Use of zebrafish embryos as avatar of patients with pancreatic cancer: A new xenotransplantation model towards personalized medicine

Gregorio Di Franco, Alice Usai, Niccola Funel, Matteo Palmeri, Ida Elena Rosamaria Montesanti, Matteo Bianchini, Desirée Gianardi, Niccolò Furbetta, Simone Guadagni, Enrico Vasile, Alfredo Falcone, Luca Emanuele Pollina, Vittoria Raffa, Luca Morelli, Gregorio Di Franco, Alice Usai, Niccola Funel, Matteo Palmeri, Ida Elena Rosamaria Montesanti, Matteo Bianchini, Desirée Gianardi, Niccolò Furbetta, Simone Guadagni, Enrico Vasile, Alfredo Falcone, Luca Emanuele Pollina, Vittoria Raffa, Luca Morelli

Abstract

Background: The response to chemotherapy treatment of patients with pancreatic ductal adenocarcinoma (PDAC) is difficult to predict and the identification of patients who most likely will benefit from aggressive chemotherapy approaches is crucial. The concept of personalized medicine has emerged in the last years with the objective to tailor the medical treatment to the individual characteristics of each patient, and particularly to the tumor biology of each patient. The need for in-vivo xenotransplantation models for cancer patients has increased exponentially, and for this reason zebrafish avatars have gained popularity. Preliminary studies were conducted also with PDAC tissue.

Aim: To develop a simple, not expensive, diffusible zebrafish embryo model as avatar for patients affected by PDAC.

Methods: Tumor tissue was taken from the surgical specimen by the histopathologist. After its fragmentation into small pieces, they are stained with CM-Dil. Small pieces of stained tissue were transplanted into the yolk of wt AB zebrafish embryos with a glass capillary needle. Embryos were incubated at 35 °C in E3 medium supplemented with 1% Pen/Strep in the presence or absence of drugs for the following days in respect of the treatment plan (Gemcitabine; Gemcitabine and Oxaliplatin; Gemcitabine and nab-Paclitaxel; 5-Fluorouracil and Folinic acid and Oxaliplatin and Irinotecan). The response of zebrafish xenografts to the chemotherapy options has been analyzed by monitoring the fluorescent stained area at 2 h post injection (hpi), 1 d and 2 d post injection (dpi). In each time point, the mean size of the stained area was measured by ImageJ and it was normalized with respect to the 1 dpi time point mean relative tumor area (RTA). We evaluated the effect of the chemotherapy exposition comparing the mean RTA of each treated subgroup and the control group and evaluating the percentage reduction of the mean RTA by comparing each treated subgroup with the control group.

Results: Between July 2018 and October 2019, a total of 15 patients with pancreatic cancer were prospectively enrolled. In all cases, it was possible to take a fragment of the tumor from the surgical specimen for the xenotransplantation in the zebrafish embryos. The histological examination confirmed the presence of a PDAC in all cases. In absence of chemotherapy (control group), over time the Dil-stained area showed a statistically significant increase in all cases. A statistically significant reduction of the mean RTA in the treated subgroups for at least one chemotherapy scheme was reported in 6/15 (40%) cases. The analysis of the percentage reduction of the RTA in treated subgroups in comparison to the control group revealed the presence of a linear relationship in each subgroup between the percentage reduction of the RTA and the number of cases reporting each percentage threshold considered for the analysis.

Conclusion: Our model seems to be effective for the xenotransplantation of PDAC tissue and evaluation of the effect of each chemotherapy scheme on the xenotransplanted tumor tissue.

Keywords: Avatar of oncological patients; Chemotherapy efficacy; Pancreatic ductal adenocarcinoma; Personalized medicine; Xenotransplantation; Zebrafish embryos.

Conflict of interest statement

Conflict-of-interest statement: The authors declare that there is no conflict of interest.

©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.

Figures

Figure 1
Figure 1
Protocol used for the evaluation of the chemotherapy drugs efficacy. A piece of pancreatic ductal adenocarcinoma tumor tissue is injected into the yolk sac of zebrafish embryos 2 dpf. At 2 h post injection (hpi) the embryos are imaged and exposed to chemotherapy for 2 d. A and B: On the right panel a representative image of control embryos after 2 hpi and 2 dpi; C and D: On the right panel a representative image of treated embryos after 2 hpi and 2 dpi. Qualitatively images show the increased of fluorescent area in control group versus the regression in xenotransplanted embryos treated with chemotherapy.
Figure 2
Figure 2
Tumor tissue was successfully engrafted in healthy zebrafish embryos in all cases. A: Surgical specimen was obtained from patient with diagnosed pancreatic ductal adenocarcinoma (PDAC); B: Two fragments of the tumor were taken; C and D: One of them was used for histological evaluation (C) revealing that the percentage of epithelial cells (mean PDAC counterpart) out of the total surface area is 31.8% ± 4.9% (n = 3) (D); E: The second fragment of the tumor was used for xenotransplantation into the yolk of zebrafish embryos 2 dpf. After 2 d post xenotransplantation, the zebrafish patient-derived xenografts (zPDX) are Formalin-Fixed Paraffin Embedded. We performed the hematoxylin and eosin staining and immunohistochemistry using anti-Human Pan-Cytokeratin antibody on zPDX sections, highlighting the presence of epithelial PDAC cells (orange arrows) and the immune-negative counterpart (black arrows) that might be associated with the microenvironment side of PDAC human tissue. PDAC: Pancreatic ductal adenocarcinoma; PDX: Patient-derived xenografts; PanCK: Pan-Cytokeratin antibody.
Figure 3
Figure 3
Representative hematoxylin and eosin stained sections of zebrafish patient-derived xenografts. Visible morphologic alteration of the human pancreatic ductal adenocarcinoma nuclei (orange arrows) in zebrafish patient-derived xenografts exposed to Gemcitabine + nab-Paclitaxel and 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan could be associated with a chemotherapy damage. Control group shows normal nuclei (black arrows). GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.
Figure 4
Figure 4
Cases with a statistically significant reduction of the mean relative tumor area for at least one chemotherapy scheme (the code below each diagram corresponds to the case number). Results are expressed as average ± SEM, aP < 0.05, 1-way ANOVA followed by Dunnett’s multiple comparisons test. Control group shows normal nuclei (black arrows). GEM: Gemcitabine; GEMOX: Gemcitabine + Oxaliplatin; GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.
Figure 5
Figure 5
Percentage of cases with a percentage reduction of mean relative tumor area equal or greater to each threshold value. RTA: relative tumor area; Control group shows normal nuclei (black arrows). k: Control group; GEM: Gemcitabine; GEMOX: Gemcitabine + Oxaliplatin; GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.

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