Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays
February 24th, 2023
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.
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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Overview
This protocol outlines methods for evaluating DNA damage repair proteins in ovarian cancer organoids derived from patients. It includes comprehensive plating and staining techniques along with objective quantification procedures.
Key Study Components
Area of Science
- Ovarian cancer research
- DNA damage response
- Organoid technology
Background
- Understanding DNA damage response is crucial for ovarian cancer pathophysiology.
- This study explores potential biomarkers for targeted therapy.
- The protocol allows examination of antigens in their functional locations.
- It is adaptable to various antigens suitable for immunofluorescence.
Purpose of Study
- To investigate DNA damage repair processes in ovarian cancer.
- To provide insights into chemotherapeutic and PARP inhibitor resistance.
- To develop methods applicable to other studies involving 3D cultures and organoids.
Methods Used
- Aspirating media from irradiated and incubated organoids without disrupting the 3D matrix.
- Utilizing immunofluorescence for antigen examination.
- Implementing quick and adaptable protocols for various antigens.
- Employing objective quantification procedures for analysis.
Main Results
- Insights into DNA damage repair mechanisms in ovarian cancer.
- Identification of resistance mechanisms to chemotherapeutics.
- Demonstration of the protocol's adaptability to different antigens.
- Potential application of methods to other areas of study.
Conclusions
- The protocol provides a valuable tool for studying DNA damage response in ovarian cancer.
- It enhances understanding of treatment resistance mechanisms.
- Methods can be broadly applied to various research contexts involving organoids.
Frequently Asked Questions
Executive Industry Relevance
Visualizing DNA damage repair proteins in patient-derived ovarian cancer organoids using immunofluorescence enables high-content, spatially resolved biomarker analysis in disease-relevant 3D models. This approach enhances predictive confidence in target validation and mechanistic de-risking for therapeutic strategies addressing chemoresistance. Integrating these assays at the discovery and preclinical interface supports risk-adjusted portfolio decisions in oncology R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of DNA repair pathways and functional target validation in clinically relevant 3D tumor models.
- Supports mechanistic de-risking by visualizing protein recruitment and colocalization after genotoxic stress.
- Facilitates identification of predictive biomarkers for chemotherapeutic and PARP inhibitor resistance.
Screening & Assay Development
- Establishes validated immunofluorescence workflows for quantitative protein foci analysis in organoids.
- Delivers reproducible, high-sensitivity readouts for downstream screening of DNA repair modulators.
- Enables standardization of imaging and automated foci quantification for scalable assay deployment.
Translational & Preclinical Research
- Aligns biomarker evaluation with disease-relevant 3D models, improving translational continuity.
- Supports preclinical assessment of drug response and resistance mechanisms in patient-derived systems.
- Provides a platform for evaluating temporal and spatial dynamics of DNA repair proteins in situ.
Pipeline & Workflow Integration
This immunofluorescence assay positions within the discovery-to-preclinical continuum, bridging mechanistic studies and translational biomarker validation in ovarian cancer research.
- Discovery Biology: Enables hypothesis testing of DNA repair function and pathway engagement in intact organoids.
- Screening: Provides quantitative, reproducible foci counts for comparative analysis of treatment conditions.
- Analytics: Supports automated image analysis and statistical comparison of protein localization and colocalization.
- Translational Research: Connects in vitro findings to patient-relevant resistance mechanisms and biomarker profiles.
- Enterprise Reuse: Adaptable to other antigens and 3D disease models for broad R&D applicability.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation and mechanistic understanding of resistance.
- Operational Value: Standardizes immunofluorescence workflows for reproducibility and scalability in 3D models.
- Strategic Value: Informs go/no-go decisions by linking biomarker dynamics to therapeutic response.
- Portfolio Impact: Enables risk-adjusted prioritization of DNA repair targets and resistance-modifying strategies.
Implementation Considerations
- Requires expertise in 3D organoid culture and immunofluorescence imaging.
- Demands access to confocal microscopy and automated image analysis software.
- Necessitates careful handling to preserve organoid integrity during processing.
- Standardization across teams is essential for reproducible quantitative outputs.
- Adaptation to other tumor types or antigens may require protocol optimization.
