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 senescence, the most commonly seen and well-studied are replicative senescence, oncogene-induced senescence (OIS), radiation-induced senescence, and therapy-induced senescence (TIS). OIS is an acute cellular response to genotoxic damage caused by replicative stress generated by aberrant oncogene activation and can to some extent prevent the pathological progression from a preneoplastic lesion to a full-blown tumor. TIS happens when tumor cells are stressed by chemotherapeutic drugs or ionizing radiation^5,6.

Senescence is considered a double-edged sword in pathology due to its highly dynamic nature. It was initially described as a beneficial tumor-suppressive mechanism to remove damaged cells from the circulating pool of dividing cells, safeguarding the normal function of organs and inhibiting tumor growth^7,8,9. However, emerging evidence has suggested a dark side of senescence. Senescent cells secrete proinflammatory cytokines, known as senescence-associated secretory phenotype (SASP), leading to fibrosis and malfunctional organs and promoting tumor initiation and progression¹⁰. Moreover, senescent cancer cells undergo epigenetic and gene-expression reprogramming in parallel with chromatin remodeling and activation of a sustained DNA-damage response (DDR)^11,12, newly acquiring new cancer-stem-cell properties³. Although senescence-capable tumors respond better to therapeutic intervention compared to senescence-incapable ones¹³, the persistence of senescent cells may lead to a poor long-term prognosis if they are not effectively identified and eliminated by senolytic drugs⁵. Either way, a reliable method to assess senescence is of significant clinical interest, not only for the prognosis of therapy treatment but also for the development of novel strategies targeting senescent cells.

Regardless of different triggers, senescent cells exhibit some common features, including enlarged, flattened, multinucleated morphology with big vacuoles, significantly expanded nuclei, formation of H3K9me3-rich senescence-associated heterochromatin (SAHF) in the nucleus, persistent accumulation of DNA damage marker γH2AX foci, activated p53-p21^CIP1 and Rb-p16^INK4a cell cycle regulatory mechanisms, stable G1 cell cycle arrest, massive induction of SASP, and elevated senescence-associated β-galactosidase (SA-β-gal) activity¹⁴. Since no single marker is sufficient to define senescence, enzymatic staining for SA-β-gal activity, which is considered the gold standard for senescence detection, is usually combined with immunohistochemical staining for H3K9me3 and Ki67 to detect TIS¹⁵. However, chemical chromogenic-based SA-β-gal is difficult to quantify. Here, we combined 5-dodecanoylaminofluorescein-di-β-D-galactopyranoside (C₁₂FDG) fluorescence-based SA-β-gal (fSA-β-gal) detection with immunofluorescent staining for γH2AX and EdU-incorporated DNA to identify C₁₂FDG⁺EdU^-γH2AX⁺ senescent cells using the advanced imaging flow cytometer system, which combines the speed, sensitivity and detailed single-cell images with spatial information that cannot be provided by flow cytometry and microscopy. This method enables rapid generation of high-resolution images allowing for the positioning and quantification of fluorescent signals within cells, while licensing the swift analysis of multiple samples by building standard pipelines.

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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 cells/mL in 4 mL of medium per well into a 6-well culture plate. Cultivate DLBCL cells in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin.
2. Add MAF (5 µg/mL) or DN (20 ng/mL) into the cell culture and gently rock the plate to mix.
   NOTE: MAF and DN are not stable. After dissolving in DMSO, the stock solution should be aliquoted and stored at -20 °C. Freeze/thaw cycles should be avoided.
3. Incubate the plate in a 5% CO₂, 37 °C incubator for 3 days.
4. After a 3 day incubation, collect the DLBCL cells in 15 mL sterile centrifuge tubes and spin at 100 × g, 4 °C for 5 min.
5. Discard the supernatant, resuspend the cell pellets with 4 mL of fresh medium, and add the suspensions back into the 6-well plate.
6. Cultivate the cell plate in a 5% CO₂, 37 °C incubator for another 2 days before harvesting for analysis.

2. Prepare solutions for staining (Table 1)

3. Stain DLBCL cells with different senescence markers

NOTE: Cell samples stained with individual markers (i.e., pacific blue-EdU, C₁₂FDG, or Alexa Fluor 647-γH2AX) are prepared to generate a compensation matrix to correct the fluorescence spillover during measurement. Although highly suggested, this step could be suspended when there is an omittable overlap (compensation coefficient value ≤ 0.1) of the emission spectra among different fluorophores. However, users must determine standardized compensation steps when using different instruments and fluorescent panels.

1. Add 10 mM EdU solution at a ratio of 1:1,000 into the DLBCL cell culture generated from step 1.6 (final EdU concentration is 10 µM). Gently rock the plate to mix and incubate in a 5% CO₂, 37 °C incubator for 3 h.
2. Take the plate out of the incubator and add 100 mM chloroquine solution to a final concentration of 75 µM (see discussion). Gently rock the plate to mix and incubate in a 5% CO₂, 37 °C incubator for 30 min.
3. Take the plate out of the incubator and add 20 mM C₁₂FDG solution at 1: 1,000 to a final C₁₂FDG concentration of 20 µM. Gently rock the plate to mix and incubate in a 5% CO₂, 37 °C incubator for 1 h.
4. Take the plate out of the incubator and add 100 mM 2-phenylethyl-β-D-thiogalactoside (PETG) solution at 1:50 to stop the fSA-β-gal staining (final PETG concentration of 2 mM). Gently rotate the plate to mix.
5. Transfer the cells to 15 mL sterile centrifuge tubes and spin at 100 × g, 4 °C for 5 min. Discard the supernatant and wash the cells with 4 mL of phosphate-buffered saline (PBS).
6. Repeat the PBS washing step. Discard the supernatant and resuspend the cell pellets in 500 µL of 4% paraformaldehyde fixation solution.
7. After 10 min incubation at room temperature, centrifuge at 250 × g, room temperature for 5 min. Discard the supernatant and wash cells with 4 mL of PBS.
8. Repeat the PBS washing step. Discard the supernatant, resuspend the cell pellet in 200 µL of saponin permeabilization buffer and transfer the suspension to a new 1.5 mL tube. After 10 min incubation at room temperature, centrifuge at 250 × g, room temperature for 5 min.
9. Discard the supernatant and resuspend the cell pellets in 200 µL of primary antibody solution (1:500 γH2AX antibody in antibody incubation solution). Incubate at 4 °C overnight in the dark.
10. Centrifuge the tubes at 250 × g, 4 °C for 5 min. Discard the supernatant and wash with 100 µL of saponin wash solution.
11. Repeat the washing step an additional two times. Discard the supernatant and resuspend the cell pellets in 500 µL of EdU detection cocktail.
12. Incubate at room temperature for 30 min in the dark. Centrifuge at 250 × g, room temperature for 5 min. Discard the supernatant and wash with 1 mL of saponin wash solution.
13. Repeat the washing step two times. Discard the supernatant and resuspend the cell pellets in 20-50 µL of PBS. Proceed to section 4 for sample measurement.

4. Imaging senescence markers using the imaging flow cytometer system

1. Empty the waste fluid bottle. Check the levels of speed beads, sterilizer, cleaner, debubbler, sheath, and rinse reagents (deionized water) to ensure sufficient fluids before turning on the instrument (see the Table of Materials).
2. Turn on the instrument and imaging software (see the software interface in Figure 1A).
3. Click on the Startup button to initialize fluidics and system calibration.
   NOTE: This procedure takes approximately 45 min.
4. Set the magnification to 40x, set fluidics speed to low, and turn on the lasers needed in the experiment. Turn on 405 nm, 488 nm, and 642 nm lasers to measure EdU-pacific blue, C₁₂FDG, and Alexa Fluor 647-γH2AX (or Alexa Fluor 647-Ki67), respectively. Set Ch6 for scatter channel, and Ch1 and Ch9 for brightfield.
5. Start with the sample expected to have the highest fluorescence to set up the intensity of the lasers. Gently flip the sample tube to mix. Open the tube lid, and insert the sample tube onto the dock. Click on Load to start.
6. Open a scatter plot and select the features Area_M01 and Aspect Ratio_M01 for the X- and Y-axes, respectively. Set a gate above aspect ratio of 0.5 to exclude doublets and cell aggregates below and on the right side, and the speed bead population on the left side (Figure 1B).
7. Open a histogram plot and select the feature Gradient RMS_M01_Ch01 for the X-axis. Choose the singlet population and set the gate to select for focused cells (Figure 1C).
   NOTE: The imaging system will automatically use speed beads to adjust the focus for imaging. However, it is recommended to select the right half of the histogram peak for the best-focused population.
8. Open a histogram plot and select Raw Max Pixel Intensities for X-axis for each color channel (Ch2, 7, and 11). Adjust the laser powers (i.e., 488 nm, 405 nm, and 642 nm lasers for Ch2, 7, and 11, respectively), so each fluorochrome has a Raw Max Pixel value between 100 and 4,000 to avoid oversaturation.
   NOTE: For this experiment, the laser power setting is 50 mW, 200 mW, and 50 mW for lasers 488 nm, 405 nm, and 642 nm, respectively.
9. Choose the focused population to record and click on Acquire to measure the DLBCL samples with consistent settings. When changing samples for measurement, click on Return to recover the sample tube. Press Load to discard the sample.
10. After all the samples are measured, turn off the brightfield and scatter laser. Measure single-color control samples to generate a compensation matrix.
11. Click on Shutdown to close the imaging system.
12. Analyze the data in the image analysis software.
    1. Use the Spot Wizard tool of the image analysis software to automatically count and quantify nuclear γH2AX foci in live-cell images. Select two cell populations (one with high and one with low spot count) to train the spot wizard for further automatic spot count analysis.

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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 c...

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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...

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Materials

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