Name: Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry (Video) | JoVE Journal
Uploaded: 2022-09-13T13:40:00.000+00:00
Duration: 14 min 1 s
Description: Learn a rapid flow cytometry protocol for detecting therapy-induced senescence in tumor cells using dual markers. This method enables reliable identification and enrichment of senescent cancer cells for downstream analysis.

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5 minutes

We thank the Cytometry and Antibody Core Facility at the University of Chicago for support on flow cytometry instrumentation. The Animal Research Center at the University of Chicago provided animal housing.

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</ol>

The authors have no conflicts of interest to declare for this study.

Over the last decade or so, flow cytometry has become a more common assay platform in cancer research due to the emerging popularity of tumor immunology, the development of lower-cost flow cytometers, and the improvement of shared instrumentation facilities at academic institutions. Multicolor assays are now standard, as most newer instruments are equipped with violet, blue-green, and red to far-red optical arrays. Thus, this DDAOG protocol is likely to be compatible with a wide array of flow cytometers. Of course, any f...

Senescent cells normally accumulate in organisms over years during normal biological aging but may also develop rapidly in tumor cells as a response to damage induced by various cancer treatments, including radiation and chemotherapy. Though no longer proliferating, therapy-induced senescent (TIS) tumor cells may contribute to treatment resistance and drive recurrence1,2,3. Factors secreted by TIS cells can exacerbate tumor malignancy by promoting immune evasion or metastasis4,5. TIS cells develop complex, context-specific phenotypes, altered metabolic profiles, and unique immune responses6,7,8. Therefore, the identification and characterization of TIS tumor cells induced by various cancer treatment approaches is a topic of ongoing interest to the cancer research community.
To detect TIS tumor cells, conventional senescence assays are widely used, primarily based on detecting increased activity of the senescence marker enzyme, the lysosomal beta-galactosidase GLB19. Detection at a near-neutral (rather than acidic) lysosomal pH allows for specific detection of senescence-associated beta-galactosidase (SA-β-Gal)10. A standard SA-β-Gal assay that has been used for several decades uses X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), a blue chromogenic beta-galactosidase substrate, to detect SA-β-Gal in fixed cells by light microscopy11. The X-Gal assay allows the qualitative visual confirmation of TIS utilizing commonly available reagents and laboratory equipment. A basic transmitted light microscope is the only instrumentation required to evaluate the presence of the blue chromogen. However, the X-Gal staining procedure can lack sensitivity, sometimes requiring more than 24 h for color to develop. Staining is followed by low-throughput, subjective scoring of individual senescent cells based on counting the cells exhibiting some level of intensity of the blue chromogen under a light microscope. As X-Gal is cell-impermeable, this assay requires solvent-fixed cells, which cannot be recovered for downstream analysis. When working with limited samples from animals or patients, this can be a major drawback.
Improved SA-β-Gal assays using cell-permeant, fluorescent enzyme substrates, including C12-FDG (5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside, green) and DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-Galactopyranoside, far-red) have previously appeared in the literature12,13,14,15. The chemical probe structure and optical characteristics of DDAOG are shown in Supplementary Figure S1. These cell-permeant probes permit the analysis of living (rather than fixed) cells, and fluorescent rather than chromogenic probes facilitate the use of rapid high-throughput fluorescent analysis platforms, including high-content screening instruments and flow cytometers. Sorting flow cytometers enable the recovery of enriched populations of living senescent cells from cell cultures or tumors for downstream analysis (e.g., western blotting, ELISA, or 'omics). Fluorescence analysis also provides a quantitative signal, allowing for more accurate determination of the percentage of senescent cells within a given sample. Additional fluorescent probes, including viability probes and fluorophore-labeled antibodies, can readily be added for multiplexed analysis of targets beyond SA-β-Gal.
Similar to DDAOG, C12-FDG is a fluorescent probe for SA-β-Gal, but its green fluorescent emission overlaps with intrinsic cellular AF, which arises during senescence due to the accumulation of lipofuscin aggregates in cells16. By utilizing the far-red DDAOG probe, green cellular AF can be used as a secondary parameter to confirm senescence17. This improves assay reliability by using a second marker in addition to SA-β-Gal, which can often be unreliable as a single marker for senescence18. As the detection of endogenous AF in senescent cells is a label-free approach, it is a rapid and simple way to expand the specificity of our DDAOG-based assay.
In this protocol, we demonstrate the use of DDAOG and AF as a rapid, dual-parameter flow cytometry assay for the identification of viable TIS tumor cells from in vitro cultures or isolated from drug-treated tumors established in mice (Figure 1). The protocol uses fluorophores compatible with a wide range of standard commercial flow cytometry analyzers and sorters (Table 1). Quantitation of the percentage of viable senescent cells using standard flow cytometry analysis is enabled. If desired, an optional immunolabeling step may be performed to evaluate cell surface antigens of interest concurrently with senescence. Identified senescent cells can also be enriched using standard fluorescence-activated cell sorting (FACS) methodology.
<img alt="Drug treatment process diagram; in vitro/in vivo; flow cytometry; TIS onset; experimental setup." class="xfigimg" src="/files/ftp_upload/64176/64176fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Experimental workflow. A schematic summarizing key points of the DDAOG assay. (A) A TIS-inducing drug is added to mammalian cultured cells or administered to tumor-bearing mice. Time is then allowed for the onset of TIS: for cells, 4 days following treatment; for mice, 22 days total, with three treatments every 5 days plus 7 days recovery. Cells are harvested or tumors are dissociated into suspension. (B) Samples are treated with Baf to adjust lysosomal pH for detection of SA-β-Gal for 30 min; then, DDAOG probe is added for 60 min to detect SA-β-Gal. Samples are washed 2x in PBS, and a viability stain is briefly added (15 min). Optionally, samples can be stained with fluorescent antibodies in open fluorescence channels and/or fixed for later analysis. (C) Samples are analyzed using a standard flow cytometer. Viable cells are visualized in dot plots showing red DDAOG (indicating SA-β-Gal) versus green autofluorescence (lipofuscin). A gate to determine the percentage of TIS cells is established based on untreated control samples (not shown). If a sorting cytometer (FACS) is used, TIS cells can be collected and placed back into culture for further in vitro assays or lysed and processed for molecular biology assays. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; TIS = therapy-induced senescence; FL-Ab = fluorophore-conjugated antibody; Baf = Bafilomycin A1; SA-β-Gal = senescence-associated beta-galactosidase; PBS = phosphate-buffered saline; FACS = fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/64176/64176fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorophore</td>
			<td>Detects</td>
			<td>Ex/Em (nm)</td>
			<td>Cytometer laser (nm)</td>
			<td>Cytometer detector / bandpass filter (nm)</td>
		</tr>
		<tr>
			<td>DDAOG</td>
			<td>SA-β-Gal</td>
			<td>645/6601</td>
			<td>640</td>
			<td>670 / 30</td>
		</tr>
		<tr>
			<td>AF</td>
			<td>Lipofuscin</td>
			<td>&lt; 600</td>
			<td>488</td>
			<td>525 / 50</td>
		</tr>
		<tr>
			<td>CV450</td>
			<td>Viability</td>
			<td>408/450</td>
			<td>405</td>
			<td>450 / 50</td>
		</tr>
		<tr>
			<td>PE</td>
			<td>Antibody/surface marker</td>
			<td>565/578</td>
			<td>561</td>
			<td>582 / 15</td>
		</tr>
	</tbody>
</table>
Table 1: Fluorophores and cytometer optical specifications. Cytometer specifications used in this protocol are listed for an instrument with a total of 4 lasers and 15 emission detectors. DDAOG detected at 645/660 nm is the form of the probe cleaved by SA-β-Gal1. Uncleaved DDAOG can exhibit low level fluorescence at 460/610 nm but is removed by wash steps in the protocol. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; AF = autofluorescence; PE = phycoerythrin; SA-β-Gal = senescence-associated beta-galactosidase.

<table><tbody><tr><td>Bafilomycin A1</td><td>Research Products International</td><td>B40500</td><td></td></tr><tr><td>Bleomycin sulfate&amp;nbsp;</td><td>Cayman</td><td>13877</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>US Biological</td><td>A1380</td><td></td></tr><tr><td>Calcein Violet 450 AM viability dye</td><td>ThermoFisher Scientific</td><td>65-0854-39</td><td>eBioscience</td></tr><tr><td>DPP4 antibody, PE conjugate</td><td>Biolegend</td><td>137803</td><td>Clone H194-112</td></tr><tr><td>Cell line: A549 human lung adenocarcinoma</td><td>American Type Culture Collection</td><td>CCL-185</td><td></td></tr><tr><td>Cell line: B16-F10 mouse melanoma</td><td>American Type Culture Collection</td><td>CRL-6475</td><td></td></tr><tr><td>Cell scraper</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell strainers, 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>DDAO-Galactoside</td><td>Life Technologies</td><td>D6488</td><td></td></tr><tr><td>DMEM medium 1x</td><td>Life Technologies</td><td>11960-069</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2438</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>Doxorubicin, hydrochloride injection (USP)</td><td>Pfizer</td><td>NDC 0069-3032-20</td><td></td></tr><tr><td>Doxorubicin, PEGylated liposomal (USP)</td><td>Sun Pharmaceutical</td><td>NDC 47335-049-40</td><td></td></tr><tr><td>EDTA 0.5 M</td><td>Life Technologies</td><td>15575-038</td><td></td></tr><tr><td>Etoposide&amp;nbsp;</td><td>Cayman</td><td>12092</td><td></td></tr><tr><td>FBS</td><td>Omega&amp;nbsp;</td><td>FB-11</td><td></td></tr><tr><td>Fc receptor blocking reagent</td><td>Biolegend</td><td>101320</td><td>Anti-mouse CD16/32</td></tr><tr><td>Flow cytometer (cell analyzer)</td><td>Becton Dickinson (BD)</td><td>Various</td><td>LSRFortessa</td></tr><tr><td>Flow cytometer (cell sorter)</td><td>Becton Dickinson (BD)</td><td>Various</td><td>FACSAria</td></tr><tr><td>GlutaMax 100x</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>HEPES 1 M</td><td>Lonza</td><td>BW17737</td><td></td></tr><tr><td>Liberase TL</td><td>Sigma</td><td>5401020001</td><td>Roche</td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 1x</td><td>Corning</td><td>MT21031CV</td><td>Dulbecco&#039;s PBS (without calcium and magnesium)</td></tr><tr><td>Rainbow calibration particles, ultra kit</td><td>SpheroTech</td><td>UCRP-38-2K</td><td>3.5-3.9 &amp;micro;m, 2E6/mL</td></tr><tr><td>RPMI-1640 medium 1x</td><td>Life Technologies</td><td>11875-119</td><td></td></tr><tr><td>Sodium chloride 0.9% (USP)</td><td>Baxter Healthcare Corporation</td><td>2B1324</td><td></td></tr><tr><td>Software for cytometer data acquisition, &quot;FACSDiva&quot;</td><td>Becton Dickinson (BD)</td><td>n/a</td><td>Contact BD for license</td></tr><tr><td>Software for cytometer data analysis, &quot;FlowJo&quot;</td><td>TreeStar</td><td>n/a</td><td>Contact TreeStar for license</td></tr><tr><td>Trypsin-EDTA 0.25%</td><td>Life Technologies</td><td>25200-114</td><td></td></tr></tbody></table>

far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Several experiments were performed to demonstrate the comparability of DDAOG to X-Gal and C12-FDG for the detection of senescence by SA-β-Gal. First, X-Gal was used to stain senescent B16-F10 melanoma cells induced by ETO (Figure 2A). An intense blue color developed in a subset of ETO-treated cells, while other cells exhibited less intense blue staining. Morphology was enlarged in most ETO-treated cells. Staining ETO-treated cells with fluorescent SA-β-Gal substrate C12...

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

far-red-fluorescent-senescence-associated-galactosidase-probe-for

Research

JoVE Journal

Cancer Research

This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a β-galactosidase substrate. The substrate is cleaved by active lysosomal β-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

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A Sensitive Method to Quantify Senescent Cancer Cells

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Senescence Detection Using Reflected Light in Adipose Stromal Vascular Fraction

This article presents a live-cell LysoTracker protocol to measure lysosomal remodeling during senescence in IMR-90 human fibroblasts.

Lysosomal Profiling With LysoTracker For Quantitative Assessment of Cellular Senescence In Human Fibroblasts

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

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The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200–600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

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Learn a rapid flow cytometry protocol for detecting therapy-induced senescence in tumor cells using dual markers. This method enables reliable identification and enrichment of senescent cancer cells for downstream analysis.

Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry (Video) | JoVE Journal

Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

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