Name: a sensitive method to quantify senescent cancer cells
Uploaded: 2013-08-02T13:00:00
Description: Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

We thank the SF 4206 ICORE (Universit&eacute; de Caen) for the use of the cytometry facility. J.C. received post-doctoral fellowships from Conseil de Radioprotection d'EDF and Conseil R&eacute;gional of Basse-Normandie. Those data were part of projects funded by the Ligue Nationale contre le Cancer - Comit&eacute;s de l'Orne et du Calvados.

1. Preparation of C12FDG, Bafilomycin A1, and Cell Culture
<ol>
	<li>Dissolve the C12FDG powder in dimethyl sulfoxide (DMSO) to a final concentration of 20 mM. Aliquot and store at -20 &deg;C. DMSO should be used with appropriate safety conditions.</li>
	<li>Dissolve the bafilomycin A1 powder in DMSO to a final concentration of 0.1 mM. Aliquot and store at - 20 &deg;C. Bafilomycin should be used with appropriate safety conditions.</li>
	<li>Cultivate RPMI 8226 cell line, or other cell lines adherent or not, ...

<ol>
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We presented here a flow cytometry-based method to quantify cellular senescence in live cancer cells. This method is based on the use of the membrane permeable compound, the C12FDG, and can therefore be used in any cell types and after various treatments as long as the compound is taken up by the cells. Upon cellular uptake, the C12FDG is hydrolyzed by the &beta;-galactosidase, enriched in lysosomes of senescent cells. After laser excitation, the compound emits green fluorescence, allowing fo...

Since early sixties and its discovery by Hayflick and Moorhead, senescence still remains a cellular curiosity 1. Human cells do not indefinitely proliferate and, by senescence, Hayflick and Moorhead coined the cellular process of ageing. Senescence is a stable cell cycle arrest in which cells remain metabolically active as opposed to cellular death. To date, four cellular mechanisms have been shown to induce senescence: telomere shortening, DNA damage, chromatin perturbation, and abnormal expression of mitogenic oncogenes 2. This indicates that not only normal cells but also cancer cells are able to undergo senescence 3. As a matter of fact, cancer cells undergoing senescence were shown to constitute a barrier to further tumor progression 4-5. However, several reports have now pointed out that, under certain circumstances, cellular senescence can also promote malignancy 6-7. Studying senescence of cancer cells is about to become an urge in cancer research as currently used chemotherapeutic drugs can lead to senescence of cancer cells not only in vitro but also in vivo 8.
The master assay to investigate for the presence of senescent cells is the &beta;-galactosidase assay at acidic pH. This histochemical assay reflects the increased in lysosomal biogenesis occurring in most senescent cells. Briefly, exogenous X-gal, applied to cells, is cleaved by &beta;-galactosidase enriched in lysosomes of senescent cells, giving rise to a blue color 9. The blue senescent cells can then be quantified under a bright field microscope. Although very popular, the &beta;-galactosidase assay presents major drawbacks. First, this assay is performed on fixed cells. Second, it is time consuming because it implies to quantify the degree of senescence by counting a significantly high number of senescent cells under a microscope. Third, this assay is not sensitive as the discrimination between senescent and non-senescent cells entirely relies on the observer.
We discuss here an unexploited, quick, and sensitive cytometry-based assay to assess for the presence of live senescent cells. This assay is based on the hydrolysis of a membrane permeable molecule, the 5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside (C12FDG), by &beta;-galactosidase enriched in senescent cells. After hydrolysis and laser excitation, the C12FDG emits green fluorescence and can therefore be detected by flow cytometry 10, 11. The detection of senescent cells via flow cytometry offers the great advantage of being rapid and sensitive. In addition, detection of senescent cells by flow cytometry can be coupled with the detection of other cellular markers. This assay can be used to detect senescent cells within a population of cancerous cells treated with chemotherapy and can be routinely set up on tissue sections, after cellular dissociation, in clinic.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside </td>
			<td>Invitrogen</td>
			<td>D-2893</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dimethyl sulfoxide </td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bafilomycin A1</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxorubicin</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640 cell culture medium</td>
			<td>Lonza</td>
			<td>BE12-702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal calf serum</td>
			<td>PAA Laboratories GmBH</td>
			<td>A15 101</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotics</td>
			<td>Gibco</td>
			<td>5290-018</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>A94291</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a sensitive method to quantify senescent cancer cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Multiple Myeloma, a hematological malignancy, was used to evaluate the senescence induced by a chemotherapeutical procedure. To do so a commercially available MM cell line (RPMI 8226) was treated for 2 days with doxorubicin, a chemotherapeutical drug. The cells were then subjected to bafilomycin A1 treatment and further incubated with C12FDG. Cells were analyzed by flow cytometry. Figure 1 illustrates the different steps and shows that the percentage of senescent cells can be determined within...

Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

A Sensitive Method to Quantify Senescent Cancer Cells

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

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