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

Summary

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.

Abstract

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-β-Gal).

Typically, the SA-β-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-β-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-β-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-β-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.

Introduction

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 recurrence^1,2,3. Factors secreted by TIS cells can exacerbate tumor malignancy by promoting immune evasion or metastasis^4,5. TIS cells develop complex, context-spe....

Protocol

All animal work described was approved by the Institutional Animal Care and Use Committee at the University of Chicago.

1. Preparation and storage of stock solutions

NOTE: If cells will be flow-sorted, all solutions should be prepared using sterile techniques and filtered through a 0.22 µm filter device.

1. Prepare a stock solution of DDAO-Galactoside at 5 mg/mL in DMSO. Aliquot at 50 µL per tube (or volume desired). Store at −20 °C in the dark for up to 1 year.
2. Prepare a stock solution of Bafilomycin A1 at 1 mM in DMSO. Aliquot at 50 µL per tube (or v....

Results

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 C

Discussion

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.......

Materials

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