Construction of Human Breast Cancer Organoids and Comet Assay–Based Quantification of Doxorubicin-Induced DNA Damage

Summary

This protocol establishes a human breast cancer organoids (BCOs) model and quantitatively evaluates doxorubicin-induced DNA damage using the comet assay. The feasibility of applying the comet assay to BCOs is demonstrated, and a reproducible, scalable, and operational workflow is provided for detecting DOX -induced DNA damage in BCOs.

Abstract

Breast cancer is the most common malignancy among women worldwide and exhibits substantial heterogeneity. Doxorubicin (DOX) is widely used in breast cancer treatment, and its primary mechanism of action involves inducing DNA double-strand breaks and triggering cell death. In this protocol, a human BCO model was established to quantitatively assess DOX-induced DNA damage. Primary breast cancer cells were isolated from patient-derived tumor tissues through mechanical and enzymatic dissociation and subsequently cultured in a three-dimensional matrix to generate BCOs. Organoids were then treated with 8 μM DOX for 2 h. DNA damage was evaluated after 2 h of treatment. Following treatment, organoids were dissociated into single cells, embedded in low-melting-point agarose, and electrophoresed under alkaline conditions. Following electrophoresis, DNA migration patterns were visualized as comet structures under fluorescence microscopy and quantitatively analyzed using OpenComet software to determine the percentage of tail DNA (% Tail DNA). The comet assay successfully detected DNA damage in BCOs. Compared to the control group, BCOs exposed to 8 μM DOX for 2 h exhibited elongated comet tails following organoid dissociation and electrophoresis. Quantitative analysis revealed that the percentage of tail DNA was significantly higher in the treated group, indicating the induction of DNA damage. This highly sensitive method supports toxicological and pharmacodynamic studies of DOX and provides a robust technical platform for assessing DNA damage in patient-derived BCO systems.

Introduction

Breast cancer is a malignant tumor that arises from breast epithelial tissues due to genetic alterations, hormonal dysregulation, and other pathogenic factors. It is the most common malignancy among women worldwide and exhibits unique epidemiological patterns and substantial intratumoral heterogeneity^1,2. DOX, a first-line chemotherapeutic agent, intercalates into DNA, inhibits topoisomerase II activity, and induces DNA strand breaks, ultimately triggering apoptosis in tumor cells³. Traditional two-dimensional cell culture systems are limited in their ability to recapitulate the complex cellular organization and heterogeneity of breast tumors. In contrast, BCOs are three-dimensional structures derived from patient tumor tissues that retain the key characteristics of the original tumor, including cellular composition, tissue architecture, and molecular phenotypes. Notably, BCO models demonstrate high concordance with clinical drug responses; for a given therapeutic agent, negative response concordance reaches 100%, and positive response concordance reaches 98% between BCOs and the corresponding patient⁴.

BCOs serve as an advanced in vitro model that recapitulates key structural and functional features of primary tumors and have been widely applied to investigate tumorigenesis, disease progression, and therapeutic resistance⁵. Tumor initiation in breast cancer typically relies on the cooperative inactivation of multiple tumor suppressor genes, such as TP53, PTEN, RB1, and NF1, as single-gene alterations are insufficient to drive malignant transformation of normal mammary epithelium. This process is accompanied by loss of cell polarity, architectural disorganization, and uncontrolled proliferation^6,7,8.

The comet assay (also referred to as single-cell gel electrophoresis, SCGE) is a classical method for assessing DNA damage at the single-cell level. The technique was first introduced by Rydberg and Johanson in 1978, and subsequently refined by Ostling and Johanson, who established the neutral comet assay for detecting DNA double-strand breaks. In 1988, Singh and colleagues incorporated alkaline electrophoresis conditions, enabling the detection of DNA single-strand breaks, abasic sites, and alkali-labile lesions, substantially expanding the assay’s applicability⁹. The fundamental principle of the comet assay is that DNA electrophoretic mobility depends on DNA integrity. Following agarose embedding and lysis to remove cellular membranes and proteins, damaged DNA fragments migrate toward the anode during electrophoresis to form a “comet tail,” whereas intact DNA remains near the loading site, forming the “comet head”¹⁰. Parameters such as tail length, percentage of tail DNA (% tail DNA), and Olive tail moment allow semi-quantitative or quantitative assessment of DNA damage, and time-course experimental designs can be used to evaluate DNA repair capacity. Due to its high sensitivity, low sample input requirements, and relative procedural simplicity, the comet assay has been widely utilized in genetic toxicology, cancer biology, and drug response studies, particularly for evaluating DNA damage induced by chemotherapeutic agents and radiotherapy, as well as tumor cell resistance mechanisms. The assay is highly sensitive to experimental conditions and procedural variability, and reproducibility and inter-laboratory comparability depend on standardized workflows and automated image analysis¹¹. In recent years, the incorporation of high-throughput imaging software, organoid models, and gene-editing systems has further enhanced the utility of the comet assay in investigating DNA damage and repair mechanisms and in assessing tumor treatment responses.

Compared with traditional DNA damage assays such as γ-H2AX immunofluorescence staining or flow cytometry, the comet assay provides a direct means to quantify physical DNA strand breaks at the single-cell level. Because this methodology operates independently of specific protein expression or antibody specificity, it effectively eliminates false-negative results arising from variations in cell-cycle states or aberrations in DNA damage response pathways¹². Furthermore, unlike conventional two-dimensional monolayer cultures, the three-dimensional organoid model used in this study more faithfully recapitulates the complex spatial architecture, extracellular matrix interactions, and drug permeation gradients of in vivo tumors. While traditional cell-line models frequently overestimate genotoxicity due to artificial drug overexposure, the organoid platform evaluates DOX-induced DNA damage within a microenvironment that closely mirrors clinical physiological conditions, thereby substantially enhancing the reliability and translational value of this screening paradigm¹³.

Despite the substantial advantages of the organoid-integrated comet assay established in this study for evaluating DNA damage, its practical implementation requires careful consideration of several technical limitations and applicability parameters related to sample input. The protocol requires thorough yet gentle enzymatic dissociation of the organoids to yield a highly viable single-cell suspension. A seeding density of 15,000–20,000 primary breast cancer cells per 100 µL of the basement membrane matrix (~200 cells/1 µL) sustains the intercellular paracrine signaling necessary for robust sphere formation while preventing central necrosis induced by hyper-density and rapid nutrient depletion. Regarding the therapeutic window, this assay requires careful calibration of DOX concentration and exposure duration to prevent excessive cell death (>20–30%). Supralethal drug exposure generates an abundance of “hedgehog” comets, which obscure bona fide DNA damage signals and confound data interpretation. Regarding assay limitations, the inherently low throughput of the standard alkaline comet assay makes it inadequate for high-throughput drug screening. Furthermore, this technique demands rigorous standardization of experimental procedures—including gel adherence stability, alkaline unwinding duration, and electrophoretic uniformity—as even minute operational deviations can introduce substantial systemic errors.

In conclusion, this protocol establishes human BCOs and demonstrates the feasibility of applying the comet assay to quantify DOX-induced DNA damage. Following 8 µM DOX treatment of fully established BCOs, DNA damage was assessed using the comet assay. This approach enables a stable and direct evaluation of DOX-induced DNA damage.

Protocol

The study was approved by the Ethics Committee of Suzhou Wuzhong People's Hospital (Approval No. 20251w03) and was conducted in accordance with medical ethical standards. All subjects in this research provided written informed consent.

1. Construction of human BCOs

1. Transport and storage of breast cancer tissue
   1. Immediately place freshly resected human breast cancer tissue into pre-chilled tissue preservation solution (4 °C).
      NOTE: Ensure the tissue remains fully submerged during transport.
2. Mechanical dissociation of breast cancer tissue
   1. Perform all procedures in a biosafety cabinet. Place the breast cancer tissue into a 6 cm culture dish and remove visible adipose tissue using sterile scalpels and forceps.
   2. Add 3 mL of PBS to the 6 cm culture dish and wash the tissue three times.
   3. Aspirate and discard the Phosphate-buffered saline (PBS).
   4. Repeat steps 1.2.2 and 1.2.3 two additional times.
   5. Transfer the breast cancer tissue into a 1.5 mL microcentrifuge tube using sterile surgical scissors and cut the tissue into small fragments approximately 2 x 5 mm in size.
3. Enzymatic digestion of breast cancer tissue
   1. Add 1 mL of organoid dissociation solution to the 1.5 mL microcentrifuge tube and transfer the suspension into a 15 mL centrifuge tube.
   2. Add an additional 8 mL of organoid dissociation solution to the 15 mL centrifuge tube.
   3. Incubate the 15 mL centrifuge tube at 37 °C for 40 min.
   4. Agitate the tube on a shaker for 1 min every 10 min during digestion.
   5. After 40 min, add 6 mL of PBS to the 15 mL centrifuge tube to terminate digestion.
      ​NOTE: Add PBS directly to the digestion mixture to stop enzymatic activity.
4. Isolation of primary breast tumor cells and organoid embedding
   1. Place a 100 µm cell strainer onto a 50 mL centrifuge tube.
   2. Add 1 mL of complete cell culture medium onto the cell strainer.
      NOTE: Prepare the complete cell culture medium using 500 mL of basal medium, 50 mL of fetal bovine serum, and 5 mL of penicillin-streptomycin.
   3. Aspirate 1 mL of the digested tissue suspension from the 15 mL centrifuge tube and apply it onto the cell strainer.
   4. Repeat step 1.4.3 until the entire suspension passes through the strainer.
   5. Rinse the cell strainer with an additional 1 mL of complete cell culture medium.
   6. Centrifuge the 50 mL centrifuge tube at 350 × g for 3 min at 4 °C.
   7. Aspirate and discard the supernatant.
   8. Resuspend the cell pellet in 1 mL of complete cell culture medium and transfer it into a 1.5 mL microcentrifuge tube.
   9. Centrifuge the suspension at 370 × g for 5 min at 4 °C.
   10. Aspirate and discard the supernatant.
   11. Add 100 µL of the basement membrane matrix to the 1.5 mL microcentrifuge tube and mix thoroughly.
       NOTE: Maintain a seeding density of 15,000–20,000 primary breast cancer cells per 100 µL of the basement membrane matrix (200 cells/1 µL). This sustains intercellular paracrine signaling for robust sphere formation and prevents central necrosis.
   12. Dispense 25 µL of the basement membrane matrix-cell mixture into each well of a 24-well plate.
   13. Invert the 24-well plate and incubate it at 37 °C for 10 min to allow basement membrane matrix polymerization.
   14. Add 500 µL of BCO culture medium to each well of the 24-well plate.
       NOTE: Culture BCOs using a commercial ready-to-use medium with a proprietary formulation.
   15. Incubate the plate in a humidified incubator at 37°C with 5% CO₂.
       NOTE: Following seeding in the extracellular matrix, the BCOs were cultured for 7–10 days to allow for proper 3D structural development. The organoids were considered fully established and ready for drug treatment when they exhibited dense, spherical 3D morphologies and reached an average diameter of approximately 50–100 µm, with no signs of central necrosis. Once these endpoint criteria were met, the BCOs were subsequently treated with DOX.

2. Detection of DOX-Induced DNA damage in BCOs using the comet assay

The experimental protocol was adapted from established guidelines and further optimized to suit specific laboratory conditions¹⁴.

CAUTION: Execute all DOX handling procedures—including dry powder weighing, stock solution preparation, and serial dilution of working solutions—exclusively within a certified Class II biological safety cabinet or a chemical fume hood. Strictly avoid any manipulation on open laboratory benches. Wear double-layered nitrile gloves and protect the drug from light throughout all procedures. Finally, segregate all DOX-contaminated consumables (e.g., pipette tips and microcentrifuge tubes) strictly as 'cytotoxic waste' for specialized disposal; never mix these items with routine biohazardous waste.

1. DOX treatment of BCOs
   1. Aspirate and discard the spent culture medium.
   2. Add 500 µL of fresh BCOs culture medium to each well
   3. Remove and discard 4 µL of the medium from each well
   4. Add 4 µL of 1 mM DOX stock solution to each well.
      NOTE: To establish a final working concentration of 8 µM, determine the required stock volume using the standard dilution equation: C₁V₁ = C₂V₂. Where C₁ represents the initial DOX stock concentration (1 mM), C₂ is the target concentration (8 µM), and V₂ is the final well volume (500 µL). Solving for V₁ yields 4 µL. Consequently, the protocol requires adding 4 µL of the 1 mM DOX stock to the remaining 496 µL of culture medium per well, then thoroughly mixing to ensure homogeneous distribution.
   5. Incubate the plate in a 37°C incubator with 5% CO₂ for 2 h.
2. Dissociation of BCOs
   1. Aspirate and discard the BCO culture medium from the 24-well plate.
   2. Add 500 µL of organoid dissociation solution to each well.
   3. Pipette up and down repeatedly to fully disrupt the basement membrane matrix and organoid structures.
   4. Incubate the plate at 37 °C with 5% CO₂ for 10 min.
   5. Transfer 500 µL of the resulting suspension from each well into a 15 mL centrifuge tube.
   6. Centrifuge the suspension at 350 × g for 3 min at 4 °C.
   7. Aspirate and discard the supernatant.
   8. Add 1 mL of complete culture medium to the 15 mL centrifuge tube to resuspend the cell pellet and subsequently transfer the resulting suspension into a 1.5 mL microcentrifuge tube.
   9. Centrifuge the suspension at 350 × g for 3 min at 4 °C.
   10. Aspirate and discard the supernatant.
       NOTE: Following centrifugation, the organoid pellet remains in the centrifuge tube.
3. Cell resuspension and agarose embedding
   1. Preheat the metal heating block to 95 °C.
   2. Place 1 mL of low-melting-point (LMP) agarose into a metal heating block and incubate for 10 min to ensure complete melting.
   3. Pipette 100 µL of molten 0.5% low-melting-point agarose into a 1.5 mL microcentrifuge tube containing BCOs.
   4. Immediately transfer the samples to a 37 °C water bath and allow them to equilibrate for 10 min.
   5. Mix thoroughly and immediately pipette 50 µL of the cell–agarose suspension onto a microscope slide.
4. Cell lysis and DNA unwinding
   1. Immerse the slides in cold lysis solution and incubate at 4 °C in the dark for 12 h.
   2. Remove the slides from the lysis solution and immediately immerse them in alkaline unwinding solution.
      NOTE: To prepare the alkaline unwinding solution, dissolve 0.6 g of NaOH in 4.975 mL of ultrapure water, then add 250 µL of 200 mM EDTA. To prepare the alkaline electrophoresis buffer, dissolve 12 g of NaOH in 995 mL of ultrapure water, then add 5 mL of 200 mM EDTA.
      ​CAUTION: NaOH in the comet assay buffer is a highly corrosive strong base capable of causing severe cutaneous and ocular burns. During solution preparation, add the solid NaOH pellets to the water slowly, incrementally, and under continuous magnetic stirring. Never pour water directly onto the dry pellets, as the violent exothermic reaction can cause the caustic liquid to boil and spatter. Furthermore, execute all dry powder weighing exclusively within a chemical fume hood to prevent the inhalation of corrosive dust.
   3. Immerse the slides in lysis solution and incubate at 4 °C in the dark for 1 h.
5. Electrophoresis
   1. Transfer the slides from the alkaline unwinding solution into the electrophoresis chamber.
   2. Add 1 L of alkaline electrophoresis buffer into the electrophoresis tank to ensure complete submersion of the slides.
   3. Perform electrophoresis at 25 V and 300 mA for 30 min.
6. Slide washing and fixation
   1. Immerse the slides in 5 mL of 70% ethanol and incubate for 5 min at room temperature.
      NOTE: Ethanol presents a severe flammability hazard. When performing large-volume preparations or tissue dehydration, maintain robust ventilation in the experimental environment. Strictly isolate the operational area from all ignition sources, including open flames (such as alcohol burners) and electrostatic sparks. Post-use, immediately seal the reagent containers and secure them within a dedicated explosion-proof flammable storage cabinet.
   2. Place the slides in a 37 °C incubator and allow them to dry for 15 min.
7. DNA staining and imaging
   1. Add 1 µL of nucleic acid fluorescent stain to 10 mL of ultrapure water.
      CAUTION: Treat these nucleic acid-intercalating agents as potential mutagens and handle them with strict precautions. To prevent cross-contamination in routine workspaces, establish a dedicated 'nucleic acid staining zone' equipped with dedicated pipettes. If supplementing molten agarose with dyes, allow the gel to cool to approximately 60 °C before adding it to prevent inhalation of hazardous, dye-laden vapors. Finally, discard all spent staining solutions and waste gels strictly into designated nucleic acid hazard receptacles.
   2. Immerse the slides in the staining solution prepared in Step 2.7.1 and incubate for 30 min at room temperature in the dark.
   3. Perform a single 5-min wash with 5 mL of ultrapure water.
   4. Air-dry the slides for 15 min in a 37 °C incubator.
   5. Visualize comet structures using a fluorescence microscope with a green excitation filter and detect red fluorescence.

3. Data processing

1. Perform automated quantitative analysis of comet assay images using the OpenComet plugin in Fiji (official distribution) to obtain DNA damage-related parameters.
   ​NOTE: Regarding the statistical analysis, this work strictly included only high-resolution, clearly imaged comets that exhibit intact morphology and no overlapping. Conversely, the screening protocol excluded 'hedgehog' or 'ghost' comets (which exhibit a diminished head and an extensive tail, indicating severe apoptosis or necrosis), as well as images with blurred edges, cell clustering, or background artifacts.
   1. Open the Fiji software (Supplementary Figure 1A).
   2. Click Plugins in the menu bar (Supplementary Figure 1A).
   3. Select OpenComet (Supplementary Figure 1B).
   4. Click Browse under Input files and select the comet assay images for analysis (Supplementary Figure 1C).
   5. Click Browse under Output directory and define the destination folder for output files (Supplementary Figure 1C).
   6. Click Run to initiate the automated analysis (Supplementary Figure 1C). This step concludes the data processing and analysis workflow.
      NOTE: For each experimental group, this study quantified the percentage of tail DNA (%Tail DNA) from a randomized selection of at least 50 cells. Subsequent statistical analysis used a one-way analysis of variance (ANOVA) to compare group means, with p < 0.05 considered statistically significant.

Results

Establishment of BCOs

This study successfully established breast cancer organoids from primary tumor tissue specimens. At the initial stage of culture, a large number of single cells or small cell clusters were clearly observed, with cells distributed in a relatively dispersed manner and lacking obvious three-dimensional spherical organization. Some cells exhibited round or near-round morphologies, indicating that tumor-derived cells survived and adapted to the extracellular matrix but had not yet established stable organoid structures (Figure 1A–C).

During the 24-day culture period, regions of increased cell density emerged within the BCOs, indicating the coexistence of distinct cellular growth states (Figure 1D).

Histopathological and immunohistochemical evaluations were performed to establish the baseline characteristics of the parental primary tumor, which exhibited a complex morphological architecture and a specific molecular subtype. Using this well-characterized clinical specimen, we successfully established a 3D organoid model that serves as a clinically relevant platform for downstream applications (Figure 1E,F).

DOX induces significant DNA damage in BCOs

The comet assay was employed to evaluate the extent of DNA damage induced by DOX in BCOs. Compared with the control group, treatment with 8 µM DOX for 2 h resulted in a marked elongation of comet tails and a pronounced increase in DNA migration toward the tail region in BCOs (Figure 2A,B). Under suboptimal conditions, low-quality comet images are frequently observed, characterized by diffuse fluorescence signals and ill-defined tail boundaries. Conversely, the optimized protocol consistently yields typical comet morphology, underscoring the critical importance of standardized procedures in the single-cell gel electrophoresis (SCGE) assay (Figure 2C).

Quantitative analysis of comet images using the OpenComet software demonstrated that the percentage of tail DNA (% Tail DNA) in the DOX-treated group was significantly higher than that in the control group (Figure 2D), with the difference reaching statistical significance (**** = p < 0.0001).

These results indicate that short-term DOX exposure induces substantial DNA damage in BCOs, confirming the feasibility of the comet assay for assessing DOX-induced DNA damage in three-dimensional organoid models.

Figure 1: Establishment of human BCOs. (A) Primary breast cancer cells isolated from human breast cancer tissue, presenting as single cells or small cell clusters (10x). (B) Representative morphology of isolated cells observed under a 20x objective. (C) Representative morphology of isolated cells observed under a 40x objective. (D) After 24 days of culture, the cells formed BCOs with a typical three-dimensional architecture and smooth outer boundaries. (E) Representative Hematoxylin and Eosin (H&E) staining of the parental tumor, displaying the typical morphological architecture of invasive breast carcinoma (High-power field). (F) Immunohistochemical (IHC) staining of primary breast cancer biomarkers (High-power field). Please click here to view a larger version of this figure.

Figure 2: Quantitative assessment of DOX-induced DNA damage in BCOs by the comet assay. (A) Representative fluorescence microscopy images of the comet assay in control BCOs. In the absence of DOX treatment, nuclei remained intact, with DNA predominantly retained in the comet head and minimal tail signals, indicating low basal DNA damage. A total of 1,043 individual comets from non-overlapping fields were analyzed. The mean % Tail DNA is 47.67% (n = 3 independent biological replicates). (B) Representative fluorescence microscopy images of the comet assay in BCOs after treatment with 8 µM DOX for 2 h. Compared with the control group, organoids in the DOX-treated group exhibited markedly elongated comet tails and increased DNA fluorescence signals in the tail region. A total of 311 individual comets from non-overlapping fields were analyzed. The mean % Tail DNA is 60.08% (n = 3 independent biological replicates). (C) Low-quality comet formations—specifically apoptotic or late-stage necrotic cells termed "hedgehogs" or "ghost cells"—exhibit an almost imperceptible head and a broad, faint, diffuse cloud-like tail; these formations require strict exclusion as outliers. (D) Quantitative analysis of comet assay results using the OpenComet software. The percentage of tail DNA (% Tail DNA) was significantly increased in DOX-treated BCOs compared with controls. **** = p < 0.0001. Please click here to view a larger version of this figure.

Supplementary Figure 1: Image processing and analysis workflow for the comet assay. (A) Navigate to the Plugins menu within the software interface. (B) Selection of the OpenComet tool. (C) Utilization of the Browse function to designate the target file directory, followed by initiation of the automated analysis via the Run command.Please click here to download this file.

Discussion

The comet assay represents a significant advancement in genotoxicity assessment methods. Owing to its single-cell resolution and the direct visualization of DNA damage patterns based on electrophoretic migration, this technique offers clear advantages over conventional genotoxicity assays¹⁵.

Several critical steps during the experimental procedure influence reproducibility and data interpretation. First, lysis conditions and lysis duration must remain consistent to minimize inter-batch variability. When handling primary breast cancer cells, investigators should avoid exposure to light to prevent additional DNA damage. The agarose gel must fully cover the slide area and remain level to prevent gel detachment. Low-melting agarose should be handled on ice to avoid premature solidification. Organoid size and variability in cell dissociation efficiency may affect electrophoretic migration and signal interpretation; strict control during sample preparation is required.

Despite its high sensitivity and operational simplicity, the comet assay presents several limitations. The assay throughput is constrained by the number of slides, limiting its suitability for large-scale screening applications. The comet assay primarily detects DNA strand breaks and does not efficiently identify base modifications or bulky DNA adducts. Investigators should combine alkaline or enzyme-modified comet assay conditions with complementary approaches, such as Western blotting, γ-H2AX detection, or LC–MS-based analyses, to achieve a more comprehensive assessment of DNA damage. Furthermore, data analysis relies largely on semi-quantitative image-based scoring systems, and variability in analytical parameters and software algorithms may affect reproducibility across experiments.

The present experimental protocol exhibits certain limitations, notably the omission of a positive control (e.g., H₂O₂, a known potent inducer of severe DNA damage). In the absence of a positive control, it remains unclear whether the DNA damage induced by 8 µM DOX over 2 h represents the theoretical maximum achievable in this experimental system. Furthermore, the current methodology relied on a single drug concentration (8 µM) and a single exposure duration (2 h). While these conditions successfully induced a robust DNA damage phenotype suitable for qualitative analysis, the lack of concentration and time-course gradients precludes a comprehensive evaluation of DOX pharmacodynamics.

To balance robust drug-induced genotoxicity with the technical feasibility of the comet assay, 8 µM DOX was selected as the optimal treatment concentration. Although clinical peak plasma concentrations of DOX typically range from 1–5 µM, the inherent physical density of organoids—characterized by tight intercellular junctions and extracellular matrix (ECM) encapsulation—significantly restricts drug penetration relative to conventional cell cultures. Consequently, concentrations slightly above the clinical peak plasma levels are required to achieve homogeneous drug penetration throughout the organoid. Consistent with published literature and our preliminary dose-response assays, 8 µM was identified as the optimal threshold within this short-term detection window¹⁶. This concentration effectively induces robust, quantifiable DNA strand breaks (characterized by a high percentage of tail DNA) while avoiding extensive acute necrosis or late apoptosis. Conversely, excessive concentrations precipitate severe DNA degradation and cell death, yielding unquantifiable 'hedgehog' or 'ghost' comets during electrophoresis that confound assay interpretation¹⁷.

This study established explicit troubleshooting strategies for critical steps. To resolve overlapping comets caused by incomplete organoid digestion, the protocol emphasizes combining adequate enzymatic incubation with gentle mechanical pipetting to ensure the purity of the single-cell suspension. To prevent gel detachment from the slides, the procedure mandates thoroughly drying the normal-melting-point agarose pre-coating and executing steady transfers when moving the slides into lysis solutions or electrophoresis buffers. Furthermore, to mitigate high background damage in control groups induced by mechanical stress and light exposure, the guidelines require performing all cell dissociation and centrifugation steps on ice at 4 °C, alongside strictly shielding the exposed DNA from light.

Whereas flow cytometry typically evaluates late-stage cytotoxic phenotypes, the comet assay acutely captures direct DNA damage at an extremely early stage—merely 2 h post-DOX treatment—preceding any significant cell death, a feat unattainable with bulk-sample averaging methods such as traditional Western blotting. By strictly standardizing operational parameters (including treatment concentration, exposure duration, and the timing of alkaline unwinding and electrophoresis), the protocol facilitates the standardized preparation of high-contrast fluorescent single-cell images. These images ensure seamless integration with automated comet image analysis software, which not only substantially reduces human bias during manual calculations of '%Tail DNA' or 'tail moment' but also vastly enhances the objectivity and efficiency of data extraction.

In summary, the comet assay offers substantial advantages for DNA damage assessment, standardized protocols, automated analysis platforms, and integrated multi-technology approaches remain essential to overcome its limitations and to enhance its translational value in cancer pharmacodynamic studies.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 μm cell strainer	BIOLOGIX	15-1100
15 mL centrifuge tubes	BIOFIL	None	15 mL centrifuge tubes
1 mL pipette tips	Zhejiang Baidi Biotechnology Co., LTD	H808633
200 μL pipette tips	Zhejiang Baidi Biotechnology Co., LTD	H808214
24-well plates	Corning Incorporated	3524
50 mL centrifuge tubes	BIOFIL	None	50 mL centrifuge tubes
Breast Cancer Organoid Kit Plus	Mogenel Biotechnology	MA-0807T011LP	BCO culture medium
Comet Slide	R&D SYSTEMS	4250-050-03
Dry-bulb thermostat	Hangzhou Ruiyi Science and Technology Trading Co., Ltd.	DH100-2	Heating block
Dulbecco’s Modified Eagle Medium	gibco	C11995500BT	Complete cell culture medium
EDTA	TREVIGEN	4250-050-04
Electric Constant-Temperature Water Bath	Shanghai Mulang Instrument Manufacturing Co., Ltd.	MSY-24	Water bath
Electrophoresis apparatus	BIO-RAD	1645052
Fetal bovine serum	EallBio	03.U16001DC
Fluorescence microscope	WEIDA	IM-300LD4
Forceps	Beyotime	FS019
GelRed	Beyotime	D0140
GraphPad Prism 8.0	GraphPad Software		Statistical analysis software
ImageJ	National Institutes of Health, NIH	1.54p	Image processing software / image
LMA garose	R&D SYSTEMS	4250-050-02	Low-melting-point agarose
Low-temperature high-speed centrifuge	Anhui Zhongke Zhongjia Scientific Instrument Co., LTD	KDC-40	Centrifuge
Lysis Solution	TREVIGEN	4250-050-01	Lysis solution
Matrigel	Mogenel Biotechnology	82755	The basement membrane matrix
NaOH	DAMAO	1588
OpenComet software	Open-source ImageJ plugin, National University of Singapore, Singapore	v1.3.1
Organoid Dissociation Solution	Mogenel Biotechnology	MB-0818L01L
Penicillin – Streptomycin	Beyotime	CO22
Phosphate-buffered saline (1x PBS)	Fdbio Science Biotech Co.,Ltd	FD7032	PBS
Pipettes	Rainin	Pipet-Lite XLS
Scissors	Beyotime	FS001
Tissue Digestion Solution	Mogenel Biotechnology	MB-0818L06L/MB-0818L06S	Organoid dissociation solution