Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA243511. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Deborah Frank for her editorial comments.

The collection of human specimens for this research was approved by the Washington University School of Medicine Institutional Review Board. All eligible patients over the age of 18 years had a diagnosis or presumed diagnosis of high-grade serous ovarian cancer and were willing and able to provide informed consent. The tumor tissue from either primary or metastatic sites, in addition to ascites and pleural fluid, were obtained from consented patients at the time of care.
1. Selection of established PDOs for viability assay
NOTE: Typically, these organoids are within the first five passages. As described previously, ovarian cancer PDOs are formed by resuspending primary cell suspension in basement membrane extract (BME) such as Cultrex or Matrigel17 (see Table of Materials).
<ol>
	<li>PDOs should have a perceived doubling time of 24-72 h for the best efficiency of the assay. Estimate the perceived doubling time by visually determining the number of days it takes for organoids to form after passaging/plating. 
	NOTE: In our experience, organoids that take longer than three days to form cannot be used for this growth inhibition assay.</li>
	<li>Identify drugs and select a range of concentrations to test (e.g., Carboplatin, 0-50 &#181;M, see Table of Materials).</li>
	<li>Determine the plate setup. This assay will be performed in triplicate, limiting the number of drugs and concentrations that can be tested on one plate. 
	NOTE: Selected PDO lines must be carefully observed before performing the assay. Established ovarian cancer PDOs form multicellular solid and hollow spheres with tumor-like budding shapes (Figure 1B). PDO morphology, genomic profile, etc., must be compared to that of the patient sample (when possible) before performing the assay29-33. Ovarian cancer PDOs can be generated from primary and metastatic tumor samples and ascites17.</li>
</ol>
2. Reagent preparation for the viability assay
<ol>
	<li>Base Media: To advanced DMEM/F12, add 1% (v/v) penicillin-streptomycin, 1x glutamax, and 1% (v/v)HEPES17 (see Table of Materials).</li>
	<li>Prepare Advance Organoid Media for ovarian cancer organoids following previously published report17.</li>
</ol>
3. Plating of organoids (~Day -2)
NOTE: This step must be performed 1-3 days before adding the drugs. Before beginning, warm all reagents (Base Media, Advance Organoid Media, and organoid dissociation reagent; see Table of Materials) to 37 &#176;C in a water bath. Thaw BME in an ice water bath.
<ol>
	<li>Use a brightfield microscope to confirm whether organoids of interest are 70%-90% confluent. 
	NOTE: It is recommended to save images of the organoids for future reference.</li>
	<li>Add 1-2 mL of warmed Base Media to the well containing organoids and pipette up and down to mechanically dissociate the BME-containing organoids. Transfer the entire solution to a 15 mL conical tube17. 
	NOTE: Typically, 1-2 mL of media is enough to properly dilute the BME. Organoids of the same patient and passage can be combined.</li>
	<li>Vortex for 5-10 s to further dissociate the cells, and centrifuge at 1107 x g for 5 min at room temperature (RT).</li>
	<li>Remove the supernatant with a single-channel pipette and discard. Resuspend organoids in 1 mL organoid dissociation reagent (see Table of Materials) and transfer to a 1.5 mL microcentrifuge tube. Incubate for 7&#160;min at 37 &#176;C.
	<ol>
		<li>If the pellet is still gelatinous because of the remaining BME, add an additional 1 mL of Base Media, vortex for 5-10 s, and repeat centrifugation.</li>
	</ol>
	</li>
	<li>Centrifuge at 1107 x g for 5 min at RT. Remove and discard the supernatant and resuspend the cell pellet in 1 mL of Base Media.</li>
	<li>Count the number of cells in each PDO culture using an automatic cell counter (see Table of Materials) or a hemocytometer.</li>
	<li>Resuspend cells at 20,000 cells per 10 &#181;L of 25% Base Media and 75% BME. Do this by resuspending the organoids in the media and mixing&#160;with BME.</li>
	<li>Plate one 3 &#181;L droplets of resuspended BME + PDO cells into one well of a black opaque 96-well plate (see Table of Materials). Place the droplets in the center of each well. Plate each drug concentration in triplicate. 
	NOTE: Since the cell viability assay is luminescence-based, it is best to use black opaque plates to avoid background. Transparent plates can be used to visualize organoids during the assay,&#160;but the bottom of the plates should be covered with opaque tape before measuring luminescence.</li>
	<li>Incubate plate in a cell culture incubator at 37 &#176;C for 15 min.</li>
	<li>Add 100 &#181;L of Advance Organoid Media to each well. Incubate plate for 24-72 h (determine according to perceived PDO doubling time). Add 100 &#181;L of Advance Organoid Media to an empty well for use as blank. 
	NOTE: The goal is to allow organoids to form.</li>
	<li>Optional: For growth rate analysis, plate additional triplicate PDOs in a separate plate. This will be used to count cells at Time = 0 and should be assayed on Day 0 using the viability assay described in Section 5.</li>
</ol>
4. Addition of drugs for the viability assay on Day 0
NOTE: Day 0 refers to the day the drugs are added to the fully formed organoids.
<ol>
	<li>Dilute selected drugs in Advance Organoid Media to desired concentrations. Dilutions can be made in 1.5 mL tubes or in a pre-set up 96-well plate, which would allow for the use of a multi-channel pipette to dispense the media. 
	NOTE: Drugs must be resuspended in the solvent suggested by the manufacturer, such as dimethyl sulfoxide. For this study, the following dilutions of carboplatin in Advance Organoid Media, were made: 1, 5, 10, 25, 50, and 75 &#181;M.</li>
	<li>Remove media from each well with a single or multi-channel pipette, being careful not to touch or disturb the PDO/BME droplet.</li>
	<li>Add 100 &#181;L of Advance Organoid Media and desired drug(s) to each well. Ensure to add fresh media to the three control wells. 
	NOTE: Drug concentrations will vary and depend on the scientific question and drug mechanism of action. When deciding what concentrations to use, we begin with previously established drug concentrations in comparable 2D cell lines (e.g., immortalized ovarian cancer cell lines). Concentrations may need to be increased if there is no observed effect on organoids.</li>
	<li>Refresh media and drugs as needed (repeat steps 4.1-4.3). Whether the media will need to be refreshed depends on the assay length, the drug&#39;s biological activity, and the drug&#39;s half-life. For assays over one week, the media will need to be refreshed at least once.</li>
</ol>
5. Ending the viability assay for readout (~Day 7)
NOTE: This step can be performed on Days 7-10. The assay length must be determined according to the half-life and pharmacodynamics of the drugs being tested.
<ol>
	<li>Allow the viability assay reagents to reach room temperature in dark. Store the reagents at 4 &#176;C overnight (in the dark) to reduce the thaw time.</li>
	<li>Remove the assay plate from cell culture incubator and allow it to acclimate to room temperature for 30 min. The plate does not have to acclimate in the dark.</li>
	<li>Add 100 &#181;L of viability assay reagent (see Table of Materials) to each well (for a total volume of 200 &#181;L), and place on a plate-shaker for 5 min (80 rpm). Remove the plate from the shaker and incubate for an additional 25 min at room temperature. Ensure the plate is always protected from light by covering it with foil or an opaque box.</li>
	<li>Turn on the bioluminescence plate reader and open the i-control software (see Table of Materials).</li>
	<li>Under Connect to: Instrument Name, select infinite 200Pro.</li>
	<li>Select Luminescence and Default Script.</li>
	<li>From the drop-down menu, choose plate type [BD96fb_Falcon-BD] Falcon 96 Flat Black.</li>
	<li>Determine which wells to read and highlight the corresponding wells under Part of Plate.</li>
	<li>Under Measurements on the left side of the screen, drag and drop Luminescence under the Part of Plate.</li>
	<li>Select the luminescence parameters: Attenuation: NONE; Integration time: 1000 ms; Settle time: 0 ms.</li>
	<li>Remove the cover from the plate and load it into the plate reader. Press Start to begin.</li>
	<li>On completion, export data and save.</li>
</ol>
6. Data analysis
<ol>
	<li>Calculate the percent cell viability.
	<ol>
		<li>Subtract the &#34;Blank&#34; from every well for the Blank Corrected readings. Next, calculate Control Average by averaging the three control wells.</li>
		<li>Use the following equation to calculate the percentage of viable cells in each well: (Experimental well / Control Average) x 100</li>
		<li>Graph the resulting data in the analysis software (see Table of Materials).</li>
	</ol>
	</li>
	<li>Examine Growth Rate (GR) metrics.
	<ol>
		<li>Manually calculate the GR metrics according to the published report34.</li>
		<li>Alternatively, use the online GR calculator (see Table of Materials) to generate the metrics35. Use the Day 0 measurements as &#34;cell_count_time0&#34;, which reflect the growth rate of the untreated PDOs.</li>
		<li>Export data and graph.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article describes a method that can be used to assess the therapeutic effects of conventional or novel drugs on ovarian cancer PDOs. Researchers must consider several issues before conducting the viability assay in the PDO model.
First, when selecting a PDO to use in the viability assay, one must determine the ideal organoid type (tumor vs. ascites) and passage number for their needs. In our experience, ascites-derived PDOs grow more rapidly and are easier to generate than tumor-...

Although rare, ovarian cancer is one of the most lethal gynecological cancers1,2. A challenge in developing new treatments is that ovarian cancer is heterogeneous, and the tumor microenvironment differs greatly among patients. Additionally, many ovarian cancers develop resistance to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors, highlighting the need for greater therapeutic options3,4,5.
One approach that may be useful in identifying new therapeutics is using patient-derived organoids (PDOs). Organoids are three-dimensional clusters of multiple cell types that self-organize and form in vitro&#160;&#34;mini-organs&#34;6,7,8,9,10. Organoids can recapitulate important tissue morphology and gene expression profiles11,12. Some of the first organoids were derived from intestinal, gastric, and colon cancer cells from both mice and humans8,9,13. Long-lived organoid cultures have been established from a wide range of benign and malignant tissues, including the bladder, colon, stomach, pancreas, brain, retina, and liver14,15,16. We previously demonstrated methods to establish PDOs from ovarian cancer tumors and ascites samples17. PDOs can be used to study molecular characteristics, cellular mechanisms, and novel drug treatments18,19,20. PDOs have several advantages over traditional two-dimensional primary cell cultures for drug screening. Although primary two-dimensional cultures are a low-cost method for drug screens, primary cell cultures are single-cell types and lack the three-dimensional architecture of tumors21,22,23. Nevertheless, PDOs are a precious resource, and cost-effective protocols are needed to optimize their use in therapeutic drug screening.
This article describes an in vitro method to use ovarian cancer PDOs to test the effects of known or candidate drugs. Whereas current medium- and high-throughput drug screens using PDOs require expensive automated dispensing instruments24,25,26, this cost-effective method uses readily available basic lab supplies and an ATP-based cell viability assay in a standard 96-well plate format (Figure 1A). This method will facilitate preliminary tests of novel ovarian cancer drugs prior to scaling up to larger screens27,28. Although ovarian cancer PDOs are used here, this method can be applied to other cancer organoid models.

<table><tbody><tr><td>1.5 mL Plastic Tubes</td><td></td><td></td><td></td></tr><tr><td>15 mL Plastic Tubes</td><td></td><td></td><td></td></tr><tr><td>96 well Flat Black Plates</td><td>MidSci</td><td>781968</td><td></td></tr><tr><td>Advance Organoid Media&amp;nbsp;</td><td></td><td></td><td>see Graham et al 2022 (Jove)</td></tr><tr><td>Advanced DMEM/F12</td><td>Thermo Fisher</td><td>12634028</td><td></td></tr><tr><td>Automated Cell Counter</td><td>Thermo Fisher</td><td>AMQAX1000</td><td></td></tr><tr><td>Brightfield Microscope</td><td></td><td></td><td></td></tr><tr><td>Carboplatin&amp;nbsp;</td><td>Teva&amp;nbsp;Pharmaceuticals&amp;nbsp;USA</td><td>NDC&amp;nbsp;00703-4246-01</td><td></td></tr><tr><td>CellTiter-Glo 3D Viability&amp;nbsp;</td><td>Promega</td><td>G9681</td><td></td></tr><tr><td>Cultrex</td><td>R &amp;amp; D Systems</td><td>3533-010-02</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2650-100ML</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>GR Calculator&amp;nbsp;</td><td>http://www.grcalculator.org</td><td></td><td>Online calculator</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630080</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Plate Rocker</td><td></td><td></td><td></td></tr><tr><td>Sterile P10, P200, and P1000 Barrier Sterile Pipette Tips</td><td></td><td></td><td></td></tr><tr><td>Sterile P10, P200, and P1000 Pipettes</td><td></td><td></td><td></td></tr><tr><td>Tecan Infinte 200Pro Plate Reader; i-Control Software</td><td>Tecan</td><td></td><td></td></tr><tr><td>TrypLE</td><td>Thermo Fisher</td><td>12605010</td><td>Organoid dissociation reagent</td></tr></tbody></table>

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 is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

These results illustrate the response of two PDOs to the chemotherapy drug carboplatin, which is used to treat ovarian cancer. Organoids were derived from tumor biopsy (PDO #1) and from ascites (PDO #2). These organoids were selected based on their perceived doubling time (1-2 days) and morphological appearance (formation of many large organoids). Both PDO #1 and PDO #2 were plated on Day -2, at passage two, and carboplatin was added on Day 0. We tested the following carboplatin concentrations diluted in Advance Organoid...

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

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

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 ...

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1.4K Views.  Washington University of Medicine in St. Louis. Dit protocol beschrijft een goedkope methode voor het uitvoeren van levensvatbaarheidstests voor geneesmiddelen met behulp van van patiënten afgeleide organoïden van eierstokkanker. De belangrijkste voordelen van deze techniek zijn het gebruik van goedkope en direct beschikbare materialen en laboratoriumapparatuur. Deze methode kan worden gebruikt voordat dure high-throughput medicijnscreenings worden uitgevoerd.Hoewel de methode zich bezighoudt met het uitvoeren van levensvatbaarhei...

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

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

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 models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

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

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

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

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 discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

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 ...

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 organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human 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 ...

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.

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.

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

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

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

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