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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

Abstract

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel α2δ1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel α2δ1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

Introduction

Radiotherapy plays an important role in cancer treatment. However, existence of radioresistant cancer stem cells (CSCs) may lead to relapse or poor outcomes after radiotherapy1,2. CSCs are characterized by their self-renewal capacity and ability to generate heterogenous cancer cells3. Armored with a more efficient DNA damage repair capacity or higher levels of free-radical scavenging systems or other mechanisms, CSCs are relatively resistant to radiotherapy4,5,6,7

Protocol

NOTE: Steps are performed under the indicated temperature. For steps in which the temperature is not mentioned, perform under room temperature (18–25 °C). Cell culture medium should be stored at 4 °C, and other reagents should be stored according to the manufacturer’s guides. Medium should be pre-warmed to 37 °C before being added to cells.

1. Cell sorting

  1. Antibody conjugation
    NOTE: Considering the shorter incubation time, direct-labeled antibody i.......

Representative Results

α2δ1-high and α2δ1-low A549 cells were sorted (Figure 1A). Some markers may show distinct populations and are easy to gate. However, some markers just show high and low expression patterns, rather than distinct positive and negative populations. In this situation, an isotype control is very important for gating. The expression of α2δ1 in sorted cells is validated by qPCR. The expression of CACNA2D1, the gene that encode α2δ1, is higher in sort.......

Discussion

This protocol describes methods to study the radiosensitivity of CSCs in cancer cell lines in vitro. In this study, the expression of α2δ1 is continuous in NSCLC cell lines. Therefore, gating is based on an isotype control. Before sorting, α2δ1 expression should be examined in multiple cell lines by flow cytometry and validated by QPCR or western blot. It is recommended to re-analyze α2δ1 expression of the sorted α2δ1-high and α2δ1-low cells by flow cytometry, by observin.......

Acknowledgements

This work was supported by National Natural Science Foundation of China (81402535 and 81672969) and National Key Research and Development Project (2016YFC0904703).

....

Materials

NameCompanyCatalog NumberComments
0.5% Trypsin-EDTA (10X), no phenol redThermo Fisher15400054Dilute in to 0.05% (1X) with autoclaved distilled water
1B50-1This antibody is produced and friendly supplied by Laboratory of Carcinogenesis and Translational Reseach (Ministry of Education/Beijing), Department of Cell Biology, Peking University Cancer Hospital and Institute. See reference 10. Alternatively, commercial antibody of calcium channel α2δ1 subunit can be used (ABCAM, ab2864) (Yu, et al., Am J Cancer Res, 2016; 6(9): 2088-2097)
4% formaldehyde solutionSolarbioG2160
A549ATCCRRID: CVCL_0023
B27Thermo Fisher17504044
Biological Safety CabinetThermo Fisher1336
CentrifugeEppendorf5910R
DMEM/F-12Thermo Fisher12500062
EGF Recombinant Human ProteinThermo FisherPHG0311
Fetal bovine serumThermo Fisher16140071
FGF-Basic (AA 1-155) Recombinant Human ProteinThermo FisherPHG0261
Flow cytometer/cell sorterBDFACSARIA III
H1299ATCCRRID: CVCL_0060
H1975ATCCRRID: CVCL_1511
Lightning-Link Fluorescein KitInnova Biosciences310-0010
linear acceleratorVARIANCLINAC 600C/D
Methyl celluloseSigma AldrichM7027
Penicillin-Streptomycin, LiquidThermo Fisher15140122
Phosphate buffered salineSolarbioP1020
RPMI-1640Thermo Fisher11875093
SYBRGREENTOYOBOQPK-201
TRIzolThermo Fisher15596026
Violet crystal staining solutionSolarbioG1062

References

  1. Brunner, T. B., Kunz-Schughart, L. A., Grosse-Gehling, P., Baumann, M. Cancer stem cells as a predictive factor in radiotherapy. Seminars in Radiation Oncology. 22 (2), 151-174 (2012).
  2. Baumann, M., Krause, M., Hill, R. Exploring the role....

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Cancer Stem CellsRadiosensitivityLung CancerFlow CytometrySphere Formation AssayColony Formation AssayRadioresistanceDNA Damage RepairReactive Oxygen SpeciesCell Cycle Arrest

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