Research Article: Development of a DIPG Orthotopic Model in Mice Using an Implantable Guide-Screw System

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Miguel Marigil, Naiara Martinez-Velez, Pablo D. Domínguez, Miguel Angel Idoate, Enric Xipell, Ana Patiño-García, Marisol Gonzalez-Huarriz, Marc García-Moure, Marie-Pierre Junier, Hervé Chneiweiss, Elías El-Habr, Ricardo Diez-Valle, Sonia Tejada-Solís, Marta M. Alonso, Maria G Castro.

http://doi.org/10.1371/journal.pone.0170501

Abstract

In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system.

It consisted of a guide-screw also called bolt, a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy, antibodies or gene/viral therapies.

The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model.

Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons.

Partial Text

Diffuse Intrinsic Pontine Gliomas (DIPGs) represent the most frequent tumor among brainstem gliomas and constitute a real challenge for everyone devoted to the treatment of pediatric brain tumors[1,2]. Since its first description in the twentieth century, therapeutic alternatives remain scarce[3]. A dismal median overall survival between 9 to 13 months has remained unchanged in spite of combination of radiotherapy with targeted therapies. Unlike other brainstem tumors that benefit from surgical treatment such as focal pontine gliomas, exophytic, tectal or cervicomedullary tumors, DIPG due to its diffuse nature and anatomic extension within the pons, remains a fatal neoplasm[4]. Up to now the diagnosis was based on specific clinical symptoms and a characteristic radiographic appearance which usually shows a diffuse enlargement of the pons along with a variable and irregular contrast enhancement pattern. Thanks to the advent of biopsies which have led to an understanding of the genetic makeup of these tumors as well as generation of cell lines, several in vivo models have been recently developed including murine models that recapitulates the genotype of theses tumors[5–8] Most of these models employ stereotaxic-guided systems. The main advantage of using stereotaxy consists of precise access to the pons region throughout a biopsy needle aimed to a specific region in the brainstem according to previous standard coordinates. However, to perform studies with a high number of animals, such as survival studies with new therapeutic strategies or delivery of gene therapy agents, which need to be injected intratumorally, stereotaxy is extremely time-consuming, even in the hands of experienced researchers. In addition, if serial subsequent injections need to be performed there is the risk that they do not fall exactly in the same place.

Several years ago tissue sampling was not thought to be neither necessary nor suitable because of the potential mortality and morbidity associated with the biopsy process. The paucity of therapeutic alternatives led to redefine DIPG patients′ management in order to obtain a better understanding of the pathobiological pathways that would allow for more adequate treatment. Fortunately, the advent of technical breakthroughs, new biopsy protocols and interinstitutional collaborations has allowed a surge in research in this devastating disease including the development of several in vivo DIPG models [13–15].

In this work we developed a preclinical in vivo DIPG model based on a guide-screw system fixed over mice skull that is feasible and allows for reproducible DIPG tumor generation in a fast and consistent fashion. This system permits the use of a considerable amount of animals for experiment and allows for the subsequent intratumoral injection of different therapeutic agents.

 

Source:

http://doi.org/10.1371/journal.pone.0170501

 

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