Name: quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence
Uploaded: 2023-02-10T14:15:00
Description: Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

PV is supported by the Inaugural Pedal the Cause Grant by the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital, Pilot Research Grant from Marsha Rivkin Center for Ovarian Cancer Research, Cancer Research Grant from Mary Kay Ash Foundation and V-Foundation. NR is supported by the NIH Cell and Molecular Biology training T32 grant to Washington University, St. Louis.

NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those derived from non-ovarian sources. A schematic of the protocol is shown in Figure 2.
1. Plating the cells
<ol>
	<li>Make poly-L-lysine coated coverslips.
	<ol>
		<li>Add autoclaved 12 mm diameter coverslips to a 50 mL conical tube with poly-L-lysine solution, and place on a rocker for 15 min.</...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+repair+in+ovarian+cancer:+Unlocking+the+heterogeneity.">DNA damage repair in ovarian cancer: Unlocking the heterogeneity.</a> Journal of Ovarian Research. 11, 50 (2018).</li><li>Labidi-Galy, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+grade+serous+ovarian+carcinomas+originate+in+the+fallopian+tube.">High grade serous ovarian carcinomas originate in the fallopian tube.</a> Nature communications. 8, 1093 (2017).</li><li>Kroeger, P. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repertoire+of+mutational+signatures+in+human+cancer.">The repertoire of mutational signatures in human cancer.</a> Nature. 578 (7793), 94-101 (2020).</li><li>Byun, T. S., Pacek, M., Yee, M. -. C., Walter, J. C., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+uncoupling+of+MCM+helicase+and+DNA+polymerase+activities+activates+the+ATR-dependent+checkpoint.">Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.</a> Genes &amp; Development. 19 (9), 1040-1052 (2005).</li><li>Toledo, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR+prohibits+replication+catastrophe+by+preventing+global+exhaustion+of+RPA.">ATR prohibits replication catastrophe by preventing global exhaustion of RPA.</a> Cell. 155 (5), 1088-1103 (2013).</li><li>Brickner, J. R., Garzon, J. L., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+a+tightrope:+The+complex+balancing+act+of+R-loops+in+genome+stability.">Walking a tightrope: The complex balancing act of R-loops in genome stability.</a> Molecular Cell. 82 (12), 2267-2297 (2022).</li><li>Cejka, P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+resection:+Mechanism+and+control.">DNA end resection: Mechanism and control.</a> Annual Review of Genetics. 55, 285-307 (2021).</li><li>Kowalczykowski, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+molecular+mechanisms+of+recombinational+DNA+repair.">An overview of the molecular mechanisms of recombinational DNA repair.</a> Cold Spring Harbor Perspectives in Biology. 7 (11), 016410 (2015).</li><li>Tirman, S., Cybulla, E., Quinet, A., Meroni, A., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMPOL+ready,+set,+reprime.">PRIMPOL ready, set, reprime.</a> Critical Reviews in Biochemistry and Molecular Biology. 56 (1), 17-30 (2021).</li><li>Taglialatela, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REV1-Pol&#950;+maintains+the+viability+of+homologous+recombination-deficient+cancer+cells+through+mutagenic+repair+of+PRIMPOL-dependent+ssDNA+gaps.">REV1-Pol&#950; maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.</a> Molecular Cell. 81 (19), 4008-4025 (2021).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+An+essential+regulator+of+genome+integrity.">ATR: An essential regulator of genome integrity.</a> Nature Reviews. Molecular Cell Biology. 9 (8), 616-627 (2008).</li><li>Tirman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+post-replicative+repair+mechanisms+fill+PRIMPOL-dependent+ssDNA+gaps+in+human+cells.">Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.</a> Molecular Cell. 81 (19), 4026-4040 (2021).</li><li>Cong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+gaps+are+a+key+determinant+of+PARP+inhibitor+synthetic+lethality+with+BRCA+deficiency.">Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.</a> Molecular Cell. 81 (15), 3128-3144 (2021).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCNE1+copy+number+is+a+biomarker+for+response+to+combination+WEE1-ATR+inhibition+in+ovarian+and+endometrial+cancer+models.">CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.</a> Cell Reports. Medicine. 2 (9), 100394 (2021).</li><li>Konstantinopoulos, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Replication+stress+biomarker+is+associated+with+response+to+gemcitabine+versus+combined+gemcitabine+and+ATR+inhibitor+therapy+in+ovarian+cancer.">A Replication stress biomarker is associated with response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in ovarian cancer.</a> Nature Communications. 12, 5574 (2021).</li><li>Buisson, R., Boisvert, J. L., Benes, C. H., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+but+concerted+roles+of+ATR,+DNA-PK,+and+Chk1+in+countering+replication+stress+during+S+phase.">Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase.</a> Molecular Cell. 59 (6), 1011-1024 (2015).</li><li>Belan, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=POLQ+seals+post-replicative+ssDNA+gaps+to+maintain+genome+stability+in+BRCA-deficient+cancer+cells.">POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.</a> Molecular Cell. 82 (24), 4664-4680 (2022).</li><li>da Costa, A. A. B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+replication+stress+in+cancer+therapy.">Targeting replication stress in cancer therapy.</a> Nature Reviews. Drug Discovery. 22 (1), 38-58 (2023).</li><li>Chen, R., Wold, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+protein+A:+Single-stranded+DNA's+first+responder:+Dynamic+DNA-interactions+allow+replication+protein+A+to+direct+single-strand+DNA+intermediates+into+different+pathways+for+synthesis+or+repair.">Replication protein A: Single-stranded DNA's first responder: Dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.</a> BioEssays. 36 (12), 1156-1161 (2014).</li><li>Bhat, K. P., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA+and+RAD51:+Fork+reversal,+fork+protection,+and+genome+stability.">RPA and RAD51: Fork reversal, fork protection, and genome stability.</a> Nature Structural &amp; Molecular Biology. 25 (6), 446-453 (2018).</li><li>Mar&#233;chal, A., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA-coated+single-stranded+DNA+as+a+platform+for+post-translational+modifications+in+the+DNA+damage+response.">RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.</a> Cell Research. 25 (1), 9-23 (2015).</li></ol>

As was mentioned in the protocol, it is valuable to include a few experimental controls to ensure that the assay is working. These include a no IdU treated sample as well as a no primary antibody treated sample. Both negative controls should yield cells that are stained by DAPI but contain no IdU signal.
Based on the experimental conditions and cell lines used, different antibody dilutions may be needed to obtain the best fluorescent signal. Too much signal may result in an inability to quanti...

Genomic DNA is frequently exposed to multiple assaults from various endogenous and exogenous sources1. The frequency of endogenous damage directly correlates with the levels of metabolic byproducts, such as reactive oxygen species or aldehydes, which are intrinsically higher in multiple cancer types, including ovarian cancers2,3. It is imperative that DNA damage is efficiently resolved; otherwise, it can foster genotoxic lesions and, consequently, mutagenesis. The ability of cells to repair genotoxic lesions is reliant on the functionality of error-free DNA repair pathways and the efficient regulation of cell cycle progression in response to DNA damage. Notably, many ovarian cancers bear functionally inactivating mutations in p53 and, thus, have a defective G1/S checkpoint, leading the cells to initiate DNA replication despite the presence of unrepaired genomic lesions4,5. The degree of DNA damage in ovarian cancers is further compounded by the observation that more than 50% of high-grade serous ovarian carcinoma (HGSOC) have defects in BRCA1- and BRCA2-mediated homologous recombination, the error-free DNA repair pathway, and around 20% have amplification in the gene CCNE1, which prematurely pushes G1 cells into the S-phase6. Together the high frequency of endogenous DNA damage, defective checkpoints, and malfunctioning repair pathways exponentially enhance the accumulation of genomic lesions in ovarian cancers. These lesions can serve as impediments to the progression of critical cellular processes such as DNA replication and transcription. As discussed below, such impediments catalyze the generation of single-stranded DNA (ssDNA) in cells.
The double helix of DNA is critical for safeguarding the genome from multiple mutagenic processes, such as spontaneous depurination and depyrimidination, the activity of cytosine deaminases, and oxidative DNA damage1,7. In contrast, ssDNA is highly vulnerable to these mutational events. Multiple processes in cells can result in the generation of ssDNA (Figure 1). These include the following:
(i) Stalling of the DNA replication machinery: This leads to an uncoupling of the DNA helicase and polymerase, leaving stretches of ssDNA8,9.
(ii) Stalling of the transcription machinery: Persistent stalling of RNA polymerase leads to the generation of three-stranded hybrid DNA/RNA structures called R-loops. R-loop formation exposes the displaced, non-transcribed DNA as a single strand10.
(iii) DNA end-resection: The initiation of homology-directed repair requires the generation of a 3&#39; ssDNA to catalyze the search for a homologous sequence11.
(iv) D-loop: Strand invasion during homologous recombination can result in the displacement of the non-template complementary strand, resulting in ssDNA12.
(v) Replication-coupled gaps: During DNA replication, lagging strand synthesis happens in a discontinuous fashion, whereby Okazaki fragments are first generated and then ligated. A delay or defect in processing the Okazaki fragments can also result in ssDNA formation. Finally, if the replication fork on a leading strand encounters a stalling lesion, DNA polymerase, and primase, PRIMPOL can reprime the synthesis downstream, leaving an ssDNA gap behind13,14.
Evidently, most of these events either happen when the DNA replication machinery faces genomic lesions or during replication-coupled repair, suggesting that higher DNA damage leads to increased levels of ssDNA. As many of these events are replication-associated, the formation of ssDNA is considered the marker of &#34;replication stress&#34; in cells15,16.
Here, we describe an assay that can be used to reliably quantify ssDNA in cells. The simplicity, reproducibility, and cost benefits of this approach make it amenable to be used for assessing the replication-stress response in cells. Emerging studies have revealed that the level of ssDNA can also be a predictor of responses to chemotherapy, such as inhibitors of PARP1/2 enzymes, ATR, and Wee1 kinase17,18,19,20,21. These inhibitors are being pursued in the treatment regimen of several HGSOCs22. Therefore, this assay can also be a useful tool to predict chemotherapeutic responses in ovarian cancer cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Paraformaldehyde (PFA)</td>
			<td>Fisher Scientific</td>
			<td>NC0179595</td>
			<td>10 g sucrose + 100 mL 10X PBS + water to make volume to 925 mL. Add 75 mL 40% Methanol free PFA, mix, and make aliquots of 50 mL before storage 
			Storage: Store in -20 &#176;C</td>
		</tr>
		<tr>
			<td>5-iodo-2&#39;-deoxyuridine (IdU)</td>
			<td>Sigma Aldrich</td>
			<td>I7125-5G</td>
			<td>MW = 354.10 g/mol.For 10 mM stock: dissolve 3.541 mg IdU to 1 mL 1 N liquid ammonia 
			Storage: Stored in -20 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-BrdU antibody</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-mouse Alexa Fluor Plus 488 secondary antibody</td>
			<td>Thermo Scientific</td>
			<td>A32766</td>
			<td>Light sensitive - keep in dark 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td>Made by adding specific mass to volume of PBS 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Circular Cover Glass&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72230-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS GA3 Software&#160;</td>
			<td>Nikon&#160;</td>
			<td>77010604</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td>Growth Media: RPMI supplemented with L-glutamine, 0.01 mg/mL bovine insulin; fetal bovine serum to a final concentration of 20% and 1X Pen Strep 
			Storage: Freezing Media: growth media + 5% DMSO and stored in -80 &#176;C</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>Sigma Aldrich</td>
			<td>P4832-50ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Thermo Scientific</td>
			<td>P36962</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Water, sterile-filtered</td>
			<td>Sigma Aldrich</td>
			<td>W3500-6X500ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Representative images and the quantification of IdU foci from the nuclei derived from the untreated cells and cells treated with 0.5 mM hydroxyurea for 24 h are shown in Figure 4. Both nuclei are stained and identifiable in the DAPI channel. The analysis of these images consists of quantifying the number of foci in each nucleus. The number of foci is proportional to the degree of replication stress.
<img alt="Figure 1" cl...

Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Our protocol presents a highly efficient and easily reproducible method to quantify single stranded DNA, which is a marker of replication stress in a variety of cell lines. Additionally, its simplicity allows for high throughput applications and screens. In particular, this method allows for rapid quantification of a replication stress, a hallmark of several ovarian cancers.It is also amenable to a host automatic analysis software, further increasing efficiency. Single stranded DNA is now being actively considered as a biomarker for response to chemotherapy targeting DNA repair pathways. Hence, this method is highly applicable in that scenario.Replication stress exists beyond ovarian cancer contexts or BRCA1, BRCA2 deficient cancers. And thus, this method can be applied to a wide array of other cell types or disease contexts. To begin, make polylysine-coated cover slips by adding autoclaved 12 millimeter diameter cover slips and polylysine solution to a 50 milliliter conical tube, and place on a rocker for 15 minutes.Aspirate the solution on a tissue culture hood. Wash the cover slips by adding sterile water, and place the tube containing the cover slips back on the rocker for five minutes. After washing the cover slips three times, aspirate water from the tube and spread the cover slips on a sterile dish.Aspirate the remaining water and let them dry in the tissue culture hood for one hour or until no water droplets remain. Once dry, seal the dish with Parafilm and place it at 4 degrees Celsius. To prevent the detachment of the cells from the cover slips during the pre-extraction step, place one polylysine-coated cover slip in each well of a 24-well plate.Once the OVCAR3 cells reach 70 to 80%confluency, aspirate the medium from the plate and wash the cells with 5 to 7 milliliters of PBS. Aspirate the PBS from the plate. After aspirating the PBS, add 1 milliliter of 0.25%trypsin and place the dish in the incubator at 37 degrees Celsius for 8 to 10 minutes or until cells lift off from the bottom of the plate.Collect the cells with 5 to 10 milliliters of medium and add the cell suspension to a conical tube. Count the cells manually with a hemocytometer using standard procedures. Next, dilute the cell suspension, and obtain a number that will yield 70 to 80%confluency after three populations.Add 1 milliliter of cell suspension on polylysine cover slips placed in the wells of a 24-well plate, and grow the cells in the culture medium under standard conditions. After one population doubling, aspirate the existing media from the plate and pulse the cells with 10 micromolar IdU for the two subsequent population doublings. To harvest the cells, replace the medium with ice cold 0.5%PBSTx on ice for five minutes.For fixation of cells, aspirate PBSTx and incubate the cells for 15 minutes with 3%paraformaldehyde at room temperature. After three to four washes with PBS, keep fixed cells at 4 degrees Celsius until further use. After fixation, permeabilize the cells using 0.5%PBSTx on ice for five minutes, ensuring to cover the entire cover slip.After washing the cells three to four times with 1 milliliter of 0.2%PBST at room temperature, aspirate the PBST and block the samples using 5%BSA made in PBS for 30 minutes at room temperature. For immunostaining, prepare a unified chamber by putting a wet paper towel on a flat bottom Tupperware. Cover the 24-well plate lid with Parafilm.Place it in the humidified chamber and lay down the cover slips on the plate lid. Add 60 microliters of diluted anti-BrdU primary mouse antibody on the top of the cover slip. Alternatively, to reduce the antibody from drying and use less volume, place a drop of diluted antibody on the Parafilm and flip the cover slip onto it.Then incubate for one hour at 37 degrees Celsius. After incubation, aspirate the primary antibody. Return the cover slips back to a 24-well plate and wash them with 0.2%PBST four times.Add diluted secondary antibody on the cover slip as demonstrated earlier, and incubate for one hour in the dark at room temperature. Label a microscope slide and mount the cover slip on the slides with DAPI mounting medium. Store slides in the dark at room temperature for 24 hours if the mounting medium needs to be cured or hardened.The nuclei derived from the untreated and treated cells with 0.5 millimolar hydroxyurea were stained and identifiable in the DAPI and IdU channels. The analysis of these images consists of quantifying the number of foci in each nucleus. The number of foci is proportional to the degree of replication stress.It is important to consider the doubling time of the cell line of choice as the time of IdU pulsing may vary for longer or shorter doubling periods. Alternatively, we can probe cells for replication protein A to confirm the results and assess various replication stress responses in cells. This technique is used for many cell lines in tandem with any DNA damage-inducing agent to examine the accumulation of single-strained DNA.Thus, this method is widely accessible and applicable.

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Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (&#60;1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (&#62;500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...