sevim-Nalkiran H, Zaman AM, Matsuda K, Khasraw M, Nowak AK, et al. Ibudilast sensitizes glioblastoma to temozolomide by targeting Macrophage Migration Inhibitory Factor (MIF). Sci Rep. 2019; 9: 2905, CrossRef.
Chen X, Liao R, Li D, Sun J. Induced cancer stem cells generated by radiochemotherapy and their therapeutic implications. Oncotarget. 2017; 8: 17301–12, CrossRef.
Francis KR, Wei L. Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning. Cell Death Dis. 2010; 1: e22–11, CrossRef.
Khong TL, Thairu N, Larsen H, Dawson PM, Kiriakidis S, Paleolog EM. Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer. BMC Cancer. 2013; 13: 518, CrossRef.
Calvani M, Rapisarda A, Uranchimeg B, Shoemaker RH, Melillo G. Hypoxic induction of an HIF-1alpha-dependent bFGF autocrine loop drives angiogenesis in human endothelial cells. Blood. 2006; 107: 2705–12, CrossRef.
Shi YH, Wang YX, Bingle L, Gong LH, Heng WJ, Li Y, et al. In vitro study of HIF-1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: Involvement of PI-3K/Akt and MEK1/ERK pathways. J Pathol. 2005; 205: 530–6, CrossRef.
Han ZB, Ren H, Zhao H, Chi Y, Chen K, Zhou B, et al. Hypoxiainducible factor (HIF)-1α directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF). Carcinogenesis. 2008; 29: 1853–61, CrossRef.
DOI: https://doi.org/10.18585/inabj.v12i2.977
Copyright (c) 2020 The Prodia Education and Research Institute
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Indexed by:
The Prodia Education and Research Institute
