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 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 β-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 β-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 β-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-β-D-galactopyranoside (C₁₂FDG), by β-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.

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. In order to record a sufficient number of events during the flow cytometry analysis, 5 x 10⁵ cells are required. To determine the percentage of senescent cells, three cell samples are required: unlabeled sample, untreated cells stained with C₁₂FDG, treated cells stained with C₁₂FDG.
4. Seed the cells 24 hr prior performing the experiment so that cells are in the log-phase of the cellular proliferation curve.

2. Induction of Senescence

1. Induce senescence of cancer cells by adding a chemotherapeutical drug. Drug concentration and duration of the treatment should be adapted to the cell type tested. Start with a 48 hr treatment at the final concentration of 50 nM doxorubicin for a concentration of cells at about 10⁶ cells/ml. Do not forget to include untreated sample (negative control).

3. Bafilomycin A1 Treatment

1. Check cell viability by mixing cells with trypan blue (v/v), as the chemotherapeutical drug used in the previous step might lead to cell apoptosis. Fill into a Malassez chamber and count the number of blue cells (dead cells) and of white cells (living cells). If the percentage of viability is less than 70%, dead cells might be removed using an appropriate kit to ensure that dead cells will not bias the subsequent analysis.
2. Remove the culture media and replace by prewarmed fresh culture media. Treat the cells with a final concentration of 100 nM of bafilomycin A1. Bafilomycin A1 is used to neutralize the acidic pH of lysosomes. Incubate 1 hr at 37 °C, 5% CO₂.

4. C₁₂FDG Staining

1. Dissolve C₁₂FDG in prewarmed fresh culture media to a final concentration of 2 mM.
2. Add the compound to cells at a final concentration of 33 μM and incubate 2 hr at 37 °C, 5% CO₂. If using non-adherent cells, centrifuge the cells at 300 x g for 5 min at 4 °C (the speed might have to be adjusted to the cell type used), wash 2x with PBS. Resuspend the cell pellet in 500 μl of cold PBS. If using adherent cells, remove the media and wash 2x with PBS. Harvest the adherent cells using a trypsin solution and centrifuge cells at 300 x g for 5 min at 4 °C (the speed might have to be adjusted to the cell type used). Resuspend the cell pellet in 500 μl of cold PBS. In both cases, the cell concentration should be about 10⁶ cells/ml.
3. Perform a co-immunolabeling to further characterize the population of senescent cells.

5. Flow Cytometry Analysis

1. Calibrate the flow cytometer using the control beads recommended by the manufacturer of the flow cytometer. For all steps, at least 10⁴ events have to be recorded.
2. Run the first sample containing unlabeled cells. Prepare a two-parameter graphic displaying Forward Scatter Channel (FSC) versus Side Scatter Channel (SSC). Set the PhotoMultiplier Tubes (PMTs) and define a region of interest excluding dead cells and cellular debris that might have appeared during the sample preparation. Gate this region of interest in a one-parameter histogram displaying FL1 on the log scale of the x-axis and the number of events in the y-axis. The total number of events represents the number of cells analyzed. Set the PMT so that unlabeled cells appear in the first decade on the log scale of the x-axis. Determine the autofluorescence of the cells if necessary.
3. Run the second sample containing untreated cells. On the one-parameter histogram displaying FL1 (fluorescence of C₁₂FDG), place the cursor after the dimly labeled population. This threshold will allow the discrimination between non-senescent cells (dimly labeled cells) and senescent cells (brightly labeled cells).
4. Run the third sample containing treated cells. Brightly labeled cells, i.e. senescent cells appear above the threshold determined in the previous step. Evaluate the percentage of senescent cells by dividing the number of bright events per the total number of events. If needed, the activity of the β-galactosidase can also be estimated using the mean fluorescence intensity.

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