Name: creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research
Uploaded: 2021-05-05T13:00:00
Description: Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

The authors would like to thank Dr. Jody Barbeau, Federica Parisi and Rajendra Kumari for critical reading and editing of the manuscript. The authors would also like to thank the Crown Bioscience Oncology in vitro and in vivo team for their great technical efforts.

All the protocols and amendment(s) or procedures involving the care and use of animals were reviewed and approved by the Crown Bioscience Institutional Animal Care and Use Committee (IACUC) prior to conducting the studies. The care and use of animals was conducted in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines as reported in the Guide for the Care and Use of Laboratory Animals, National Research Council (2011). All animal experimental procedures...

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The preliminary data for PDX-/PDXO-CR2110 in this report supports the biological equivalence between PDX and its derivative, PDXO, with regards to genomics, histopathology and pharmacology, since both models represent the disease forms from the original CSC of patient. Both models are patient-derived disease models, potentially predictive of the clinical response of patients10,11,12,21. The mat...

Cancers are a collection of diverse genetic and immunological disorders. Successful development of effective treatments is highly dependent on experimental models that effectively predict clinical outcomes. Large libraries of well-characterized patient-derived xenografts (PDXs) have long been viewed as the translational in vivo system of choice to test chemo- and/or targeted therapies due to their ability to recapitulate patient tumor characteristics, heterogeneity and patient drug response1, thus enabling Phase II-like mouse clinical trials to improve clinical success2,3. PDXs are generally considered as cancer stem cell diseases, featuring genetic stability, in contrast to cell line derived xenografts2. Over the last few decades, large collections of PDXs have been created throughout the world, becoming the workhorse of cancer drug development today. Although widely used and with great translational value, these animal models are intrinsically costly, time consuming and low throughput, therefore inadequate for large scale screening. PDX are also undesirable for immuno-oncology (IO) testing due to an immune-compromised nature4. It is thus impractical to take full advantage of the available large library of PDXs.
Recent discoveries, pioneered by the Hans Clevers&#8217; laboratory5, have led to the establishment of in vitro cultures of organoids generated from adult stem cells in most human organs of epithelial origin5. These protocols have been further refined to allow the growth of organoids from assumed CSCs in human carcinomas of various indications6,7. These patient-derived organoids (PDOs) are genomically-stable8,9 and have been shown to be highly predictive of clinical treatment outcomes10,11,12. In addition, the in vitro nature of PDOs enables high-throughput screening (HTS)13, thus potentially offering an advantage over in vivo models and leveraging large organoid libraries as a surrogate of the patient population. PDOs are poised to become an important discovery and translational platform, overcoming the many limitations of PDXs described above.
Both PDO and PDX are patient-derived and CSC-driven models, with the ability to evaluate therapeutics in the context of either personalized treatment or clinical trial format. Existing large libraries of PDXs, like the proprietary collection of &#62;3000 PDXs14,15,16,17, are therefore suitable for the rapid generation of libraries of tumor organoids (PDX-derived organoids, or PDXO), resulting in a matched library of paired PDX and PDXO models. This report describes the procedure to create and characterize colorectal cancer PDXO-CR2110 in relation to its parental PDX-CR2110 model16.

<table>
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634028</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td>Washing medium</td>
		</tr>
		<tr>
			<td>Collagenese type II</td>
			<td>Invitrogen</td>
			<td>17101015</td>
			<td>Digest tumor</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Organoid culture matrix (Basement Membrane Extract, growth factor reduced)</td>
		</tr>
		<tr>
			<td>N-Ac</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma</td>
			<td>S7076</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Sigma</td>
			<td>G9145</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Rspondin</td>
			<td>Peprotech</td>
			<td>120-38-1000</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Abmole</td>
			<td>M1817</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td>Screening assay</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D</td>
			<td>Promega</td>
			<td>G9683</td>
			<td>Screening assay (luminescent ATP indicator)</td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher</td>
			<td>Multidrop combi</td>
			<td>Plating organoids/CellTiter-Glo 3D addition</td>
		</tr>
		<tr>
			<td>Digital dispener</td>
			<td>Tecan</td>
			<td>D300e</td>
			<td>Compound addition</td>
		</tr>
		<tr>
			<td>Envision Plate reader</td>
			<td>Perkin Elmer</td>
			<td>2104</td>
			<td>Luminescence reading</td>
		</tr>
		<tr>
			<td>Balb/c nude mice</td>
			<td>Beijing HFK Bio-Technology Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>tRNA purification kit</td>
		</tr>
		<tr>
			<td>DNAeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermo Fisher</td>
			<td>HG-4000-012</td>
			<td>Organoid embedding</td>
		</tr>
	</tbody>
</table>

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.

Morphology of PDXOs, typical of organoids under light microcopy, and consistent with parental PDX per H&#38;E staining 
Under light microscopy, PDXO-CR2110 demonstrates typical cystic morphology (Figure 1A), as described previously for patient-derived organoids (PDO), evidence supporting the similarity between PDXO and PDO under the same culture conditions.
Histopathological examination by H&#38;E staining reveals that the tissue structures a...

Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

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

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) ...

This protocol can be used to establish a matched library of paired patient-derived xenografts, or PDX, and corresponding patient-derived organoid lines. The advantage of this method is that it can be used to create organoids using PDX for in vitro screening, resulting in matched pairs of in vivo/in vitro models. Begin by harvesting the tumors and processing the tissue as described in the text manuscript.After tissue digestion, pipette the tissue up and down with a five milliliter sterile plastic pipette. Then add 20 milliliters of AD+to the homogenate and filter it with a 100 micrometer cell strainer. Wash the filtrate twice with AD+then transfer it to a plastic tube and centrifuge it at 450 times G for five minutes.Resuspend the cells in BME and keep them on ice. To prepare the organoid culture, transfer 200 microliters of the cell suspension to each well of a six-well plate and incubate the plate at 37 degrees Celsius for 30 minutes. Add two milliliters of organoid media to each well and image the representative drops under a microscope.Maintain the organoid cultures at 37 degrees Celsius and 5%carbon dioxide with medium changes every three to four days and passage at a one-to-two ratio every seven days. After dissociating the organoids, run them through a 70 micrometer filter into a 50 milliliter plastic tube. Count the organoids under a microscope and resuspend them in BME on ice for a final concentration of 5%Add 50 microliters of the organoid suspension into each 384-well plate with a liquid dispenser for a seeding density of 200 CR2110 PDXOs per well.Use the digital dispenser to add SN38 to each well in serial dilution. Create a plate map using the digital dispenser software tool. The treatments should include a negative control vehicle with 100%viability and positive control of five micromolar Staurosporine with 0%viability.When finished, place the drug-treated 384-well plates back into 37 degree Celsius incubator. Under light microscopy, PDXO CR2110 shows typical cystic morphology demonstrating the similarity between PDX-derived organoids and patient-derived organoids under the same culture conditions. Histopathological examination by H&E staining reveals that the tissue structures and cell types of PDXO CR2110 are similar to the original PDX CR2110.Genomic profile comparisons of PDX and PDXO demonstrate a 94.92%correlation of transcriptome expression and a 97.67%concordance of DNA mutations, suggesting an overall genomic similarity between this pair of models. Drug sensitivity assays were performed on PDXO CR2110 in 384-well plates. PDXO CR2110 was sensitive to irinotecan and resistant to cisplatin, consistent with PDX treatment results.The matched pair of in vitro and in vivo models can compliment each other for in vitro screening and validation in vivo, improving the success rate of drug discovery and potentially reducing attrition rates in clinical development.

creating-matched-vivoin-vitro-patient-derived-model-pairs-pdx-pdx

Research

JoVE Journal

Cancer Research

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

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 ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

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 ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

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.

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 ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...