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 were under sterile conditions at SPF (specific pathogen-free) facilities and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals from different government institutions (e.g., The National Institutes of Health). The protocols were approved by the Committee on the Ethics of Animal Experiments at the facility institution (e.g., institutional IACUC Committee).
1. Preparation for tumor transplantation
<ol>
	<li>Animal housing
	<ol>
		<li>House Balb/c nude mice (n=5) in individual ventilated cages, at 20-26 &#176;C, 30-70% humidity, and a lighting cycle of 12-h light/12-h dark, with corn cob bedding changed weekly, and irradiation sterilized dry granule food plus sterile drinking water ad libitum.</li>
	</ol>
	</li>
	<li>Donor tumor fragment preparation
	<ol>
		<li>Closely monitor tumor-bearing donor mice for body weight (BW, via weighing balance), and tumor volume (TV, by caliper measurement).</li>
		<li>When TV reaches 800-1000 mm3, euthanize the tumor-bearing animals in a biohazard hood.
		<ol>
			<li>Place mice in a euthanasia chamber with a lid that delivers CO2 gas into the chamber. Discharge gas into the chamber at a flow rate that produces rapid unconsciousness with minimal distress to the animal. The optimum flow rate for CO2 is around 2-2.5 Lpm.</li>
			<li>Ensure euthanasia by observing that the animals do not regain consciousness.</li>
			<li>After apparent clinical death, maintain gas flow for &#62;1 min to minimize the possibility that an animal may recover.</li>
		</ol>
		</li>
		<li>Sterilize the skin around the tumor using iodophor swabs. Collect tumors by removing adjacent non-tumor tissues and placing in a Petri dish containing 20 mL of PBS, pre-chilled (4 &#176;C) prior to euthanasia.</li>
		<li>Wash the tumors with PBS in another Petri dish to remove blood components followed by cutting in half and removing any extra skin, blood vessels, calcification and/or necrosis.</li>
		<li>Place only intact tumor pieces into a sterile 50 mL centrifuge tube with 20 mL of PBS, prior to transporting to a separate animal room for transplantation.</li>
	</ol>
	</li>
</ol>
2. Subcutaneous tumor growth
<ol>
	<li>Subcutaneous inoculation of tumor piece into immunocompromised mice
	<ol>
		<li>Cut the PDX tumor into 2 mm pieces with a scalpel, and place each into trocars for subcutaneous (SC) implantation.</li>
		<li>Anesthetize the recipient animals with 5% isoflurane (maintained by a nose cone at 1%). The animals relax, losing their righting reflex and eventually becoming immobile and unresponsive to pain. Fix the anesthetized mice onto an experiment board in the right lateral position and sterilize with iodophor swabs, particularly the areas surrounding the site of tumor inoculation.</li>
		<li>On left flank skin just cranial to the hip, make a 0.5 cm incision with a scalpel and create a tunnel under the skin towards the forelimb,&#160;2-3 cm, using blunt forceps.</li>
		<li>Aseptically transfer one cube of tumor fragment per inoculation site from the medium and place deep inside the subcutaneous tunnel. Visually confirm position of the fragment prior to closure of wound with wound clips.</li>
		<li>Monitor the tumor implanted mice (5) until they become conscious to maintain sternal recumbence, and then return them to their cage only after their full recovery from anesthesia.</li>
	</ol>
	</li>
	<li>Tumor-bearing mice health monitoring
	<ol>
		<li>Check water and food consumption daily and record body weight weekly.</li>
		<li>Check the mice during routine monitoring. Record any effects on mobility, breathing, grooming and general appearance, food and water consumption, BW gain/loss, ascites, etc.</li>
		<li>Measure TV twice weekly using calipers and express in mm3 per: TV = 0.5 a &#215; b2, where a and b are the length and width of the tumor, respectively.</li>
		<li>Sacrifice animals to collect samples when any of the following signs appear: BW loss &#62;20%; impaired mobility (not able to eat or drink); unable to move normally due to significant ascites or enlarged abdomen; effort in respiration; death.</li>
	</ol>
	</li>
</ol>
3. Necropsy and tumor harvest
<ol>
	<li>When tumor volume reaches 200-800 mm3, euthanize mice per approved protocols (see step 1.2.2).</li>
	<li>Collect tumor tissues by removing adjacent non-tumor tissues, followed by placing the tissue in a 50 mL plastic tube with AD+++ (on ice) before dissociation.</li>
</ol>
4. Preparation for PDX-derived organoid culture
NOTE: All the following steps were performed inside a biosafety cabinet per tissue culture standard guidelines. Keep pre-warmed stocks of 96-, 24-, and 6-well plates in a 37 &#176;C incubator before use.
<ol>
	<li>Reagent preparations
	<ol>
		<li>Prepare basement membrane extract (BME) solution (growth factor reduced, Phenol Red-free). Keep 10 mL of BME in a 4 &#176;C refrigerator overnight. Once thawed, swirl the BME bottle to ensure dispersion.</li>
		<li>Prepare base media/washing buffer AD+++. Add 5 mL of 200 mM L-glutamine, 1 M HEPES and Pen/Strep to Advanced DMEM/F12 media by pipetting with a 5 mL pipette.</li>
		<li>Prepare colon organoid medium as described by Sato et al.18 through supplementing base media with N-Ac (1 mM), A83-01 (500 nM), B27 (1x), EGF (50 ng/mL), Noggin (100 ng/mL), Nicotinamide (10 mM), SB202190 (10 nM), R-spondin (500 ng/mL), L-glutamine (2 mM), HEPES (10 &#181;M), penicillin-streptomycin (1x) and Y-27623 (10 &#181;M)19.</li>
		<li>Prepare AD+++Digestion medium: 10 mL of 1x organoid culture media with 500 &#181;L of collagenase type II (20 mg/mL) and 10 &#181;L of RhoKI Y-27632 (10 mM).</li>
	</ol>
	</li>
	<li>Tumor Dissociation20
	<ol>
		<li>Transfer the tumor in a 50 mL plastic tube to a 10 cm Petri dish. Take a macroscopic photograph alongside a ruler and record a description of its conditions (i.e., size, fat tissues, vascularisation, necrosis, etc.).</li>
		<li>Remove excess AD+++ by aspiration, and cut the tumor tissue into small pieces by scissors followed by transferral of 1-2 pieces into a 2 mL microtube for snap freezing on dry ice. Store at -80 &#176;C for genomic profiling. Mince the remaining pieces into finer pieces by scissors before transferral to a 50 mL plastic tube using AD+++.</li>
		<li>Wash minced tissue 2-3 times by 35 mL of AD+++, followed by addition of 10 mL of digestion medium (see step 4.1.4) and placement on an orbital shaker at 4 x g for 1 h at 37 &#176;C.</li>
		<li>Homogenize the digested tissue by pipetting up and down using a 5 mL sterile plastic pipette, followed by addition of 20 mL of AD+++ and filtration by 100 &#181;m cell strainer.</li>
		<li>Wash the pass-through twice with AD+++ (spin at 450 x g for 5 min), followed by resuspension in BME (add 4x volume of BME to the pellet for suspension) and keep on ice19.</li>
	</ol>
	</li>
	<li>Preparation of organoid culture
	<ol>
		<li>Add the BME cell suspension into 6-well plate in multiple drops to a total of 200 &#181;L per well.</li>
		<li>Transfer the plate to a 37 &#176;C incubator. After 30 min, the gel drops solidify.</li>
		<li>Add 2 mL of organoid media to each well, with the representative drops recorded by microscopic photography before transferring to an incubator (37 &#176;C and 5% CO2).</li>
		<li>Maintain the organoid cultures with medium change every 3-4 days, and passage at a 1:2 ratio every 7 days or depending on their growth and density.</li>
	</ol>
	</li>
</ol>
5. Histopathology and next generation sequencing (NGS) analysis
<ol>
	<li>Histopathology
	<ol>
		<li>Collect organoids from the well with the existing medium using a P1000 pipette, followed by centrifugation (spin at 450 x g for 5 min), washing with PBS and fixation in 10% formalin for 1 h.</li>
		<li>Place the fixed organoids in 100% gelatin at the bottom of 50 mL conical tube, followed by routine tissue processing and embedding.</li>
		<li>Perform haematoxylin&#8211;eosin (H&#38;E) staining using standard protocols on 4 mm paraffin sections.</li>
	</ol>
	</li>
	<li>RNAseq and whole exome sequencing
	<ol>
		<li>Collect organoids from the well with the existing culture medium using a P1000 pipette, followed by centrifugation using a microcentrifuge at maximum speed (12,000 x g) for 5 min at 4 &#176;C.</li>
		<li>Collect the pellet by removing the medium supernatant to ensure no visible BME was present, followed by snap freezing in a microtube (dry ice) and then transfer to a -80 &#176;C freezer.</li>
		<li>Extract RNA or DNA using standard procedure from manufacturers and perform NGS analysis for both RNAseq and whole exome sequencing (WES).</li>
	</ol>
	</li>
</ol>
6. IC50 assay20
<ol>
	<li>Organoid seeding in 384-well plate for IC50 assay
	<ol>
		<li>Dissociate organoids in BME drops from each well (digesting BME) by adding 20 &#181;L of 100x dispase solution to each well (6-well plate), which contained 2 mL of organoid medium, followed by 30 min of incubation at 37 &#176;C.</li>
		<li>Pipette digested organoids from all wells through a 70 &#181;m filter into a 50 mL plastic tube to collect the organoids.</li>
		<li>Count the organoids under microscopy for the determination of organoid concentration. Suspend the organoids using culture medium, before adding BME to reach final concentration of 5% (v/v) on ice.</li>
		<li>Add 50 &#181;L of the organoid suspension into each 384-well plate with the liquid dispenser, according to the plate map with a seeding density of 200 CR2110 PDXOs per well in corresponding organoid culture medium.</li>
	</ol>
	</li>
	<li>Cisplatin and irinotecan treatment
	<ol>
		<li>Use cisplatin (with the highest concentration of 100 &#181;M) and &#8220;irinotecan&#8221; (with the highest concentration of 10 &#181;M). Add SN-38 (a metabolite of irinotecan, as opposed to irinotecan which is usually used for in vivo studies) to each well according to the drug dilution scheme for 9 doses, in serial dilution by digital dispener.</li>
		<li>Create the platemap using the digital dispenser software tool. Include a negative control vehicle with 100% viability and postive control of 5 &#181;M staurosporine, which showed 0% viability.</li>
		<li>Place the drug-treated 384-well plates back into 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Determination of organoid cell viability after drug treatment
	<ol>
		<li>At the end of the 5 days of drug treatment, determine organoid cell viability using luminescent cell viability reagents as per the manufacturer&#8217;s recommended procedure. Add luminescent reagent into each well with the liquid dispener and mix for 5 min on a plate shaker, followed by a 30 min incubation at room temperature in dark.</li>
		<li>Record the luminescent signal on a luminescence multi-well plate reader.</li>
		<li>Calculate the normalized viabilities of each well using the raw readings from the plate reader and create a dose-response curve and IC50 values by nonlinear curve fitting.</li>
	</ol>
	</li>
</ol>

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All authors are or were current full-time employees of Crown Bioscience, Inc.

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) ...

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Research

JoVE Journal

Cancer Research

6.2K Views.  Crown Bioscience Inc., Changping District, Beijing, China. 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’ method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

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

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 ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...