Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Summary

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Abstract

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated β-galactosidase (SA-β-gal), the DNA synthesis marker 5-ethynyl-2′-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Introduction

A variety of stimuli can trigger cellular senescence, causing cells to enter a state of stable cell cycle arrest. These stimuli include intrinsic signaling changes or extrinsic stresses. Intrinsic signals include progressive telomere shortening, changes in telomere structure, epigenetic modification, proteostasis disorders, mitochondrial dysfunction, and activation of oncogenes. Extrinsic stresses include inflammatory and/or tissue damage signals, radiation or chemical treatment, and nutritional deprivation^1,2,3,4. Among distinct types of se....

Protocol

1. DLBCL cell lines with mafosfamide or daunorubicin treatment to induce cellular senescence

NOTE: The protocol also works for adherent cancer cells. Depending on cell size, seed 1-2 × 10⁵ cells into one well of a 6-well plate and incubate the plate in a 5% CO₂, 37 °C incubator overnight before treatment. The protocol steps are the same as for suspension cells but with two exceptions. First, cells need to be trypsinized off the plate after step 3.4. Second, wash steps are performed without centrifugation before trypsinization.

1. Count DLBCL cells and seed 1 × 10⁶ cel....

Results

A compensation matrix was generated using image analysis software by loading recorded data of single-color control samples. As shown in Supplemental Figure S1, a non-negligible (coefficient value ≥ 0.1) light spillover from EdU to C₁₂FDG was detected with crosstalk coefficient value 0.248, while the crosstalk among other channels was not significant. Four different DLBCL cell lines were treated with 5 µg/mL MAF or 20 ng/mL DN to induce cellular senescence and analyzed using either conventional.......

Discussion

This method examined the senescence-entering capability of four different DLBCL cell lines upon chemotherapy treatment, with bright-field imaging and flow cytometry-based quantification. On a single-cell level, we successfully detected major C₁₂FDG⁺EdU^-Ki67⁺ senescent populations in treated KARPAS422 and WSU-DLCL2 cells, and to a lesser extent in OCI-LY1 cells, while the SU-DHL6 cell line was resistant to the treatment. The difference in senescence-entering capability among cel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody	Biolegend	613407
Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)	Biolegend	350509
C12FDG (5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside)	Fisher Scientific	11590276
Chloroquin -diphosphat	Sigma aldrich	C6628
Cleanser (Coulter Clenz)	Beckman Coulter	8546929
Click-iT EdU Pacific Blue Flow Cytometry Assay Kit	Thermo Scientific	C10418
Daunorubicin	Medchemexpress	HY-13062A
Debubbler (70% Isopropanol)	Millipore	1.3704
Image Analysis software (Amnis IDEAS 6.3)	Luminex	CN-SW69-12
Instrument and imaging software (Amnis ImageStreamX Mk II Imaging Flow Cytometer System and INSPIRE software)	Luminex	100220
KARPAS	DSMZ	ACC 31
mafosfamide cyclohexylamine	Niomech	D-17272
OCI-LY1	DSMZ	ACC 722
Paraformaldehyde	Fisher Scientific	11473704
PETG (2-Phenylethyl-β-D-thiogalactosid)	Sigma aldrich	P4902
saponin	Sigma aldrich	47036
Sheath	Millipore	BSS-1006-B
SpeedBead Kit for ImageStream	Luminex	400041
Sterilizer (0.4-0.7% Hypochlorite)	VWR	JT9416-1
SU-DHL6	DSMZ	ACC 572
WSU-DLCL2	DSMZ	ACC 575