Name: creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research
Uploaded: 2021-05-05T13:00:00.000+00:00
Duration: 4 min 49 s
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 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 &amp;amp; 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...

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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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JoVE Journal

Cancer Research

6.2K Views.  Crown Bioscience Inc., Changping District, Beijing, China. Dit protocol kan worden gebruikt om een overeenkomende bibliotheek van gekoppelde patiënt-afgeleide xenografts, of PDX, en overeenkomstige patiënt-afgeleide organoïde lijnen vast te stellen. Het voordeel van deze methode is dat het kan worden gebruikt om organoïden te maken met PDX voor in vitro screening, wat resulteert in overeenkomende paren in vivo / in vitro modellen. Begin met het oogsten van de tumoren en het verwerken van het weefsel zoals beschreven in het teksthandschrift.

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

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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 ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

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 ...

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 ...

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 ...

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. ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

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

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