Name: simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells
Uploaded: 2022-07-12T14:30:00
Description: Watch this Scientific Journal Video about Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells at JoVE.com

This work was supported by a grant to Yong Yu from Johannes Kepler University Linz (BERM16108001).

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 &#215; 105 cells into one well of a 6-well plate and incubate the plate in a 5% CO2, 37 &#176;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....

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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...

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 deprivation1,2,3,4. 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 radiation5,6.
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 growth7,8,9. 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 &#947;H2AX foci, activated p53-p21CIP1 and Rb-p16INK4a cell cycle regulatory mechanisms, stable G1 cell cycle arrest, massive induction of SASP, and elevated senescence-associated &#946;-galactosidase (SA-&#946;-gal) activity14. Since no single marker is sufficient to define senescence, enzymatic staining for SA-&#946;-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-&#946;-gal is difficult to quantify. Here, we combined 5-dodecanoylaminofluorescein-di-&#946;-D-galactopyranoside (C12FDG) fluorescence-based SA-&#946;-gal (fSA-&#946;-gal) detection with immunofluorescent staining for &#947;H2AX and EdU-incorporated DNA to identify C12FDG+EdU-&#947;H2AX+ 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody</td>
			<td>Biolegend</td>
			<td>613407</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)</td>
			<td>Biolegend</td>
			<td>350509</td>
			<td></td>
		</tr>
		<tr>
			<td>C12FDG (5-Dodecanoylaminofluorescein Di-&#946;-D-Galactopyranoside)</td>
			<td>Fisher Scientific</td>
			<td>11590276</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroquin -diphosphat</td>
			<td>Sigma aldrich</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanser (Coulter Clenz)</td>
			<td>Beckman Coulter</td>
			<td>8546929</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Pacific Blue Flow Cytometry Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>C10418</td>
			<td></td>
		</tr>
		<tr>
			<td>Daunorubicin</td>
			<td>Medchemexpress</td>
			<td>HY-13062A</td>
			<td></td>
		</tr>
		<tr>
			<td>Debubbler (70% Isopropanol)</td>
			<td>Millipore</td>
			<td>1.3704</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis software (Amnis&#160;IDEAS&#160;6.3)</td>
			<td>Luminex</td>
			<td>CN-SW69-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument and imaging software (Amnis&#160;ImageStreamX&#160;Mk II Imaging Flow Cytometer System and INSPIRE software)</td>
			<td>Luminex</td>
			<td>100220</td>
			<td></td>
		</tr>
		<tr>
			<td>KARPAS</td>
			<td>DSMZ</td>
			<td>ACC 31</td>
			<td></td>
		</tr>
		<tr>
			<td>mafosfamide cyclohexylamine</td>
			<td>Niomech</td>
			<td>D-17272</td>
			<td></td>
		</tr>
		<tr>
			<td>OCI-LY1</td>
			<td>DSMZ</td>
			<td>ACC 722</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>11473704</td>
			<td></td>
		</tr>
		<tr>
			<td>PETG (2-Phenylethyl-&#946;-D-thiogalactosid)&#160;</td>
			<td>Sigma aldrich</td>
			<td>P4902</td>
			<td></td>
		</tr>
		<tr>
			<td>saponin</td>
			<td>Sigma aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath</td>
			<td>Millipore</td>
			<td>BSS-1006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedBead Kit for ImageStream</td>
			<td>Luminex</td>
			<td>400041</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer (0.4-0.7% Hypochlorite)</td>
			<td>VWR</td>
			<td>JT9416-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-DHL6</td>
			<td>DSMZ</td>
			<td>ACC 572</td>
			<td></td>
		</tr>
		<tr>
			<td>WSU-DLCL2</td>
			<td>DSMZ</td>
			<td>ACC 575</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells

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&#160;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 &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). 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.

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 &#8805; 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 &#181;g/mL MAF or 20 ng/mL DN to induce cellular senescence and analyzed using either c...

Watch this Scientific Journal Video about Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells at JoVE.com

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

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

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

This method can rapidly generate high resolution images that allow an analysis of spatial distribution and quantification of fluorescent signals within cells while allowing rapid analysis of multiple samples. Cells are measured using the advanced imaging flow cytometry system, combining speed, sensitivity and detailed single cell images with spatial information yielding unique data that cannot be provided by flows optometry or microscopy. One important advice is to provide enough cells to establish the instrument settings and we suspend the cells at the high density for the measurements.Apart from this data acquisition is straightforward and similar to conventional flow cytometry. First generate the DLBCL cell lines as described in the manuscript. Then add 10 millimolar EdU solution at a ratio of one to 1000 into the DLBCL cell culture and incubate the plate in a 5%carbon dioxide incubator at 37 degrees Celsius for three hours after mixing the plate by gentle rocking.Take the plate out after incubation and add 100 millimolar chloroquine solution to a final concentration of 75 micromolar. Mix by rocking gently and incubate for 30 minutes. Then add 20 millimolar C12 FDG solutions to a final concentration of 20 micromolar.After gentle mixing, incubate the plate for one hour as demonstrated previously. Next, add 100 millimolar 2-phenylethyl-beta-d-thiogalactoside solution at one to 50 to stop the FSA beta-gal staining. And gently rotate the plate to mix.Transfer the cells to 15 milliliter sterile centrifuge tubes and spin at 100 times G for five minutes at four degree Celsius. Once the supernatant is discarded, wash the cells with four milliliters of PBS and centrifuge at 100 times G for five minutes at four degrees Celsius. Then re-suspend the cell pellets in 500 microliters at 4%paraformaldehyde fixation solution.After 10 minute incubation at room temperature, centrifuge at 250 times G for five minutes at room temperature. Discard the supernatant and wash the cells with four milliliters of PBS twice and centrifuge at 100 times G for five minutes at room temperature. After washing, discard the supernatant and re-suspend the cell pellet in 200 microliters of saponin permeabilization buffer.Now transfer the suspension to a new 1.5 milliliter tube and incubate for 10 minutes at room temperature. Pellet the cells at 250 times G for five minutes at room temperature. Then re-suspend the cells in 200 microliters of primary antibody solution and incubate at four degree Celsius overnight, in the dark.Centrifuge the tubes at 250 times G for five minutes at four degrees Celsius. Discard the supernatant and wash with 100 microliters of saponin wash solution. After washing the cells twice, discard the supernatant and re-suspend the cell pellets in 500 microliters of EdU detection cocktail.Incubate at room temperature for 30 minutes in the dark and centrifuge at 250 times G for five minutes at room temperature. Discard the supernatant and wash with one milliliter of saponin wash solution. Repeat the washing two times, discard the supernatant and re-suspend the cell pellets in 20 to 50 microliters of PBS.Before turning on the instrument, empty the waste fluid bottle and check the levels of SpeedBeads, sterilizer, cleaner, debubbler and sheath sufficient fluids. After turning on the instrument and imaging software, click on the startup button to initialize fluidics and system calibration. Set the magnification to 40 times and set the fluidic speed to low.After switching on the lasers, turn on a 405 nanometers laser to measure EdU Pacific Blue, 488 nanometers to measure C12-FDG and 642 nanometers to measure Alexa-Fluor-647-gamma-H2AX. Set channel six for the scatter channel, channel one and channel nine for the bright-field. Then start with the sample expected to have the highest fluorescence to set up the intensity of the lasers and gently flip the sample tube to mix.After opening the tube lid, insert the sample tube onto the dock. Click on Load to start and open a scatter plot. Next, select the Features area, underscore MO1 and aspect ratio underscore MO1 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 SpeedBead population on the left side. Now, open Histogram Plot and select the feature gradient root mean square of mask one and channel one for the X axis. Choose the singlet population and set the gate to select for focused cells.Open a histogram plot and select raw max pixel intensities for X axis channel two, seven, and 11. Then adjust the the laser powers of 488 nanometers, 405 nanometers and 642 nanometers lasers for channel two, seven and 11 respectively so each fluorochrome has a raw max pixel value between 100 and 4, 000 to avoid oversaturation. 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. After all the samples are measured, turn off the bright-field and scatter laser.Measure single color control samples to generate a compensation matrix. Click on Shutdown to close the imaging system. Analyze the data in the image analysis software.Use the Spot Wizard tool of the image analysis software to automatically count and quantify nuclear gamma H2AX foci and 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. The flow cytometry method in single cell images showed increased C12-FDG positive, EdU negative, gamma H2AX positive, senesced population and KARPAS422.WSU-DLCL2 and OCI-LY1 cells, but not in SU-DHL6 cells. The imaging-based analysis presented significantly higher numbers of gamma H2AX foci and KARPAS422, WSU-DLCL2 and OCI-LY1, but not in SU-DHL6 cells. Similar results were represented by frequency and quantification data.Adding saponin to the cell suspension is crucial, otherwise the antibodies cannot reach the cell internal antigens and harsh detergents lead to loss of C12-FTG signal Imaging cytometry is optimal for analyzing changes in marker localization like translocation of a transcription factor to the nucleus in response to certain stimuli. Such an analysis can also be achieved with our protocol. This method allows to visualization of multiple fluorescent markers on a single-cell level which helps detect the presence, intensity and localization of the individual signals.

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