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
NOTE: See Table 1 for the solutions and media to be prepared.
<ol>
	<li>Agarose gel support
	<ol>
		<li>Weigh the agarose powder in a microwavable flask and dissolve it in a given volume of E3 zebrafish medium&#160;to make a 1% gel. Heat in a microwave until the agarose has completely dissolved. 
		NOTE: Do not overboil the solution.</li>
		<li>Pour the melted agarose into a Petri dish and wait until the gel has completely solidified.</li>
		<li>Make small agarose cylinders (~5 mm high) using a plastic Pasteur pipette with the tip cut off. Once prepared, store in a Petri dish at 4 &#176;C wrapped in aluminum foil.</li>
	</ol>
	</li>
	<li>Glass microneedles
	<ol>
		<li>Pull the borosilicate glass capillaries with a puller to obtain fine needles (settings: HEAT 990, PULL 550). 
		&#8203;NOTE: From one capillary, it is possible to obtain two fine needles with a tip diameter of 10 &#181;m.</li>
	</ol>
	</li>
</ol>
2. Fish crossing and egg collection
<ol>
	<li>Transfer adult fish in breeding tanks 3 days prior to tissue implantation, as described by Avdesh et al.17.</li>
	<li>&#8203;NOTE: A ratio of 1:1 or 2:3 males to females is recommended. The fish density should be a maximum five fish per liter of water. Keep males and females separated with a barrier overnight.</li>
	<li>The next day, remove the barrier and allow the fish to mate.</li>
	<li>Remove the fish from the breeding tanks and return them to their housing tanks.</li>
	<li>Pour the water from the breeding tank through a fine mesh net. Transfer the fertilized eggs to a Petri dish with E3 zebrafish medium.</li>
	<li>Check the Petri dish with a stereomicroscope and discard the cloudy eggs. Keep the fertilized eggs in fresh E3 zebrafish medium at 28 &#176;C.</li>
</ol>
3. Specimen collection
NOTE: Autoclave forceps and a scalpel handle.
<ol>
	<li>Immediate processing
	<ol>
		<li>Collect the surgical specimen of tumor in 10 mL of tumor medium at 4 &#176;C (tumor specimen ranging from 5 mm to 10 mm in diameter). Transfer the sample at 4 &#176;C from the desired location for immediate processing.</li>
	</ol>
	</li>
	<li>Storage overnight (optional)
	<ol>
		<li>Collect the surgical tumor specimen in 10 mL of tumor medium and store the sample overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Storage at -80 &#176;C (optional, least recommended)
	<ol>
		<li>Store the sample at -80 &#176;C in a cryogenic vial with tumor medium supplemented with 5% dimethyl sulfoxide (DMSO).</li>
	</ol>
	</li>
</ol>
4. Sample processing
NOTE: Perform the steps under a sterile tissue culture laminar flow hood.
<ol>
	<li>Wash the whole tumor tissue with 5 mL of fresh tumor medium, pipetting up and down 10x using a plastic Pasteur pipette. Aspirate and discard the washing medium. Repeat this step 3x. 
	NOTE: Avoid aspiration of the tumor tissue as it could remain attached to the plastic Pasteur pipette.</li>
	<li>Transfer the sample in a Petri dish and immerge it in 1-2 mL of fresh tumor medium. Cut the tumor sample into small pieces (1-2 mm3) using a scalpel blade and place them in a sterile 5 mL plastic tube with tumor medium.</li>
	<li>Set the McIlwain tissue chopper to 100 &#181;m thickness. Place the specimen fragments on the circular plastic table of the chopper and chop them. Rotate the table by 90&#176; and repeat the chopping.</li>
	<li>Centrifuge the fragments at 300 &#215; g for 3 min. Then, carefully aspirate the supernatant and discard it.</li>
	<li>Incubate the fragments with a fluorescent cell tracker, CM-DiI (final concentration of 10 &#181;g/mL in Dulbecco&#39;s phosphate buffered saline [DPBS]), Deep Red (final concentration of 1 &#181;L/mL in DPBS), or CellTrace (final concentration of 5 &#181;M in DPBS) for 30 min, placing the tube in a 37 &#176;C water bath.</li>
	<li>Resuspend the fragments by gently pipetting up and down every 10 min. 
	NOTE:&#160;In case of CellTrace, add medium containing at least 1% protein at the end of the incubation, according to the manufacturer&#39;s instructions.</li>
	<li>Centrifuge at 300 x g for 3 min and discard the supernatant. Repeat this step 3x with 1 mL of DPBS to remove unincorporated dye.</li>
	<li>Suspend the fragments in 5 mL of DPBS in a 60 mm Petri dish. 
	NOTE: Make sure that the tissue does not dry out.</li>
</ol>
5. Establishment of zPDX
NOTE: Perform the steps under a sterile tissue culture laminar flow hood.
<ol>
	<li>Anesthetize the 2 days post fertilization (dpf) embryos with 0.16 mg/mL tricaine in E3 zebrafish medium.</li>
	<li>Put three agarose cylinders (step 1.1.3) in a Petri dish and lay a zebrafish embryo on a cylinder, exposing one side. Remove the excess solution to keep the embryo in just a thin film.</li>
	<li>Transfer the piece of stained tissue with sterile forceps from the Petri dish to the 1% agarose support where the embryo is lying. Pick up the tissue, put it on top of the embryo yolk, and then push it into the perivitelline space using the heat-pulled glass microneedle (step 1.2.1).</li>
	<li>Gently add some drops of E3 1% penicillin-streptomycin (Pen-Strep) to the embryo to bring it back into the liquid.</li>
	<li>Repeat steps 5.2-5.4 for all the embryos, and finally, remove the agarose supports from the Petri dish and incubate the embryos at 35 &#176;C.</li>
	<li>Check the embryos for the correct xenografts (positive staining) 2 h post implantation using a fluorescent stereomicroscope. Discard the embryos with tumor fragments that are not completely inside the perivitelline space, as well as the dead embryos. Randomly distribute the embryos in six multi-well plates (maximum n = 20 embryos/well), equally divided into groups according to the experimental plan (e.g., control and FOLFOXIRI).</li>
</ol>
6. Treatment
<ol>
	<li>Dilute the drug (e.g., 5-fluorouracil, oxaliplatin, irinotecan) into E3 1% Pen-Strep, mixing thoroughly by pipetting up and down several times. As proposed by Usai et al.12, use a fivefold dilution of the drug in the fish water, with respect to the equivalent plasma concentration (EPC).</li>
	<li>Mix the drugs to prepare the cocktail (e.g., FOLFOXIRI).</li>
	<li>Remove the media from each well and add the drug cocktail 2 h after implantation.</li>
	<li>Treat the embryos for 3 days. Renew the drug cocktail every day.</li>
</ol>
7. Whole-mount immunofluorescent staining
NOTE: Before starting, place acetone at -20 &#176;C and prepare the solutions listed in Table 1.
<ol>
	<li>Day 1:
	<ol>
		<li>Fix the larvae with 1 mL of 4% paraformaldehyde in glass vials at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Day 2:
	<ol>
		<li>Wash the larvae 3 x 5 min with 1 mL of PBS, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Store in 1 mL of 100% methanol at -20 &#176;C overnight (or for long-term storage).</li>
		<li>Rehydrate 3 x 10 min with 1 mL of PTw (0.1% tween in PBS), gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Permeabilize with 1 mL of 150 mM Tris-HCl at pH 8.8 for 5 min at RT, followed by heating for 15 min at 70 &#176;C.</li>
		<li>Wash 2 x 10 min with 1 mL of PTw, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Wash 2 x 5 min with 1 mL of dH2O, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Permeabilize with 1 mL of ice-cold acetone for 20 min at -20 &#176;C.</li>
		<li>Wash 2 x 5 min with 1 mL of dH2O, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Wash 2 x 5 min with 1 mL of PTw, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Incubate the larvae in 1 mL of blocking buffer for 3 h at 4 &#176;C, gently agitating on a laboratory platform rocker (400 rpm).</li>
		<li>Place the larvae in well plates divided per group as follows: 10 larvae in 50 &#181;L of volume/well in a 96-well plate or 20 larvae in 100 &#181;L of volume/well in a 48-well plate.</li>
		<li>Discard the blocking buffer, incubate the larvae with primary antibody solution (e.g., rabbit anti-human cleaved caspase-3, 1:250) diluted in incubation buffer overnight at 4 &#176;C in the dark, and gently rock on a shaker plate (400 rpm). See step 7.2.11 for recommended volumes.</li>
	</ol>
	</li>
	<li>Day 3:
	<ol>
		<li>Wash the larvae sequentially 3 x 1 h with 1 mL of PBS-TS (10% goat serum, 1% Triton X-100 in PBS) and then, with 2 x 10 min with 1 mL of PBS-T (1% Triton X-100 in PBS)&#160;and 2 x 1 h with 1 mL of PBS-TS. In each wash, gently agitate the plates containing the larvae on a shaker plate (400 rpm).</li>
		<li>Incubate the larvae with fluorescent-dye conjugated secondary antibodies (e.g., Goat anti-Rabbit IgG [H+L]&#160;Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, 1:500) and 100 &#181;g/mL Hoechst 33258 diluted in incubation buffer in the dark overnight at 4 &#176;C, with gentle agitation on a shaker plate (400 rpm). See step 7.2.11 for recommended volumes of secondary antibody solution.</li>
	</ol>
	</li>
	<li>Day 4:
	<ol>
		<li>Wash 3 x 1 h with 1 mL of PBS-TS and 2 x 1 h with 1 mL of PTw, with gentle agitation on a shaker plate (400 rpm).</li>
		<li>Create a circular layer with the enamel (thickness of ~0.5-1 mm) on microscope slides. Let the enamel dry out and place the larvae in the center of the circular layer, exposing the side of the xenograft.</li>
		<li>Dry the excess solution and mount the glass coverslip with a water-soluble, non-fluorescing mounting medium.</li>
	</ol>
	</li>
</ol>
8. Imaging
<ol>
	<li>Capture images under confocal microscopy with a 40x objective. Use the following acquisition parameters: a resolution of 1024 x 512 pixels with a Z-spacing of 5 &#181;m.</li>
</ol>
9. Analysis of apoptosis by ImageJ
<ol>
	<li>Load (Fiji Is Just) ImageJ software (https://imagej.net/Fiji/Downloads) and open the Z-stack file image (click File | Open). In the pop-up window, select Stack viewing/Hyperstack and click OK.</li>
	<li>Overlay the different channels by selecting Image | Color | Make Composite.</li>
	<li>Drag the Z bar at the bottom of the image to browse through the Z-stack image and identify the xenograft area (cells that are fluorescent cell tracker positive. See step 4.5) in the zebrafish perivitelline space.</li>
	<li>Select Point Tool and count the number of apoptotic human cells (positive to fluorescent cell tracker and cleaved caspase-3), as shown in Supplementary Video S1.</li>
	<li>Double click on the Point Tool icon, change the counter, and count the total number of human cell nuclei (nuclei of CM-DiI positive cells).</li>
</ol>

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The authors have no conflicts of interest to declare.

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.
<p class="jove_...

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 ...

establishment-zebrafish-patient-derived-xenografts-from-pancreatic

Research

JoVE Journal

Cancer Research

999 Views.  University of Pisa. 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. 

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

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 ...

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. ...

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 ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.