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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>C7001</td><td></td></tr><tr><td>CellTracker Deep Red&amp;nbsp;</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)&amp;nbsp;</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 -&amp;nbsp; Lederfolin</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Glass capillaries, 3.5&quot;</td><td>Drummond Scientific Company</td><td>3-000-203-G/X</td><td>Outer diameter = 1.14 mm. Inner diameter = 0.53 mm.&amp;nbsp;</td></tr><tr><td>Glass vials&amp;nbsp;</td><td>VWR International</td><td>WHEAW224581</td><td></td></tr><tr><td>Goat anti-Rabbit&amp;nbsp;IgG&amp;nbsp;(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>A-21244&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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...

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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 ...

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Cancer Research

1.1K Views.  University of Pisa. Zebravis patiënt-afgeleide xenograft is een krachtig preklinisch model om het chemosensitiviteitsprofiel van kankerpatiënten in vivo te voorspellen. Het protocol beschrijft de generatie van het model uitgaande van een chirurgisch monster van de patiënt. Door een klein weefselfragment te xenograferen in plaats van cellen te isoleren in zebravisembryo's, is het mogelijk om de micro-omgeving van de tumor te behouden, wat de cruciale stap is voor tumorprogressie en de reactie op chemotherapie....

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 ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

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 ...

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 ...

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 ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Rapid in vivo Drug Response Prediction Using Leukemia Cell Grafts in Zebrafish Embryos

Zebrafish xenotransplantation is a pivotal technique for investigating human cancer pathogenesis and predicting individual drug responses. This document introduces a streamlined protocol (ZefiX) for expanding primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patient samples or immortalized cell lines in transiently immunosuppressed zebrafish embryos, utilizing flow cytometry for high-resolution single-cell analysis of treatment responses. Compared to solid tumor engraftments, leukemia cells profit significantly from a morpholino antisense oligonucleotide-based suppression of macrophage and neutrophil differentiating factors during the assay. Flow cytometry analysis of dissociated graft cells enables precise evaluation of cell count, proliferation rate, and vitality after treatment on a per-cell basis. This approach has been validated using targeted therapeutics such as venetoclax and dasatinib, with treatment outcomes compared to clinical records of related patient samples and traditional 2D culture controls. Notably, the protocol&#160;is completed within 7&#160;days, aligning with clinical decision-making timelines. The methodology is adaptable for testing selected drugs in various cancer types, including solid tumors, thereby supporting personalized therapeutic strategies. However, limitations on the number of drugs that can be assessed, likely due to pharmacokinetic constraints in zebrafish embryos, should be considered.

This protocol provides step-by-step instructions for generating and troubleshooting human acute lymphoblastic leukemia (ALL) xenografts from cell lines and fresh patient material in transiently immunosuppressed zebrafish embryos, along with guidelines for drug response assessment using flow cytometry. The experimental pipeline can also be adapted for solid tumors.

Zebrafish xenotransplantation is a pivotal technique for investigating human cancer pathogenesis and predicting individual drug responses. This document ...

Zebrafish Xenograft Assay: A Method to Assess the Anti-cancer Efficacy of Therapeutics on Tumorigenicity in Zebrafish In Vivo

Source: Lee, J. Y et al.,<a href="https://www.jove.com/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp. (2018)
This video describes the zebrafish xenograft assay to assess in vivo tumor formation and study the effect of therapeutic agents.

Source: Lee, J. Y et al.,Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...

JoVE Encyclopedia of Experiments

Lung Cancer

In vivo study

Zebrafish Patient-Derived Xenograft (zPDX): A Cancer Model

Source: Wang L. et. al., <a href="https://www.jove.com/v/59507/" target="_blank">Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment</a>. J. Vis. Exp. (2019).
This video describes the technique to create a zebrafish patient derived xenograft model. In the sample protocol, we assess the efficiency of the drugs Gemcitabine and Navitoclax on the pancreatic tumor xenograft model.

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

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