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

Summary

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.

Abstract

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.

Introduction

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 medicine^4,5,6. 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 recurrence^7,8,9.

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 research^11,12. 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 interplay^15,16. 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.

Protocol

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.

1. Agarose gel support
   1. Weigh the agarose powder in a microwavable flask and dissolve it in a given volume of E3 zebrafish medium to make a 1% gel. Heat in a microwave until the agarose has completely dissolved.
      NOTE: Do not overboil the solution.
   2. Pour the melted agarose into a Petri dish and wait until the gel has completely solidified.
   3. 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 °C wrapped in aluminum foil.
2. Glass microneedles
   1. Pull the borosilicate glass capillaries with a puller to obtain fine needles (settings: HEAT 990, PULL 550).
      ​NOTE: From one capillary, it is possible to obtain two fine needles with a tip diameter of 10 µm.

2. Fish crossing and egg collection

1. Transfer adult fish in breeding tanks 3 days prior to tissue implantation, as described by Avdesh et al.¹⁷.
2. ​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.
3. The next day, remove the barrier and allow the fish to mate.
4. Remove the fish from the breeding tanks and return them to their housing tanks.
5. Pour the water from the breeding tank through a fine mesh net. Transfer the fertilized eggs to a Petri dish with E3 zebrafish medium.
6. Check the Petri dish with a stereomicroscope and discard the cloudy eggs. Keep the fertilized eggs in fresh E3 zebrafish medium at 28 °C.

3. Specimen collection

NOTE: Autoclave forceps and a scalpel handle.

1. Immediate processing
   1. Collect the surgical specimen of tumor in 10 mL of tumor medium at 4 °C (tumor specimen ranging from 5 mm to 10 mm in diameter). Transfer the sample at 4 °C from the desired location for immediate processing.
2. Storage overnight (optional)
   1. Collect the surgical tumor specimen in 10 mL of tumor medium and store the sample overnight at 4 °C.
3. Storage at -80 °C (optional, least recommended)
   1. Store the sample at -80 °C in a cryogenic vial with tumor medium supplemented with 5% dimethyl sulfoxide (DMSO).

4. Sample processing

NOTE: Perform the steps under a sterile tissue culture laminar flow hood.

1. 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.
2. 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.
3. Set the McIlwain tissue chopper to 100 µm thickness. Place the specimen fragments on the circular plastic table of the chopper and chop them. Rotate the table by 90° and repeat the chopping.
4. Centrifuge the fragments at 300 × g for 3 min. Then, carefully aspirate the supernatant and discard it.
5. Incubate the fragments with a fluorescent cell tracker, CM-DiI (final concentration of 10 µg/mL in Dulbecco's phosphate buffered saline [DPBS]), Deep Red (final concentration of 1 µL/mL in DPBS), or CellTrace (final concentration of 5 µM in DPBS) for 30 min, placing the tube in a 37 °C water bath.
6. Resuspend the fragments by gently pipetting up and down every 10 min.
   NOTE: In case of CellTrace, add medium containing at least 1% protein at the end of the incubation, according to the manufacturer's instructions.
7. 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.
8. Suspend the fragments in 5 mL of DPBS in a 60 mm Petri dish.
   NOTE: Make sure that the tissue does not dry out.

5. Establishment of zPDX

NOTE: Perform the steps under a sterile tissue culture laminar flow hood.

1. Anesthetize the 2 days post fertilization (dpf) embryos with 0.16 mg/mL tricaine in E3 zebrafish medium.
2. 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.
3. 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).
4. Gently add some drops of E3 1% penicillin-streptomycin (Pen-Strep) to the embryo to bring it back into the liquid.
5. 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 °C.
6. 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).

6. Treatment

1. 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).
2. Mix the drugs to prepare the cocktail (e.g., FOLFOXIRI).
3. Remove the media from each well and add the drug cocktail 2 h after implantation.
4. Treat the embryos for 3 days. Renew the drug cocktail every day.

7. Whole-mount immunofluorescent staining

NOTE: Before starting, place acetone at -20 °C and prepare the solutions listed in Table 1.

1. Day 1:
   1. Fix the larvae with 1 mL of 4% paraformaldehyde in glass vials at 4 °C overnight.
2. Day 2:
   1. Wash the larvae 3 x 5 min with 1 mL of PBS, gently agitating on a laboratory platform rocker (400 rpm).
   2. Store in 1 mL of 100% methanol at -20 °C overnight (or for long-term storage).
   3. Rehydrate 3 x 10 min with 1 mL of PTw (0.1% tween in PBS), gently agitating on a laboratory platform rocker (400 rpm).
   4. 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 °C.
   5. Wash 2 x 10 min with 1 mL of PTw, gently agitating on a laboratory platform rocker (400 rpm).
   6. Wash 2 x 5 min with 1 mL of dH₂O, gently agitating on a laboratory platform rocker (400 rpm).
   7. Permeabilize with 1 mL of ice-cold acetone for 20 min at -20 °C.
   8. Wash 2 x 5 min with 1 mL of dH₂O, gently agitating on a laboratory platform rocker (400 rpm).
   9. Wash 2 x 5 min with 1 mL of PTw, gently agitating on a laboratory platform rocker (400 rpm).
   10. Incubate the larvae in 1 mL of blocking buffer for 3 h at 4 °C, gently agitating on a laboratory platform rocker (400 rpm).
   11. Place the larvae in well plates divided per group as follows: 10 larvae in 50 µL of volume/well in a 96-well plate or 20 larvae in 100 µL of volume/well in a 48-well plate.
   12. 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 °C in the dark, and gently rock on a shaker plate (400 rpm). See step 7.2.11 for recommended volumes.
3. Day 3:
   1. 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) 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).
   2. Incubate the larvae with fluorescent-dye conjugated secondary antibodies (e.g., Goat anti-Rabbit IgG [H+L] Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, 1:500) and 100 µg/mL Hoechst 33258 diluted in incubation buffer in the dark overnight at 4 °C, with gentle agitation on a shaker plate (400 rpm). See step 7.2.11 for recommended volumes of secondary antibody solution.
4. Day 4:
   1. 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).
   2. 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.
   3. Dry the excess solution and mount the glass coverslip with a water-soluble, non-fluorescing mounting medium.

8. Imaging

1. 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 µm.

9. Analysis of apoptosis by ImageJ

1. 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.
2. Overlay the different channels by selecting Image | Color | Make Composite.
3. 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.
4. 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.
5. 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).

Results

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...

Discussion

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.

Materials

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