Name: establishment of zebrafish patient-derived xenografts from pancreatic cancer for chemosensitivity testing
Uploaded: 2023-05-12T12:00:00
Description: Watch this Scientific Journal Video about Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing at JoVE.com

This work was funded by Fondazione Pisa (project 114/16). The authors would like to thank Raffaele Gaeta from the Histopathology Unit of Azienda Ospedaliera Pisana for the patient sample selection and pathology support. We also thank Alessia Galante for the technical support in the experiments.&#160;This article is based upon work from COST Action TRANSPAN, CA21116, supported by COST (European Cooperation in Science and Technology).

The Italian Ministry of Public Health approved all the animal experiments described, in conformity with the Directive 2010/63/EU on the use and care of animals. The local Ethical Committee approved the study, under registration number 70213. Informed consent was obtained from all subjects involved. Before starting, all the solutions and the equipment should be prepared (section 1) and the fish should be crossed (section 2).
1. Preparation of solutions and equipment
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<ol>
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In vivo models in cancer research provide invaluable tools to understand cancer biology and predict the cancer treatment response. Currently, different in vivo models are available, for example, genetically modified animals (transgenic and knockout mice) or patient-derived xenografts from human primary cells. Despite many optimal features, each one has various limitations. In particular, the aforementioned models lack a reliable way to mimic the patient tumor tissue microenvironment.
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One of the problems of clinical cancer research is that cancer is not a single disease, but a variety of different diseases that can evolve over time, requiring specific treatments depending on the characteristics of the tumor itself and the patient1. Consequently, the challenge is to move toward patient-oriented cancer research, in order to identify new personalized strategies for the early prediction of cancer treatment outcomes2. This is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), since it is considered a hard-to-treat cancer, with a 5-year survival rate of 11%3.
The late diagnosis, rapid progression, and lack of effective therapies remain the most pressing clinical problems of PDAC. The main challenge is, therefore, to model the patient and identify biomarkers that can be applied in the clinic to select the most effective therapy in line with personalized medicine4,5,6. Over time, novel approaches have been proposed to model cancer diseases: patient-derived organoids (PDOs) and mouse patient-derived xenografts (mPDXs) originated from a source of human tumor tissue. They have been used to reproduce the disease to study the response and the resistance to therapy, as well as disease recurrence7,8,9.
Similarly, interest in zebrafish-based patient-derived xenograft (zPDX) models has increased, thanks to their unique and promising characteristics10, representing a quick and low-cost tool for cancer research11,12. zPDX models require only a small tumor sample size, which makes high-throughput screening of chemotherapy feasible13. The most common technique used for zPDX models is based on complete sample digestion and implantation of the primary cell populations, which partially reproduces the tumor, but has the disadvantages of a lack of tumor microenvironment and crosstalk between malignant and healthy cells14.
This work shows how zPDXs can be used as a preclinical model to identify the chemosensitivity profile of pancreatic cancer patients. The valuable strategy facilitates the xenograft process, since there is no need for cell expansion, allowing for the acceleration of the chemotherapy screening. The strength of the model is that all the microenvironment components are maintained as they are in the patient cancer tissue, because, as it is well known, the behavior of the tumor depends on their interplay15,16. This is highly favorable over alternative methods in the literature, as it is possible to preserve the tumor heterogeneity and contribute to improving the predictability of the treatment outcome and relapse in a patient-specific manner, thus enabling the zPDX model to be used in co-clinical trials. This manuscript describes the steps involved in making the zPDX model, starting with a piece of patient tumor resection and treating it to analyze the response to chemotherapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-fluorouracil</td>
			<td>Teva Pharma AG</td>
			<td>SMP 1532755</td>
			<td></td>
		</tr>
		<tr>
			<td>48 multiwell plate</td>
			<td>Sarstedt</td>
			<td>83 3923</td>
			<td></td>
		</tr>
		<tr>
			<td>96 multiwell plate</td>
			<td>Sarstedt</td>
			<td>82.1581.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose powder&#160;</td>
			<td>Merck</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nuclei Antibody, clone 235-1</td>
			<td>Merck</td>
			<td>MAB1281&#160;</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Aquarium net QN6</td>
			<td>Penn-plax</td>
			<td>0-30172-23006-6</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34567</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker CM-DiI&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C7001</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Deep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34565</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>9661S</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)&#160;</td>
			<td>PanReac AppliChem ITW Reagents</td>
			<td>A3672,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid -&#160; Lederfolin</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries, 3.5&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>Outer diameter = 1.14 mm. Inner diameter = 0.53 mm.&#160;</td>
		</tr>
		<tr>
			<td>Glass vials&#160;</td>
			<td>VWR International</td>
			<td>WHEAW224581</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit&#160;IgG&#160;(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21244&#160;&#160;</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>31872</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Irinotecan</td>
			<td>Hospira</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Temperature Freezer Vials</td>
			<td>VWR International</td>
			<td>479-1220</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Mixer</td>
			<td>SCILOGEX</td>
			<td>822000049999</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Teva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 100 mm</td>
			<td>Sarstedt</td>
			<td>83 3902500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 mm</td>
			<td>Sarstedt</td>
			<td>83 3901</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td>Sarstedt</td>
			<td>86.1171.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Mount</td>
			<td>Tebu-bio</td>
			<td>18606-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Merck</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade No 10 Sterile Stainless Steel</td>
			<td>VWR International</td>
			<td>SWAN3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Merck</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Micropipette Puller</td>
			<td>Shutter instrument</td>
			<td>P-30&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This protocol describes the experimental approach for establishing zPDXs from primary human pancreatic adenocarcinoma. A tumor sample was collected, minced, and stained using fluorescent dye, as described in protocol section 4. zPDXs were then successfully established by implantation of a piece of tumor into the perivitelline space of 2 dpf zebrafish embryos, as described in protocol section 5. As described in protocol section 6, the zPDXs were further screened to identify the chemotherapy sensitivity profiles of patient...

Watch this Scientific Journal Video about Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing at JoVE.com

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

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 ...

Zebrafish patient-derived xenograft is a powerful preclinical model to predict cancer patient chemosensitivity profile in vivo. The protocol describe the generation of the model starting from a patient surgical specimen. By xenografting a small tissue fragment rather than isolate cells into zebrafish embryos, it's possible to maintain the tumor microenvironment which is the crucial step for tumor progression and the response to chemotherapy.The big advantage of the model is given by its potential to predict the patient's prognosis in a clinical relevant timeframe of just one week acting as a tool for personalized medicine. Begin the sample processing by washing the whole tumor tissue collected from the patient with five milliliters of fresh tumor medium. Pipette it up and down 10 times using a plastic Pasteur pipette.Carefully aspirate and discard the washing medium before repeating the wash three times. Immerse the sample in one to two milliliters of fresh tumor medium. Cut the tumor sample into one to two millimeter cube pieces using a scalpel blade, and then place them in a sterile five milliliter plastic tube with tumor medium.After setting the McIlwain Tissue Chopper to 100 micrometer thickness, place the tumor pieces on the circular plastic table of the chopper and chop them. Rotate the table by 90 degrees and repeat the chopping. Collect the chopped tumor fragments in a 15 milliliter plastic tube containing the tumor medium.Centrifuge the chopped tumor fragments at 300 G for three minutes before aspirating and discarding the supernatant. Incubate the fragments with the cell trace solution or with an alternative fluorescent cell tracker such as CM-DiI or Deep Red for 30 minutes in a 37 degrees Celsius water bath. During incubation, gently resuspend the fragments every 10 minutes.At the end of the staining, remove any free dye by adding a tumor medium supplemented with at least 1%of protein. The volume will be five times the staining volume. Centrifuge the fragments at 300 G for three minutes and discard the supernatant.Add one milliliter of Dulbecco's Phosphate Buffered Saline or DPBS and repeat this washing step three times. Once done, suspend the fragments in five milliliters of DPBS in a 60 millimeter Petri dish. To establish the zebrafish-based patient-derived xenografts or zPDX, anesthetize the two days post fertilization embryos.In a Petri dish having three agarose cylinders, lay a zebrafish embryo on a cylinder exposing one side. Remove the excess solution to keep the embryo in a thin film. Using sterile forceps, transfer the piece of stained tumor tissue fragments from a 60 millimeter Petri dish to the 1%agarose support where the embryo is laid.Then pick up the tissue and place it on the embryo yolk before pushing it into the perivitelline space using the heat pulled glass microneedle. Add some drops of E3 1%penicillin streptomycin to the embryo to bring it back into the liquid. After repeating the implantation of tumor tissue in all the embryos, remove the agarose supports from the Petri dish and incubate the embryos at 35 degrees Celsius.Two hours post implantation, check the embryos for the correct xenografts with positive staining using a fluorescent stereo microscope. Discard dead embryos and the embryos without the tumor fragments completely inside the perivitelline space. Once done, randomly distribute the embryos in six multi-well plates, equally divided into groups according to the experimental plan.Dilute the drugs by mixing them into E3 1%pen-strep to prepare the FOLFOXIRI cocktail. After two hours of implantation, remove the media from each well and add the drug cocktail. Place the embryos into the incubator at 35 degrees Celsius for three days with daily changes of the medium to renew the drugs.For imaging, capture images under a 40X objective of the confocal microscope. Use the resolution of 1024 by 512 pixels with a Z spacing of five micrometers. The whole mount images of zPDXs acquired to analyze the apoptosis detected in cyano showed that the combined therapy increased cell apoptosis compared to the control group group.The case study reported that a statistically significant increase in the fraction of apoptotic cells in implanted xenografts was identified for the FOLFOXIRI-treated group compared to the control group. The protocol show an example using pancreatic cancer tissue, but zebrafish patient-derived xenografts can be established from different types of cancer. In additions, since the model has a low ethical impact, it is easily exploitable for in vivo screening of new drugs.The method is technically challenging and requires an expert operator. Prior expertise in zebrafish embryo manipulation and microinjection will speed up the training of the technique.

Research

JoVE Journal

Cancer Research

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