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

Summary

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.

Abstract

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.

Introduction

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) inhibitors^1,2. 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 und....

Protocol

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 °C.

1. Organoid generation

1. Generate the organoids following the previously published report²⁰.

Results

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: ɣ-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

Discussion

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 .......

Materials

List of materials used in this article

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