Name: visualizing dna damage repair proteins in patient-derived ovarian cancer organoids via immunofluorescence assays
Uploaded: 2023-02-24T14:20:00
Description: Watch this Scientific Journal Video about Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays at JoVE.com

We are grateful for the guidance of Pavel Lobachevsky, PhD in establishing this protocol. We&#39;d also like to acknowledge Washington University&#39;s School of Medicine in St. Louis&#39;s Department of Obstetrics and Gynecology and Division of Gynecologic Oncology, Washington University&#39;s Dean&#39;s Scholar Program, Gynecologic Oncology Group Foundation, and the Reproductive Scientist Development Program for their support of this project.

Tumor tissue and ascites were obtained after obtaining patient consent as part of a gynecologic oncology biorepository that was approved by the Washington University in St. Louis Institutional Review Board (IRB). Patients were included if they had advanced stage high-grade serous ovarian cancer (HGSOC). All procedures were performed at room temperature on the bench unless otherwise specified. All reagents were prepared at room temperature (unless otherwise indicated) and stored at 4 &#176;C.
<st...

<ol>
	<li>Lheureux, S., Gourley, C., Vergote, I., Oza, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+ovarian+cancer.">Epithelial ovarian cancer.</a> Lancet Oncology. 393 (10177), 1240-1253 (2019).</li><li>Tew, W. P., 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=PARP+inhibitors+in+the+management+of+ovarian+cancer:+ASCO+guideline.">PARP inhibitors in the management of ovarian cancer: ASCO guideline.</a> Journal of Clinical Oncology. 38 (30), 3468-3493 (2020).</li><li>Tomasova, 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=DNA+repair+and+ovarian+carcinogenesis:+impact+on+risk,+prognosis+and+therapy+outcome.">DNA repair and ovarian carcinogenesis: impact on risk, prognosis and therapy outcome.</a> Cancers. 12 (7), 1713 (2020).</li><li>Mukhopadhyay, 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=Development+of+a+functional+assay+for+homologous+recombination+status+in+primary+cultures+of+epithelial+ovarian+tumor+and+correlation+with+sensitivity+to+poly(ADP-Ribose)+polymerase+inhibitors.">Development of a functional assay for homologous recombination status in primary cultures of epithelial ovarian tumor and correlation with sensitivity to poly(ADP-Ribose) polymerase inhibitors.</a> Clinical Cancer Research. 16 (8), 2344-2351 (2010).</li><li>Graeser, M., 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+marker+of+homologous+recombination+predicts+pathologic+complete+response+to+neoadjuvant+chemotherapy+in+primary+breast+cancer.">A marker of homologous recombination predicts pathologic complete response to neoadjuvant chemotherapy in primary breast cancer.</a> Clinical Cancer Research. 16 (24), 6159-6168 (2010).</li><li>Hill, S. J., 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=Prediction+of+DNA+repair+inhibitor+response+in+short-term+patient-derived+ovarian+cancer+organoids.">Prediction of DNA repair inhibitor response in short-term patient-derived ovarian cancer organoids.</a> Cancer Discovery. 8 (11), 1404-1421 (2018).</li><li>Naipal, K. A. T., 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=Functional+ex+vivo+assay+to+select+homologous+recombination-deficient+breast+tumors+for+PARP+inhibitor+treatment.">Functional ex vivo assay to select homologous recombination-deficient breast tumors for PARP inhibitor treatment.</a> Clinical Cancer Research. 20 (18), 4816-4826 (2014).</li><li>Tumiati, M., 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+functional+homologous+recombination+assay+predicts+primary+chemotherapy+response+and+long-term+survival+in+ovarian+cancer+patients.">A functional homologous recombination assay predicts primary chemotherapy response and long-term survival in ovarian cancer patients.</a> Clinical Cancer Research. 24 (18), 4482-4493 (2018).</li><li>Mukhopadhyay, 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=Clinicopathological+features+of+homologous+recombination-deficient+epithelial+ovarian+cancers:+sensitivity+to+PARP+inhibitors,+platinum,+and+survival.">Clinicopathological features of homologous recombination-deficient epithelial ovarian cancers: sensitivity to PARP inhibitors, platinum, and survival.</a> Cancer Research. 72 (22), 5675-5682 (2012).</li><li>Johnson, S. W., Laub, P. B., Beesley, J. S., Ozols, R. F., Hamilton, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+platinum-DNA+damage+tolerance+is+associated+with+cisplatin+resistance+and+cross-resistance+to+various+chemotherapeutic+agents+in+unrelated+human+ovarian+cancer+cell+lines.">Increased platinum-DNA damage tolerance is associated with cisplatin resistance and cross-resistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines.</a> Cancer Research. 57 (5), 850-856 (1997).</li><li>Stefanou, D. T., 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=Aberrant+DNA+damage+response+pathways+may+predict+the+outcome+of+platinum+chemotherapy+in+ovarian+cancer.">Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.</a> PLoS One. 10 (2), 0117654 (2015).</li><li>Helleday, T., Petermann, E., Lundin, C., Hodgson, B., Sharma, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+pathways+as+targets+for+cancer+therapy.">DNA repair pathways as targets for cancer therapy.</a> Nature Reviews Cancer. 8 (3), 193-204 (2008).</li><li>Ivashkevich, A., Redon, C. E., Nakamura, A. J., Martin, R. F., Martin, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+&#947;-H2AX+assay+to+monitor+DNA+damage+and+repair+in+translational+cancer+research.">Use of the &#947;-H2AX assay to monitor DNA damage and repair in translational cancer research.</a> Cancer Letters. 327 (1-2), 123-133 (2012).</li><li>Fuh, 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=Homologous+recombination+deficiency+real-time+clinical+assays,+ready+or+not.">Homologous recombination deficiency real-time clinical assays, ready or not.</a> Gynecologic Oncology. 159 (3), 877-886 (2020).</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>Lee, E. K., Matulonis, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+inhibitor+resistance+mechanisms+and+implications+for+post-progression+combination+therapies.">PARP inhibitor resistance mechanisms and implications for post-progression combination therapies.</a> Cancers. 12 (8), 2054 (2020).</li><li>Panzarino, N. J., 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+underlie+BRCA+deficiency+and+therapy+response.">Replication gaps underlie BRCA deficiency and therapy response.</a> Cancer Research. 81 (5), 1388-1397 (2021).</li><li>Yee, C., Dickson, K. A., Muntasir, M. N., Ma, Y., Marsh, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+modelling+of+ovarian+cancer:+from+cell+lines+to+organoids+for+discovery+and+personalized+medicine.">Three-dimensional modelling of ovarian cancer: from cell lines to organoids for discovery and personalized medicine.</a> Frontiers in Bioengineering and Biotechnology. 10, 836984 (2022).</li><li>Regan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+staining+of+colorectal+cancer+patient-derived+organoids.">Immunofluorescence staining of colorectal cancer patient-derived organoids.</a> Methods Cell Biology. 171, 163-171 (2022).</li><li>Graham, 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=Generation+and+culturing+of+high-grade+serous+ovarian+cancer+patient+derived+organoids.">Generation and culturing of high-grade serous ovarian cancer patient derived organoids.</a> Journal of Visualized Experiments. (191), (2023).</li><li>Jakl, L., 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=Validation+of+JCountPro+software+for+efficient+assessment+of+ionizing+radiation-induced+foci+in+human+lymphocytes.">Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes.</a> International Journal of Radiation Biology. 92 (12), 766-773 (2016).</li><li>Mullen, M. M., 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=GAS6/AXL+inhibition+enhances+ovarian+cancer+sensitivity+to+chemotherapy+and+PARP+inhibition+through+increased+DNA+damage+and+enhanced+replication+stress.">GAS6/AXL inhibition enhances ovarian cancer sensitivity to chemotherapy and PARP inhibition through increased DNA damage and enhanced replication stress.</a> Molecular Cancer Research. 20 (2), 265-279 (2022).</li><li>van Ineveld, R. L., Ariese, H. C. R., Wehrens, E. J., Dekkers, J. F., Rios, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+resolution+three-dimensional+imaging+of+intact+organoids.">Single-cell resolution three-dimensional imaging of intact organoids.</a> Journal of Visualized Experiments. (160), e60709 (2020).</li><li>Dekkers, J. F., 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-resolution+3D+imaging+of+fixed+and+cleared+organoids.">High-resolution 3D imaging of fixed and cleared organoids.</a> Nature Protocols. 14 (6), 1756-1771 (2019).</li><li>O'Rourke, K. P., Dow, L. E., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+staining+of+mouse+intestinal+stem+cells.">Immunofluorescent staining of mouse intestinal stem cells.</a> Bio Protocols. 6 (4), 1732 (2016).</li><li>Nwani, N. G., Sima, L. E., Nieves-Neira, W., Matei, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+microenvironment+in+high+grade+serous+ovarian+cancer.">Targeting the microenvironment in high grade serous ovarian cancer.</a> Cancers. 10 (8), 266 (2018).</li><li>Ghoneum, 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=Exploring+the+clinical+value+of+tumor+microenvironment+in+platinum-resistant+ovarian+cancer.">Exploring the clinical value of tumor microenvironment in platinum-resistant ovarian cancer.</a> Seminars in Cancer Biology. 77, 83-98 (2021).</li><li>Yang, Y., Yang, Y., Yang, J., Zhao, X., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+in+ovarian+cancer:+function+and+therapeutic+strategy.">Tumor microenvironment in ovarian cancer: function and therapeutic strategy.</a> Frontiers in Cell and Developmental Biology. 8, 758 (2020).</li><li>van de Wetering, M., 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=Prospective+derivation+of+a+living+organoid+biobank+of+colorectal+cancer+patients.">Prospective derivation of a living organoid biobank of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li><li>Broutier, L., 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=Human+primary+liver+cancer-derived+organoid+cultures+for+disease+modeling+and+drug+screening.">Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.</a> Nature Medicine. 23 (12), 1424-1435 (2017).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</li><li>Cybulla, E., 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=Leveraging+the+replication+stress+response+to+optimize+cancer+therapy.">Leveraging the replication stress response to optimize cancer therapy.</a> Nature Reviews. , (2022).</li></ol>

The DNA damage response plays an integral role in both the development of ovarian cancer and chemotherapy resistance. Therefore, a thorough understanding of DNA repair mechanisms is imperative. Here, a methodology is presented to study DNA damage repair proteins in 3D, intact organoids. A reproducible, reliable protocol is developed utilizing hallmark antibodies to evaluate DNA damage, homologous recombination, non-homologous end joining, and replication stress. Importantly, these methods are validated using genetically ...

Ovarian cancer is the leading cause of death due to gynecologic malignancy. The majority of patients are treated with DNA-damaging drugs such as carboplatin, and those with homologous recombination repair (HRR)-deficient tumors can be given poly (ADP-ribose) polymerase (PARP) inhibitors1,2. However, most patients develop resistance to these therapies and die within 5 years of diagnosis. Dysregulation of the DNA damage response (DDR) has been associated with the development of ovarian cancer and both chemotherapy and PARP inhibitor resistance3. Thus, study of the DDR is imperative in understanding the pathophysiology of ovarian cancer, potential biomarkers, and novel targeted therapies.
Current methods to evaluate the DDR utilize immunofluorescence (IF), as this allows for the accurate identification and localization of DNA damage proteins and nucleotide analogs. Once there is a double-stranded break (DSB) in the DNA, the histone protein H2AX is rapidly phosphorylated, forming a focus where DNA damage repair proteins congregate4. This phosphorylation can be easily identified utilizing IF; in fact, the &#611;-H2AX assay has commonly been employed to confirm the induction of a DSB5,6,7,8,9. Increased DNA damage has been associated with platinum sensitivity and efficacy of DNA damaging agents10,11,12, and &#611;-H2AX has been proposed as a biomarker associated with chemotherapy response in other cancer treatments13. Upon a DSB, a cell proficient in HRR performs a series of events that leads to BRCA1 and BRCA2 recruiting RAD51 to replace replication protein A (RPA) and bind to the DNA. HRR repair uses a DNA template to faithfully repair the DSB. However, when tumors are deficient in HRR, they rely on alternative repair pathways such as non-homologous end joining (NHEJ). NHEJ is known to be error-prone and creates a high mutational burden on the cell, which uses 53BP1 as a positive regulator14. These DNA damage proteins can all be accurately identified as foci using IF. In addition to staining for proteins, IF can be used to study fork protection and single-stranded DNA gap formation. The ability to have stable forks has been correlated with platinum response, and recently, gap assays have shown the potential to predict the response to PARP inhibitors6,15,16,17. Therefore, staining for the nucleotide analogs after introduction into the genome is another way to study the DDR.
To date, evaluation of the DDR in ovarian cancer has been largely limited to homogenous 2D cell lines that do not recapitulate the clonal heterogeneity, microenvironment, or architecture of in vivo tumors18,19. Recent research suggests organoids are superior to 2D cell lines in studying complex biologic processes such as DDR mechanisms6. The present methodology evaluates RAD51, &#611;-H2AX, 53BP1, RPA, and geminin in PDOs. These methods assess the intact organoid and allow for the study of DDR mechanisms in a setting more similar to the in vivo tumor microenvironment. Together with confocal microscopy and automated foci counting, this methodology can aid in understanding the DDR pathway in ovarian cancer and personalizing treatment plans for patients.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x phosphate buffered saline with calcium and magnesium (PBS++)</td>
			<td>Sigma</td>
			<td>14-040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1x phosphate buffered saline without calcium and magnesium (PBS)</td>
			<td>Fisher Scientific&#160;</td>
			<td>ICN1860454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ant-53BP1 Antibody&#160;</td>
			<td>BD Biosciences</td>
			<td>612522</td>
			<td>diluted to 1:500 in staining buffer</td>
		</tr>
		<tr>
			<td>Ant-Geminin Antibody&#160;</td>
			<td>Abcam</td>
			<td>ab104306</td>
			<td>diluted to 1:200 in staining buffer</td>
		</tr>
		<tr>
			<td>Anti-Geminin Antibody&#160;</td>
			<td>ProteinTech</td>
			<td>10802-1-AP</td>
			<td>diluted to 1:400 in staining buffer</td>
		</tr>
		<tr>
			<td>Anti-RAD51 Antibody&#160;</td>
			<td>Abcam</td>
			<td>ab133534</td>
			<td>diluted to 1:1000 in staining buffer</td>
		</tr>
		<tr>
			<td>Anti-yH2AX Antibody&#160;</td>
			<td>Millipore-Sigma</td>
			<td>05-636</td>
			<td>diluted to 1:500 in staining buffer</td>
		</tr>
		<tr>
			<td>Ant-phospho-RPA32 (S4/S8) Antibody</td>
			<td>Bethyl Laboratories&#160;</td>
			<td>A300-245A-M</td>
			<td>diluted to 1:200 in staining buffer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP1605 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge; Sorvall St 16R Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>75004240</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope, Leica SP5 confocal system DMI4000</td>
			<td>Leica&#160;</td>
			<td>389584</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes, 15 mL</td>
			<td>Corning&#160;</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess 3 FL Automated Cell Counter (Cell Counting Machine)&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>AMQAF2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Scientific&#160;</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slip</td>
			<td>LA Colors</td>
			<td></td>
			<td>Any clear nail polish will suffice</td>
		</tr>
		<tr>
			<td>Cultrex&#160;RGF Basement Membrane Extract, Type 2</td>
			<td>&#160;R&#38;D Systems&#160;</td>
			<td>3533-010-02</td>
			<td>Could probably use Matrigel or other BME Matrix&#160;</td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>&#160;R37606</td>
			<td>NucBlue Fixed Cell ReadyProbes Reagent, Diluted in 1x PBS&#160;</td>
		</tr>
		<tr>
			<td>Glycine&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>NC0756056</td>
			<td></td>
		</tr>
		<tr>
			<td>JCountPro</td>
			<td>JCountPro</td>
			<td></td>
			<td>For access to the software, Email: jcountpro@gmail.com&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>07-000-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish&#160;</td>
			<td>StatLab</td>
			<td>SL102450</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafomraldehyde (PFA), 2%&#160;</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>157-4</td>
			<td>Dilute to 4% PFA in PBS++ to obtain 2% PFA</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer</td>
			<td>Made in Lab</td>
			<td></td>
			<td>0.2% X-100 Triton in PBS++&#160;</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips&#160;</td>
			<td>Rainin&#160;</td>
			<td>17014967</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Scientific&#160;</td>
			<td>&#160;P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Buffer&#160;</td>
			<td>Made in Lab</td>
			<td></td>
			<td>0.5% BSA, 0.15% Glycine, 0.1% X-100 Triton in PBS++&#160;</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Lab-Tek II Chamber Slide System&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>12-565-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Alderich&#160;</td>
			<td>11332481001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Scientific&#160;</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12604013</td>
			<td>animal origin-free, recombinant enzyme</td>
		</tr>
		<tr>
			<td>X-RAD 320 Biological Irradiator&#160;</td>
			<td>Precision X-Ray Irradiation&#160;</td>
			<td>X-RAD320</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing dna damage repair proteins in patient-derived ovarian cancer organoids via immunofluorescence assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

The presented protocol can successfully stain, visualize, and quantify nuclear DNA damage repair proteins in organoids. This technique was used to stain and evaluate PDOs both before and after irradiation. PDOs were exposed to 10 Gy of radiation and evaluated for the following biomarkers: &#611;-H2AX (Figure 1), a marker of DNA damage; RAD51 (Figure 2), a marker for HRR; 53BP1, a marker of NHEJ; RPA, a marker of replication stress (Figure 3<...

Watch this Scientific Journal Video about Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays at JoVE.com

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Research

JoVE Journal

Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

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 ...

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

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 ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

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 ...

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, ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...