A Sensitive Method to Quantify Senescent Cancer Cells

Summary

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 β-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Abstract

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 β-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.

Introduction

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....

Protocol

1. Preparation of C12FDG, Bafilomycin A1, and Cell Culture

1. Dissolve the C₁₂FDG powder in dimethyl sulfoxide (DMSO) to a final concentration of 20 mM. Aliquot and store at -20 °C. DMSO should be used with appropriate safety conditions.
2. Dissolve the bafilomycin A1 powder in DMSO to a final concentration of 0.1 mM. Aliquot and store at - 20 °C. Bafilomycin should be used with appropriate safety conditions.
3. Cultivate RPMI 8226 cell line, or other cell lines adherent or not, in media containing 10% fetal bovine serum and 1% antibiotics, at 37 °C, 5% CO₂. Estimate the number of cells needed for the experiment....

Results

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.......

Discussion

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 β-galactosidase, enriched in lysosomes of senescent cells. After laser excitation, the compound emits green fluorescence, allowing fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-dodecanoylaminofluorescein di-β-D-galactopyranoside	Invitrogen	D-2893
Dimethyl sulfoxide	Sigma	D2650
Bafilomycin A1	Sigma	B1793
Doxorubicin	Sandoz
Trypan blue	Sigma	T8154
RPMI 1640 cell culture medium	Lonza	BE12-702
Fetal calf serum	PAA Laboratories GmBH	A15 101
Antibiotics	Gibco	5290-018
Gallios flow cytometer	Beckman Coulter	A94291