Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

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 radiotherapy^1,2. 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 radiotherapy^4,5,6,7

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 is preferred for cell sorting. If no commercial direct-labeled antibody is available, use fluorescein-conjugating reagents to conjugate the antibody to....

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.......

Materials

Name	Company	Catalog Number	Comments
0.5% Trypsin-EDTA (10X), no phenol red	Thermo Fisher	15400054	Dilute in to 0.05% (1X) with autoclaved distilled water
1B50-1			This 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 solution	Solarbio	G2160
A549	ATCC		RRID: CVCL_0023
B27	Thermo Fisher	17504044
Biological Safety Cabinet	Thermo Fisher	1336
Centrifuge	Eppendorf	5910R
DMEM/F-12	Thermo Fisher	12500062
EGF Recombinant Human Protein	Thermo Fisher	PHG0311
Fetal bovine serum	Thermo Fisher	16140071
FGF-Basic (AA 1-155) Recombinant Human Protein	Thermo Fisher	PHG0261
Flow cytometer/cell sorter	BD	FACSARIA III
H1299	ATCC		RRID: CVCL_0060
H1975	ATCC		RRID: CVCL_1511
Lightning-Link Fluorescein Kit	Innova Biosciences	310-0010
linear accelerator	VARIAN	CLINAC 600C/D
Methyl cellulose	Sigma Aldrich	M7027
Penicillin-Streptomycin, Liquid	Thermo Fisher	15140122
Phosphate buffered saline	Solarbio	P1020
RPMI-1640	Thermo Fisher	11875093
SYBRGREEN	TOYOBO	QPK-201
TRIzol	Thermo Fisher	15596026
Violet crystal staining solution	Solarbio	G1062