167CSCs are responsible for intrinsic and acquired chemotherapy
resistant to 5-fluorouracil (5-FU) and cisplatin in esophageal
adenocarcinoma [ 1 ]. This therapy resistance is also associated with
the changes of epithelial mesenchymal transition (EMT) regula-
tion in esophageal adenocarcinoma [ 1 ]. Isolated (CD44+/
CD24−) CSCs subpopulation from esophageal adenocarcinoma
had higher proliferation and spheres formation capacity and were
exhibited more resistant to radiation therapy when compared to
that of non- CSCs cells [ 7 ]. In addition, patients with esophageal
adenocarcinoma in the CD44+/CD24− subpopulation had shown
reduced response to the neoadjuvant chemoradiotherapy [ 7 ].
Thus, CSCs targeted therapy can increase the sensitivity of cur-
rently available chemoradiotherapy and can improve the clinical
outcome. For example, inhibition of Notch signaling depletes
CSCs in patients-derived xenograft of esophageal adenocarcinoma
origin will sensitize the cancer to chemotherapy and thereby help
in downsizing the cancer growth [ 8 ].
In this chapter, we describe the identification of CSCs from a
cell line of esophageal adenocarcinoma using a combination of
multiple techniques including: (1) cell staining with Hoechst
33342 followed by fluorescence-activated cell sorting (FACS), (2)
in vitro spheres formation assay, and (3) in vivo mouse transplanta-
tion methods (Fig. 1 ). In the first step, side population (SP) cells
from the cancer cells were sorted out with fluorescence- activated
cell sorting based on ATP-binding cassette- (ABC) transporters
expression using Hoechst 33342 dye-staining. This method is
based on differential efflux of fluorescent DNA-binding dye
Hoechst 33342 in cancer stem cells relative to the non-stem cells.
The poorly stained cells, called side population (SP), have CSCs
properties [ 1 ]. The sorted SP cells were tested for tumor spheres
formation capacity in vitro. Finally, the tumorigenic properties of
the sorted CSCs of esophageal cancer cells were confirmed with
the xenotransplantation model in mouse (Fig. 1 ).2 Materials
Prepare all the reagents and solutions using ultrapure water and
chemicals of analytical grade. Unless otherwise indicated, prepare
and store all the reagents and solution at room temperature. Follow
the rules and regulations of waste disposal during disposing the
waste materials. Appropriate personal protective equipment must
be worn during experiments to escape the laboratory hazards.- Esophageal adenocarcinoma cells: OE33 cell line.
- Cell culture media: Roswell Park Memorial Institute (RPMI)
1640, l-glutamine, pH 7.2. Add about 900 mL water to a
glass beaker. With gentle stir, add 10.4 g RPMI1640 powder
2.1 Cell Culture
Cancer Stem Cells in Esophageal Adenocarcinoma