Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 21, 2018; 24(7): 794-809
Published online Feb 21, 2018. doi: 10.3748/wjg.v24.i7.794
Translational pancreatic cancer research: A comparative study on patient-derived xenograft models
Mercedes Rubio-Manzanares Dorado, Luis Miguel Marín Gómez, Daniel Aparicio Sánchez, Sheila Pereira Arenas, Juan Manuel Praena-Fernández, Juan Jose Borrero Martín, Francisco Farfán López, Miguel Ángel Gómez Bravo, Jordi Muntané Relat, Javier Padillo Ruiz
Mercedes Rubio-Manzanares Dorado, Luis Miguel Marín Gómez, Daniel Aparicio Sánchez, Miguel Ángel Gómez Bravo, Javier Padillo Ruiz, Department of Hepatobiliary and Pancreatic Surgery, Virgen del Rocio University Hospital, Seville 41013, Spain
Sheila Pereira Arenas, Jordi Muntané Relat, Oncology Surgery, Cell Therapy, and Organ Transplantation Group, Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital, University of Seville, Seville 41013, Spain
Juan Manuel Praena-Fernández, Statistics, Methodology and Evaluation of Research Unit, Virgen del Rocio University Hospital, Seville 41013, Spain
Juan Jose Borrero Martín, Francisco Farfán López, Pathology Department, Virgen del Rocio University Hospital, Seville 41013, Spain
Author contributions: Rubio-Manzanares Dorado M, Marín Gómez LM, Aparicio Sánchez D, and Pereira Arenas S contributed substantially to the conception and design of the study and the acquisition of data; Praena-Fernández JM analysed and interpreted the data; Farfán López F and Borrero Martín JJ analysed the anatomopathological samples; Rubio-Manzanares Dorado M wrote the manuscript; and Padillo Ruiz J, Gómez Bravo MÁ and Muntané Relat J drafted the article and made critical revisions related to the intellectual content of the manuscript. All authors approved the final version of the manuscript to be published.
Supported by the Andalusian Public Foundation for the Management of Health Research in Seville (FISEVI).
Institutional review board statement: The study was reviewed and approved by the Ethics Committee of The Seville University of Medicine, Virgen del Rocío Universitary Hospital, Protocol number: CEEA-US2014-013/5.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exist.
Data sharing statement: Datasets are available from the corresponding author via email. Participants gave informed consent for data sharing.
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 which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (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:
Correspondence to: Mercedes Rubio-Manzanares Dorado, MD, PhD, Staff Physician, Department of Hepatobiliary and Pancreatic Surgery, Virgen del Rocio University Hospital, Manuel Siurot St., Seville 41013, Spain.
Telephone: +34-609-074420 Fax: +34-955-012317
Received: December 2, 2017
Peer-review started: December 2, 2017
First decision: December 20, 2017
Revised: January 14, 2018
Accepted: January 18, 2018
Article in press: January 18, 2018
Published online: February 21, 2018
Research background

Currently, a dozen experimental models are available. The molecular characteristics of these models can vary substantially with the morphology of the lesion, and thus, the selection of the model is not trivial. Multiple laboratories have established models of breast xenografts, head and neck cancer, and hepatocellular tumours that maintain the characteristics of the primary tumour from which they come. Some of these models have predicted the clinical response of a specific type of tumour to different chemotherapeutic agents. The main advantage of human tumour xenografts in the mouse is that they seem to preserve the pathological and immunohistochemical characteristics of the primary tumour, which may allow us to experiment with drugs with human-like pancreatic cancer models. However, the degree to which periampullary carcinoma implants and ductal adenocarcinoma (DAC) reflect the morphological and histological characteristics of their tumours of origin have been poorly studied and there are few publications on the subject.

Research motivation

Periampullary carcinomas, and especially DAC, are characterised by early vascular, lymphatic and perineural dissemination, so that some authors consider it to be a systemic disease from the beginning. This implies criteria of unresectability and justifies the ominous prognosis of the disease. At the time of diagnosis, 85% of patients present a macroscopic disease beyond the limits of the organ. The large epidemiological series approximate the incidence of pancreas cancer to their annual mortality. Given the unfortunate prognosis of this disease, the development of new chemotherapy drugs with systemic action that complement the local surgical treatment is fundamental. One of the main problems that researchers find in developing new molecules is the lack of animal models that faithfully reproduce the characteristics of human pancreatic cancer. Both the models developed in genetically modified mice (GEMM) and those induced by carcinogenic substances have facilitated the understanding at the molecular level and the appearance of new anti-tumour drugs with in vitro activity. Unfortunately, these treatment lines are often ineffective in humans.

The appearance of immunocompromised nude mice has allowed the resumption of animal models with human xenografts, whose main limitation was the high rejection rate. In this way, we can develop experimental models of human periampullary tumours that preserve the original genotypic and phenotypic characteristics.

The morphological and histological characteristics derived from the xenografts of pancreatic cancer in the experimental models have been poorly studied. In order to make animal models that are more similar to cancer in humans, we have developed this study.

Research objectives

Following this line of work, we have developed three experimental models through the use of xenografts: subcutaneous, intraperitoneal and pancreatic. The main objective of this study is to assess the viability of orthotopic (intrapancreatic) and heterotopic (intraabdominal and subcutaneous) xenografts of human pancreas cancers implanted in nude mice.

This work is part of a more ambitious line of research that in the future intends to identify molecules or combinations of these with the help of these models to rescue patients for surgery.

Research methods

A prospective experimental analytical follow-up of the development of tumours in mice upon implantation of human pancreatic adenocarcinoma samples was presents. Specimens surgically from patients with a pathological diagnosis of pancreas adenocarcinoma were obtained. Human cancer samples were implanted as tumour xenografts in three experimental models. The surgical samples were divided into five equally sized portions of 3 mm × 3 mm × 3 mm. Three specimens were used as tumour xenograft implants at the subcutaneous, pancreatic and intraperitoneal locations in nude mice. To date, no study comparing the implantation of a heterotopic pancreatic cancer xenograft with an orthotopic tumour xenograft has been published.

Histological analysis and immunohistochemical assessment of apoptosis, proliferation, angiogenesis and fibrogenesis were performed. When a tumour xenograft got the target size, it was re-implanted in a new nude mouse. Three sequential tumour xenograft generations (F1, F2 and F3) were generated.

Research results

The main findings of this study were: (1) the engraftment rate between mice was higher than the engraftment rate from human to mouse; (2) the subcutaneous model developed a higher number of implants, although there were no significant differences in favour of any model; and (3) the tumour xenograft of the three models maintained some human characteristics including differentiation and cell proliferation. While these tumour xenografts presented reduced fibrosis and fibrogenesis, two other features of pancreatic cancer, hypovascularisation and apoptosis, were enhanced.

The practical utility of tumour grafts needs to be addressed in terms of engraftment speed and reproducibility. In fact, tumour xenografts show a rather limited engraftment rate and slow tumour growth. However, those models are readily applicable for drug experiment because, once a xenograft has been successfully engrafted, it can be used after several freeze-thaw cycles and several passages.

Although these models can develop tumours that are very similar to humans, they are not an exact reflection of them. In this way, chemotherapeutic agents could be more effective in pancreatic PDX models than in human tumours. For this reason, new preclinical studies with chemotherapeutic agents are mandatory in those models.

Research conclusions

In the establishment of patient-derived xenograft models, samples of primary tumours were implanted in immunodepressed mice subcutaneously, intraperitoneally or orthotopically, with no intermediate step of in vitro propagation. Although the subcutaneous model is easy to perform through an incision with the scalpel on the back of the mouse, the microenvironment of the tumour is not exactly the same. We have considered one step further by designing a new intraperitoneal and pancreatic model that may reproduce the natural conditions of human pancreatic cancer. However, in our study, implanted subcutaneous xenografts maintain pathological and immunohistochemical characteristics of the primary tumour from which they derive similarly to the other two developed models.

To date, there has been no study on pancreatic cancer that has determined the best location to develop xenografts in animal models. In our study, the detection of tumour development is earlier in the subcutaneous model, which implies a lower cost compared to the other models. In addition, the subcutaneous model is the one with the highest number of viable xenografts developed throughout the different re-implantations. Taking into account that the three models developed have similar anatomopathological and immunohistochemical characteristics, the subcutaneous model could be the best option for the investigation of anticancer drugs with xenografts. However, more studies are needed to confirm this theory.

Research perspectives

The majority of works that strive to broaden our knowledge about the diagnosis and treatment of pancreas cancer, are based on xenografts from cell lines cultured in vitro. Chemotherapy agents which have good results in these experimental models do not have the same results when they are used in human tumours. In our case, we have established three models of pancreas tumours directly derived from patients and we have compared the morphological and immunological characteristics of the xenografts with the human tumours in order to establish which is the model that most faithfully reflected human tumour characteristics.

In our experience, due to the earliest development and the highest number of viable xenografts, as well as being the experimental model with the lowest morbidity, the subcutaneous model may be the best model for experimentation in pancreatic cancer.

Our intention is to select the best implant route in order to use these models in the future to detect biomarkers of pancreatic cancer and to develop specific chemotherapeutic regimens for each patient.