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

Summary

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.

Abstract

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 °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.

Introduction

Precision oncology aims to find the most beneficial therapeutic strategies for individual patient¹. Currently, numerous preclinical models such as in vitro primary culture, in vitro organoid culture², and patient-derived xenografts (PDX) in mice before or after organoid culture are proposed for diagnosis and to screen/assess the potential therapeutic choices³. PDX model generated by the injection of human primary cancer cells into immune-compromised mice, is one of the most promising tools for personalized drug screening in clinical oncology^3,4. Unlike the cultured cell line in vitro, PDX models usually preserve the integrity and heterogeneity of the in vivo tumor environment, better mimicking the diversity and idiosyncratic characteristics of different tumor patients, and therefore, may predict the potential medical outcome of patients⁴. However, the generation of PDX models in mice requires high quality patient samples and months of time to gather sufficient cells and models for multi group experiments, and the cellular/genetic compositions of the xenograft may drift from those of the original patient^’s biopsy. The success rate for establishing mice PDX model is also low, making it difficult to be broadly implemented in clinical practice. For the patients carrying rapidly progressed cancers like pancreatic cancer, they may not be able to obtain valuable information from the PDX experiments in time. 

In the past few years, zebrafish has been reported to be potential hosts for not only CDX (cell-derived tumor xenograft) models, but also PDX models^5,6,7,8,9,10. As a vertebrate model animal, zebrafish harbors sufficient similarities with mammals in both genetics and physiology, with two significant advantages: transparency and small in size¹¹. Zebrafish is also highly fecundity, and hundreds of inbred larvae can be obtained within a few days from a single pair of adults¹². Several studies have employed zebrafish to generate both transgenic and xenograft models of cancer diseases^13,14. Compared to mice xenografts, zebrafish xenografts allow tracking at single cell resolution. A certain amount of human tissues is capable of generating hundreds of zebrafish PDX models (zPDXs), while may only be sufficient to generate a couple of mice PDX models^15,16. Besides, the zebrafish larvae at 2-5 dpf already develop complete circulatory systems and metabolic organs such as liver and kidney, but not the immune system¹⁷, while the remaining yolk sac is a natural 3D medium, ideal for drug screening, drug resistance tests and tumor migration observations^{6,18,19,20,21}.

With an ultimate attempt to use zPDX as a screening/testing platform for clinical use, here, we describe an optimized proposal for zPDX model of pancreatic cancer, which allows the in vivo candidate drug assessment within a short time using fewer cells at lower costs. Compared to the previous references about zPDX^6,9,10, we introduced several optimizations to make the system more feasible and reliable for clinical personalized diagnosis: 1) pre-sorting different cell groups in the primary tumor tissues and stabilizing primary cells for one week before further experiments; 2) labeling the human cells and enhancing the cell viability in xenograft via lentivirus-based genetic modification; 3) optimizing the zebrafish culture condition in both nutriment supplements (glucose and glutamine) and temperature; 4) quantifying the drug responses of different cell types in a comparative manner. We also made changes to the injection solution by adding several supplementary materials. Altogether, those improvements provide the possibility to quickly generate a more patient-like xenograft in zebrafish hosts that can be used as a reliable tool to assess the response of candidate drugs.

Protocol

All animal procedures were approved and followed the guidelines of the Animal Ethics Committee at Fudan University and all pancreatic cancer specimens were obtained from Fudan University Shanghai Cancer Center. Ethical approval was obtained from the FUSCC Ethics Committee, and written informed consent was obtained from each patient.

1. Preparing the Equipment for the Microinjection

1. Preparing the injection plate.
   1. Prepare a 50 mL solution of 1% agarose dissolved in E3 solution (0.6 g/L aquarium salt in double distilled water + 0.01 mg/L methylene blue). Boil the solution until the agarose dissolves.
   2. Pour 50 mL of the agarose solution into a 10 cm Petri dish and then place the zebrafish embryo fixation mold on the surface. Remove the mold when the agarose solution becomes solidified.
   3. Add 20 mL of E3 solution to the injection plate and maintain it at 4 °C for long-term storage.
2. Preparing the injection needles.
   1. Pull a 10 cm glass capillary with an inner dimension of 0.9 mm into two needles on a needle puller.
   2. Use forceps to cut the end of the needle to create an opening under the microscope.

2. Preparing Embryos for Transplantation

1. Place 1 to 2 pairs of adult zebrafish in a mating tank at 7-9 pm and collect the fertilized eggs at around 8 am of the next morning.
2. Transfer the fertilized eggs from the mating tank to a Petri dish containing 40 mL of fresh E3 solution and incubate at 28.5 °C.
3. After 8 h of incubation in E3 solution, add 0.03% 1-phenyl-2-thiourea (PTU) into E3 solution to inhibit pigmentation. Incubate the embryos in E3 solution with 0.03% PTU at 28.5 °C until 48 hpf. This step can be omitted if using in-bred Casper mutant zebrafish.

3. Isolation and Culture of Primary Human Cells from Fresh Surgical Pancreatic Cancer Specimen or Frozen Tissue

1. Obtain specimens of human pancreatic cancer tissue of the size around 1 cm³ during an abdominal surgery, and immediately transfer the tissue into growth media (DMEM with 10% fetal bovine serum (FBS), 10 μM Y-27632, 100 μg/mL primocin, 10 μg/mL putrescine dihydrochloride, 10 mM nicotinamide and 1% penicillin streptomycin).
2. Transfer the pancreatic cancer sample into a Petri dish and remove the surrounding necrotic tissue, adipose tissue and connective tissue.
3. Rinse the cancer tissue for 5-6 times with phosphate buffer (PBS) and cut the tissue into 1 mm³ pieces using scalpels.
4. Transfer the shredded tissues into 5 mL of HBSS in a 50 mL tube and add collagenase type IV, hyaluronidase and DNase I at final concentrations of 200 units/mL, 100 mg/L and 20 mg/L, respectively. Pipette the mixture up and down to mix well.
5. Incubate the mixture at 37 °C in a 5% carbon dioxide incubator for 15-20 min. Pipette the mixture up and down a few times every 5 min.
6. Add 7 mL of DMEM to the tube and centrifuge at 110 x g for 5 min at 4 °C when digestion is complete.
7. Decant the supernatant and re-suspend the tumor mixture in DMEM.
8. Plate the mixture into a 6 cm Petri dish in 3 mL of full growth media (DMEM with 10% FBS, 20 μg/mL insulin, 100 ng/mL bFGF, 10 ng/mL EGF, 10 μM Y-27632, 100 μg/mL primocin, 10 μg/mL putrescine dihydrochloride, 10 mM nicotinamide, 1% penicillin streptomycin). Separate the cells into two groups.
9. In group I, add 100x inhibitor of pancreatic cancer fibroblasts into the medium after 48 h to remove the overgrown fibroblasts, leaving the cancer cells as the major cell types;. In group II, the fibroblasts will outgrow the cancer cells within a week.
10. Culture both cell groups for 1-2 week depending on the cell densities/purities and change the media every three days. The expected cell types in both group I & II can occupy over 98% in proportion in a typic successful experiment.

4. Labeling the Cells with Lentivirus Expressing Anti-apoptosis Gene BCL2L1 (BCL-XL) and Different Fluorescent Proteins Separately

1. Lentivirus production
   1. Plate 3 x 10⁶ HEK 293T cells with complete DMEM medium (DMEM supplied with 10% FBS) in 10 cm dishes and culture overnight at 37 °C in a 5% carbon dioxide incubator. Replace the medium with 6 mL of serum-free media before transfection.
   2. Prepare solution A: 8 μg of BCL2L1-containing lentiviral vectors (pCDH-EF1α-mKate2-E2A-BCL2L1-WPRE or pCDH-EF1α-eGFP-E2A-BCL2L1-WPRE), 2.4 μg of pVSVG, 4 μg pMDL(Gag/Pol), 1.6 μg of pREV and serum-free DMEM in a total volume of 500 µL. Gently pipet the mixture several times, and place it at room temperature for 5 min.
   3. Prepare solution B: 40 µL of PEI (polyethyleneimine) in 460 µL serum-free DMEM. Place it at room temperature for 5 min.
   4. Slowly add solution B into solution A and leave the tube at room temperature for 30 min.
   5. Add the final mixture into the HEK 293T cell culture dish prepared in step 4.1.1 and incubate at 37 °C in a 5% carbon dioxide incubator. After 12 h, add an additional 5 mL of the complete DMEM medium. After 48 h, harvest the medium containing the lentivirus.
   6. Filter the supernatant using a 0.45 μm sterile filter, add the supernatant into a concentration column, centrifuge at 6,000 x g for 25-30 min at 4 °C. The lentivirus aliquots of 100 µL per tube are made and stored at -80 °C.
2. Infection of the primary cells
   1. Seed the cells (group I & II) to be infected in a 12-well plate with 30-40% density and culture the cells overnight at 37 °C in a 5% carbon dioxide incubator.
   2. Replace the medium with 500 µL of serum-free medium containing 8 μg/mL of polybrene for 4 h. Then, add an additional 100 µL of the lentivirus into the medium (eGFP-E2A-BCL2L1 for Group I or mKate2-E2A-BCL2L1 for Group II). After 12 h, replace the medium with 1 mL of complete medium.
   3. Check the fluorescence markers after 48 h.
   4. Harvest the infected cells and mix them at 1:1 ratio with a final concentration of 10⁶/mL.
   5. Centrifuge the cells at 110 x g for 5 min and re-suspend the cell mixture in 50 µL of injection solution (1640 medium with 10% FBS, 0.05% hyaluronic acid sodium salt, 0.05% methylcellulose).

5. Injecting Mixed Cell Suspension into the Zebrafish

1. Add 10x tricaine solution into E3 water to anesthetize zebrafish larvae and transfer the larvae (from step 2.3) to the injection plate filled by modified E3 (E3 with 1 g/L glucose and 5 mmol/L L-glutamine).
2. Fill 25 µL of mixed cell suspension into micro capillaries needle and insert the needle into the micro-injection manipulator.
3. Set injection pressure and time. Inject 50-80 cells (~8 nL) into the yolk sac of 48 hpf zebrafish.

6. Culture of the Xenografted Zebrafish (zPDX Model)

1. Transfer the post-xenografted zebrafish larvae into 40 mL of mix solution (E3 solution with 1 g/L glucose and 5 mmol/L L-glutamine) at 32 °C.

7. Drug Administration on the Xenografted Zebrafish and the Assessment of Tumor Cells/Fibroblasts Viabilities

1. Determining the optimal concentration of gemcitabine/navitoclax.
   1. Place 10 wildtype zebrafish embryos at 48 hpf into each well of a 12-well plate.
   2. Add different concentrations of gemcitabine or navitoclax in each well and incubate at 32 °C for two days.
   3. After 2 days, calculate the maximal tolerance dosage (MTD) of gemcitabine and navitoclax at which the zebrafish larvae do not shown significant malformation and abnormal behavior, and the working concentrations are set below the MTD.
2. Treatment of zPDX models with gemcitabine/navitoclax.
   1. Place 10 xenografted larvae into each well of a 12 well plate.
   2. Divide the larvae into four groups, treat the control group in E3 containing 0.1% DMSO, and treat the other groups with 5 μg/mL gemcitabine and/or 50 μM navitoclax, and incubate at 32 °C for two days.
3. Assessment of the cell viabilities and cellular composition in zPDX models.
   1. Anesthetize the xenografted larvae post-treatment and place them in 3% methylcellulose.
   2. Image the larvae from the lateral view using a fluorescence microscope or confocal microscope.
   3. Quantify the intensity of red and green fluorescence signals using ImageJ and GraphPad software.

Results

A schematized outline of the procedure is represented in Figure 1. In short, the primary cancer tissue cells were seeded into the complete medium after digestion with or without the addition of pancreatic cancer fibroblast inhibitors. Cancer cells and fibroblasts were enriched as two distinct populations that fibroblasts dominated without inhibitors, and cancer cell growth prevailed after the addition of inhibitors (Figure 2). Tw...

Discussion

Both PDX and CDX models are vital platforms in the field of tumor biology²², and the critical step of a successful inter-species transplantation is to improve the survival of the xenograft.  Recently, some studies have shown that transient expression of BCL2L1 (BCL-XL) or BCL2 may significantly improve the viability of human embryonic stem cells in mice hosts without affecting the cell identities and fates^23,24

Materials

Name	Company	Catalog Number	Comments
DMEM	GIBCO	C11995500BT
FBS	Hyclone	sv30087.03
Y-27632	Cliniscience	Y0503	Rho kinase inhibitor
Primocin	invivogen	ant-pm-1	an antibiotic for primary cell cultures
Putrescine dihydrochloride	Sigma	P5780
Nicotinamide	Sigma	N3376
penicillin streptomycin	GIBCO	15140122.00
phosphate buffer (PBS)	GIBCO	C10010500CP
HBSS	GIBCO	14170112.00
collagenase type IV	GIBCO	17104019.00
hyaluronidase	Sigma	H3884
DnaseⅠ	Sigma	D5025
insulin	Sigma	I9278
b-FGF	GIBCO	PHG0264
EGF	GIBCO	PHG0314
pancreatic cancer fibroblasts inhibitor	CHI Scientific	FibrOUT
0.45 μm sterile filter	Millipore	SLHV033RB
concentration column	Millipore	Millipore UFC910008	Concentrate the virus
polybrene	Sigma	H9268
Hyaluronic Acid Sodium Salt	Sigma	H7630
L-glutamine	GIBCO	21051024.00
gemcitabine	Gemzan
methylcellulose	Sigma	M0262
Navitoclax(ABT-263)	Selleck	S1001	Bcl-xL inhibitor
Equipment
Microinjector	NARISHIGE
stereomicroscope	OLYMPUS	MVX10
Confocal Microscope	LEICA	SP8	0.00