Name: radiosensitivity of cancer stem cells in lung cancer cell lines
Uploaded: 2019-08-21T14:30:00
Description: Watch this Scientific Journal Video about Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines at JoVE.com

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

NOTE: Steps are performed under the indicated temperature. For steps in which the temperature is not mentioned, perform under room temperature (18&#8211;25 &#176;C). Cell culture medium should be stored at 4 &#176;C, and other reagents should be stored according to the manufacturer&#8217;s guides. Medium should be pre-warmed to 37 &#176;C before being added to cells.
1. Cell sorting
<ol>
	<li>Antibody conjugation 
	NOTE: Considering the shorter incubation time, direct-labeled antibody i...

<ol>
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This protocol describes methods to study the radiosensitivity of CSCs in cancer cell lines in vitro. In this study, the expression of &#945;2&#948;1 is continuous in NSCLC cell lines. Therefore, gating is based on an isotype control. Before sorting, &#945;2&#948;1 expression should be examined in multiple cell lines by flow cytometry and validated by QPCR or western blot. It is recommended to re-analyze &#945;2&#948;1 expression of the sorted &#945;2&#948;1-high and &#945;2&#948;1-low cells by flow cytometry, by observin...

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 radiotherapy4,5,6,7,8. Identifying CSC markers and exploring their mechanisms will facilitate the development of drugs that will overcome radioresistance without increasing normal tissue damage.
Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in NSCLC cell lines9. &#945;2&#948;1 was originally identified as a CSC marker for hepatocellular carcinoma (HCC)10. Using subtractive immunization with a pair of HCC cell lines derived from the primary and recurrent tumors in the same patient, an antibody named 1B50-1 was identified to target recurrent HCC cells specifically. 1B50-1-positive cells showed high sphere formation efficiency in vitro and high tumorigenicity in vivo. Its antigen was identified by mass spectrometry, as calcium channel &#945;2&#948;1 subunit isoform 5. &#945;2&#948;1 specifically expresses in CSCs and is undetectable in most normal tissues, making it a potential candidate for targeting CSCs10. &#945;2&#948;1 can also serve as a CSC marker for NSCLC cell lines, and it has been shown to impart radioresistance to NSCLC cells partially by enhancing the efficiency of DNA damage repair in response to radiation9.
Studying radioresistant CSCs may provide clues to overcoming radioresistance. Using &#945;2&#948;1 in NSCLC as an example, major methods to study the radiosensitivity of CSCs are presented. Usually, CSCs are isolated with a putative surface marker, and the stem cell characteristics and radiosensitivity of the positive and negative cell populations are compared. Sphere formation in a serum-free medium supplemented with growth factors that support self-renewal is a useful assay to evaluate the stemness of cells in vitro. Cells with high sphere formation capacity are likely to show high tumorigenicity when injected into immunodeficient mice10,11,12. Colony formation assay is then used to assess the radiosensitivity of cells, which determines how many have lost the ability to generate descendants forming the colony after a dose of radiation13.

<table>
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA (10X), no phenol red</td>
			<td>Thermo Fisher</td>
			<td>15400054</td>
			<td>Dilute in to 0.05% (1X) with autoclaved distilled water</td>
		</tr>
		<tr>
			<td>1B50-1</td>
			<td></td>
			<td></td>
			<td>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 &#945;2&#948;1 subunit can be used (ABCAM, ab2864) (Yu, et al., Am J Cancer Res, 2016; 6(9): 2088-2097)</td>
		</tr>
		<tr>
			<td>4% formaldehyde solution</td>
			<td>Solarbio</td>
			<td>G2160</td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td></td>
			<td>RRID: CVCL_0023</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>Thermo Fisher</td>
			<td>1336</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5910R</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher</td>
			<td>12500062</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF Recombinant Human Protein</td>
			<td>Thermo Fisher</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-Basic (AA 1-155) Recombinant Human Protein</td>
			<td>Thermo Fisher</td>
			<td>PHG0261</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer/cell sorter</td>
			<td>BD</td>
			<td>FACSARIA III</td>
			<td></td>
		</tr>
		<tr>
			<td>H1299</td>
			<td>ATCC</td>
			<td></td>
			<td>RRID: CVCL_0060</td>
		</tr>
		<tr>
			<td>H1975</td>
			<td>ATCC</td>
			<td></td>
			<td>RRID: CVCL_1511</td>
		</tr>
		<tr>
			<td>Lightning-Link Fluorescein Kit</td>
			<td>Innova Biosciences</td>
			<td>310-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>linear accelerator</td>
			<td>VARIAN</td>
			<td>CLINAC 600C/D</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma Aldrich</td>
			<td>M7027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBRGREEN</td>
			<td>TOYOBO</td>
			<td>QPK-201</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Thermo Fisher</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Violet crystal staining solution</td>
			<td>Solarbio</td>
			<td>G1062</td>
			<td></td>
		</tr>
	</tbody>
</table>

radiosensitivity of cancer stem cells in lung cancer cell lines

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 &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 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.

&#945;2&#948;1-high and &#945;2&#948;1-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 &#945;2&#948;1 in sorted cells is validated by qPCR. The expression of CACNA2D1, the gene that encode &#945;2&#948;1, is higher in sort...

Watch this Scientific Journal Video about Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines at JoVE.com

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

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.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

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