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>
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The authors have nothing to disclose.

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>
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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<...

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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 ...

The study of the DNA damage response is necessary in understanding the pathophysiology of ovarian cancer potential biomarkers in novel targeted therapy. This technique allows for the sample to stay intact and allows the examination of antigens in their location of action. This protocol is quick and can be adapted to any antigen amenable to immunofluorescence.This method gives insight into ovarian cancer and DNA damage repair processes involved in chemotherapeutic and PARP inhibitor resistance. These methods can be applied to other areas of study involving the use of 3D cultures and organoids. To begin, aspirate the media from the irradiated and incubated organoids without disrupting the 3D matrix.Then wash with 300 microliters of PBS. Fix the organoids in 300 microliters of 2%paraformaldehyde for 10 minutes. After fixing, wash the organoids with 300 microliters of staining buffer and place them on a shaker for five minutes.Then permeabilize the organoid by gently adding 300 microliters of permeabilization buffer. After 20 minutes of incubation, remove the buffer and wash with 300 microliters of staining buffer. Place the organoids on a shaker for five minutes.Next, add 300 microliters of staining buffer to block the permeabilization step. Place on the shaker for 30 minutes and aspirate the staining buffer using a pipette. Then add 300 microliters of primary antibodies diluted in staining buffer.Incubate at four degrees Celsius for 16 hours. After incubation, remove the primary antibody solution. Perform three washes with 300 microliters of staining buffer for five minutes each on the shaker.Add 300 microliters of secondary antibody solution diluted in staining buffer, and incubate for one hour in the dark. After one hour, aspirate the secondary antibody solution and add 300 microliters of diluted DAPI. Place on the shaker for five minutes.Perform three washes with 300 microliters of staining buffer for five minutes each on the shaker. Remove the staining buffer and detach the chambers using the removal kit included with the chamber slides. Cut the tip of a P200 pipette tip and use this to add mounting medium to cover each well containing an organoid tab.Place a cover slip over the specimen while avoiding bubbles. Take clear nail polish and paint it onto the sides of the cover glass to seal the slide. Let it harden for one hour and then place it at 20 degrees Celsius.Acquire images using a confocal microscope using a 63X objective with immersion oil. Using this protocol, patient derived ovarian cancer organoids were stained for DNA damage repair proteins before and after irradiation, and evaluated for biomarkers such as gamma H2AX, a marker of DNA damage, and RAD51, a marker for homologous recombination repair. RPA, a marker of replication stress, 53BP1, a marker of non-homologous enjoining, and Geminin, a GS2 phase cell cycle marker were also evaluated using this technique.The BME tabs tend to depolymerize, so paying close attention after fixing and aspirating is vital. Analyzing the antigens to determine the organoids damage and the ability to mount a DNA damage response could help assist in the evaluation of chemotherapeutic resistance.

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Research

JoVE Journal

Cancer Research

1.5K vues.  Washington University in St. Louis. L’étude de la réponse aux dommages de l’ADN est nécessaire pour comprendre la physiopathologie des biomarqueurs potentiels du cancer de l’ovaire dans le cadre d’une nouvelle thérapie ciblée. Cette technique permet à l’échantillon de rester intact et permet l’examen des antigènes dans leur lieu d’action. Ce protocole est rapide et peut être adapté à tout antigène susceptible d’immunofluorescence.Cette méthode donne un aperçu du cancer de l’ovaire et des p...