Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 7, 2020; 26(21): 2792-2809
Published online Jun 7, 2020. doi: 10.3748/wjg.v26.i21.2792
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, Matteo Palmeri, Matteo Bianchini, Desirée Gianardi, Niccolò Furbetta, Simone Guadagni, Luca Morelli, General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy
Alice Usai, Ida Elena Rosamaria Montesanti, Vittoria Raffa, Department of Biology, University of Pisa, Pisa 56127, Italy
Niccola Funel, Luca Emanuele Pollina, Division of Surgical Pathology, Department of Laboratory of Medicine, Hospital-University of Pisa, Pisa 56124, Italy
Enrico Vasile, Division of Medical Oncology, Pisa University Hospital, Pisa 56124, Italy
Alfredo Falcone, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy
Luca Morelli, EndoCAS (Center for Computer Assisted Surgery), University of Pisa, Pisa 56124, Italy
Author contributions: Di Franco G and Usai A contributed equally to this work; Raffa V and Morelli L contributed equally to this work; Di Franco G, Usai A, Funel N, Palmeri M, Bianchini M, Gianardi D, Furbetta N, Guadagni S, Vasile E, Falcone A, Pollina LE, Raffa V and Morelli L contributed to the study conception and design; Di Franco G, Usai A, Funel N, Palmeri M, Montesanti IER, Bianchini M, Gianardi D, Furbetta N, Guadagni S, Pollina LE and Enrico Vasile E contributed to the data acquisition; Di Franco G, Usai A, Funel N, Palmeri M, Montesanti IER, Bianchini M, Gianardi D, Furbetta N, Guadagni S, Raffa V and Morelli L contributed to the data analysis and interpretation; Di Franco G, Usai A, Funel N, Palmeri M, Montesanti IER, Bianchini M, Gianardi D, Furbetta N, Guadagni S and Vasile E drafted the manuscript; Falcone A, Pollina LE, Raffa V and Morelli L contributed to the critical revision; all authors made the study final approval.
Supported by the Fondazione Pisa, No. 114/16.
Institutional review board statement: The study was approved by Ethics committee of “Area Vasta Nord Ovest (CEAVNO)” (protocol number 70213).
Institutional animal care and use committee statement: Zebrafish (Danio Rerio) were handled in compliance with local animal welfare regulations (authorization n. 99/2012-A, 19.04.2012) and standard protocols approved by Italian Ministry of Public Health, in conformity with the Directive 2010/63/EU.
Conflict-of-interest statement: The authors declare that there is no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Luca Morelli, FACS, MD, Associate Professor, Surgeon, General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via Paradisa 2, Pisa 56124, Italy. luca.morelli@unipi.it
Received: December 31, 2019
Peer-review started: December 31, 2019
First decision: February 24, 2020
Revised: March 27, 2020
Accepted: May 27, 2020
Article in press: May 27, 2020
Published online: June 7, 2020
ARTICLE HIGHLIGHTS
Research background

The response of patients with pancreatic ductal adenocarcinoma (PDAC) to the different chemotherapy schemes is difficult to predict. The concept of precision medicine has emerged recently with the objective to tailor the medical treatment to the individual characteristics of each patient, and particularly to the tumor biology of each patient.

Research motivation

Recently, the use of zebrafish as avatar for oncological patients has gained popularity. However, only preliminary studies were conducted with patient-derived pancreatic cancer cells or tissue.

Research objectives

The aim of this study is to evaluate the usability of zebrafish embryos as a model possibly simple, not expensive and diffusible, that could be used as avatar for patients affected by PDAC, to predict the efficacy of the different chemotherapy schemes and the clinical response to the treatment.

Research methods

A fragment of the tumor was taken from the surgical specimen and sectioned into about 3 mm3 pieces for DiI staining. Small pieces of stained tissue were xenotransplanted into the yolk of n = 100 zebrafish embryos 2 dpf. The zebrafish xenografts were incubated at 35°C with the presence or absence of drugs (GEM, GEMOX, GEM/nab-P, FOLFOXIRI) dissolved in fish water, using the equivalent dose (ED = 5). Firstly, we compared the mean relative tumor area (RTA) between each treated subgroup and the control group. Secondly, we evaluated the percentage reduction of the mean RTA (PRmRTA) in each treated subgroup in comparison to the control group, evaluating the presence of a linear relationship between each threshold value of the PRmRTA and the number of cases reporting a PRmRTA equal or greater to each threshold value. Using the linear regression line equation, we calculated for each protocol the expected percentage RTA reduction with the following formula: Expected percentage reduction of the mean RTA = (percentage of PR reported in literature – qlinear regression line equation)/mlinear regression line equation. For each chemotherapy protocol, we calculated the mean value of the expected PRmRTA and compared each other’s.

Research results

In the control group the Dil-stained areas showed a statistically significant increase over time in all cases, while a tendency to a reduction of the mean RTA was observed in treated subgroups, with a statistically significant reduction of the mean RTA for at least one chemotherapy scheme in 6/15 (40%) cases. The presence of a linear relation between the percentage reduction of the RTA and the number of cases reporting at least each percentage threshold was observed in each subgroup. The mean conversion factor was -60.4% for FOLFOXIRI, -59.3% for GEM-nab/P, -62.4% for GEMOX, and -63.7% for GEM (P = 0.626).

Research conclusions

This study provides a simple, reliant and not expensive PDAC patients-derived xenograft model for the rapid pre-clinical evaluation of the efficacy of different chemotherapy schemes available for the treatment of each individual PDAC patient’s case.

Research perspectives

Our model seems to reflect the clinical response rate reported in literature. However, to determinate the possible capability of our model in predicting the efficacy of the chemotherapy scheme administered to patients with PDAC, it is necessary to evaluate the correlation between these data and the real clinical response to adjuvant chemotherapy on patients. A co-clinical trial (NCT00070213) is underway in our institution and will be object of further publication.