verifying the identity of early-passage mpnst cells and allograft of early-passage tumor cells to demonstrate tumorigenicity

Avaliação patológica da progressão de PN-MPNST no modelo de camundongo P 0-GGFβ3

Over the last three decades, numerous laboratories have attempted to create mouse models of human cancers by introducing human cancer-associated mutations into the mouse genome or by overexpressing a gene product that is overexpressed in human cancers. The resulting genetically engineered mouse (GEM) models can be used for a variety of purposes such as establishing that the newly introduced genomic modification initiates tumorigenesis, identifying other subsequently occurring genetic or epigenetic changes that contribute to tumor progression, and defining the key signaling pathways that drive tumor initiation and progression. Unlike orthotopic xenograft models, which rely on the use of immunodeficient mice, GEM cancer models have a fully functional immune system and so more accurately model responses to candidate therapeutic agents. However, when using GEM cancer models for purposes such as these, it is essential that investigators confirm that observations made with GEM neoplasms are relevant to their human counterparts. This validation should include a thorough assessment of the pathology of the GEM neoplasms and a determination as to whether the pathologic features of the GEM neoplasms recapitulate the pathology of the corresponding human tumor type.
The tumor susceptibility syndrome neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the human nervous system, occurring in approximately 1 in every 3,000-3,500 live births1,2,3. Individuals afflicted with NF1 develop multiple benign peripheral nerve sheath tumors known as neurofibromas in their skin (dermal neurofibromas) and in large nerves and nerve plexuses (plexiform neurofibromas). While both dermal and plexiform neurofibromas worsen the patient&#39;s quality of life by producing physical, behavioral, and/or social impairment, plexiform neurofibromas (PNs) are particularly dangerous4,5.&#160;This is because PNs frequently transform into malignant peripheral nerve sheath tumors (MPNSTs), which are aggressive spindle cell neoplasms with an exceptionally low survival rate1,2. In large part, this low survival rate is because the radio- and chemotherapeutic regimens that are currently used to treat MPNSTs are ineffective. However, developing new, more effective therapies has been challenging. This is because, despite how commonly MPNSTs occur in NF1 patients, they are still rare neoplasms. As a result, it is very difficult to obtain large numbers of human tumors for study; it is also challenging to recruit enough patients with MPNSTs for clinical trials. To overcome these limitations, several GEM models have been generated with the goal of gaining further insights into the abnormalities driving neurofibroma pathogenesis and PN-MPNST progression and to facilitate preclinical trials with candidate therapeutic agents.
NF1 patients have inactivating mutations in one copy of the NF1 gene. Neurofibroma pathogenesis is triggered when an inactivating mutation in the remaining functional NF1 gene occurs in a cell in the Schwann cell lineage. Surprisingly, however, when mice were generated with germline inactivating Nf1 mutations, they did not develop neurofibromas6,7. The subsequent demonstration that mice with Nf1-null Schwann cells and Nf1 haploinsufficiency in all other cell types (Krox20-Cre;Nf1flox/- mice) developed plexiform neurofibromas suggested that reduced Nf1 gene dosage in additional cell types was required for neurofibroma pathogenesis8. Even then, the plexiform neurofibromas in Krox20-Cre;Nf1flox/- mice did not progress to become MPNSTs and so only partially mimicked the biology of their human counterparts. MPNST pathogenesis did occur when Nf1 mutations were partnered with mutations in additional tumor suppressor genes such as Trp539 or Cdkn2a10, but MPNSTs in these GEM models developed de novo or from atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs)11,12, rather than from preexisting benign plexiform neurofibromas (see13,14 for excellent reviews of these models as well as other models introducing additional MPNST-associated loss of function mutations in genes such as Suz12 and Pten15).
These mouse models have been invaluable for establishing the role that genes such as NF1, TP53, and CDKN2A play in the pathogenesis of NF1-associated peripheral nervous system neoplasms and for preclinical trials testing candidate therapeutic agents. However, we still have an incomplete understanding of the process by which plexiform neurofibromas progress to become atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs16) and then MPNSTs. Some progress has recently been made in understanding this process with the recent report that mice with deletions in Nf1 and Arf develop ANNUBPs that progress to become MPNSTs11. However, Nf1 mutation-based mouse models that fully recapitulate the process of plexiform neurofibroma-MPNST progression seen in humans do not yet exist. In addition, it is not clear whether there are multiple distinct pathways that lead to the development of MPNSTs. Given this, it is possible that the GEMs described above only model a subset of several different pathways that lead to neurofibroma-MPNST progression and MPNST pathogenesis. This point is emphasized by the fact that MPNSTs also occur sporadically and that some sporadic MPNSTs apparently do not have NF1 mutations17,18.
Although this latter point has been challenged by Magollon-Lorenz et al.&#39;s recent suggestion that at least some sporadic MPNSTs lacking NF1 mutations are melanomas or a different type of sarcoma19, we have recently reported a sporadic MPNST and a cell line derived from this tumor (2XSB cells) that was NF1 wild-type20. During the characterization of the parent tumor and the 2XSB cell line, we systematically ruled out alternative diagnostic possibilities, including melanoma and the multiple other sarcoma types that are routinely considered in the differential diagnosis of a sporadic malignant peripheral nerve sheath tumor20. In addition, we note that Magollon-Lorenz et al. acknowledged that their findings in the three sporadic MPNST cell lines that they studied could not be generalized to indicate that all tumors identified as sporadic MPNSTs are not MPNSTs.
To construct a GEM model in which neurofibroma and MPNST pathogenesis were not necessarily dependent upon specific tumor suppressor gene mutations, we generated transgenic mice in which overexpression of the potent Schwann cell mitogen neuregulin-1 (NRG1) was driven by the Schwann cell-specific myelin protein zero (P0) promoter (P0-GGF&#946;3 mice)21. We have previously shown that human neurofibromas, MPNSTs, and MPNST cell lines express several NRG1 isoforms together with the erbB receptor tyrosine kinases (erbB2, erbB3, and erbB4) that mediate NRG1 signaling and that these erbB receptors are constitutively activated22. We have also demonstrated that pharmacologic inhibitors of the erbB kinases potently inhibit MPNST proliferation22, survival23, and migration24. In keeping with our observations in humans, P0-GGF&#946;3 mice develop plexiform neurofibromas25 that progress to become MPNSTs at a high frequency21,25. We have shown that P0-GGF&#946;3 MPNSTs, like their human counterparts, commonly develop mutations of Trp53 and Cdkn2a, as well as numerous other genomic abnormalities that potentially contribute to tumorigenesis25. MPNSTs arising in P0-GGF&#946;3 mice do not have inactivating Nf1 mutations. However, using genetic complementation, we showed that NRG1 promotes tumorigenesis in P0-GGF&#946;3 mice predominantly through the same signaling cascades that are altered by Nf1 loss26; this conclusion is based on our finding that substituting NRG1 overexpression for Nf1 loss in the presence of Trp53 haploinsufficiency (P0-GGF&#946;3;Trp53+/- mice) produces animals in which MPNSTs develop de novo, as is seen in cis-Nf1+/-;Trp53+/- mice27.
To obtain this and other information demonstrating that P0-GGF&#946;3 mice accurately model the processes of neurofibroma pathogenesis and neurofibroma-MPNST progression seen in humans with NF1, we have developed specialized methodologies for processing tissues from these animals, accurately diagnosing their tumors, grading the MPNSTs arising in these mice, establishing and characterizing early-passage P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- MPNST cultures and critically comparing the pathology of P0-GGF&#946;3 PNs and MPNSTs and P0-GGF&#946;3;Trp53+/- MPNSTs to that of their human counterparts. Many of these methodologies are generalizable to other GEM models of nervous system neoplasia. Additionally, several of these methodologies are more broadly applicable to GEM models in which neoplasms arise in other organ sites. Consequently, here we present a detailed description of these methodologies.

Autor em destaque: Modelos de camundongos geneticamente modificados e caracterização patológica de tumores associados à neurofibromatose tipo 1

Identificação, diagnóstico e classificação de tumores malignos da bainha do nervo periférico em modelos de camundongos geneticamente modificados

Our group is focused on neurofibromatosis type one and understanding the pathology of the cancers associated with the disease, plexiform neurofibromas and malignant peripheral nerve sheath tumors. To do so, we employ several techniques, including the use of a genetically engineered mouse model, P0GGF beta three, Our lab and others studying plexiform neurofibromas and malignant peripheral nerve sheath tumors face similar challenges. Limited access to patient-derived tumor samples hinders our work.As a result, creating and characterizing genetically engineered mouse models is crucial for ongoing research into disease mechanisms and the potential development of treatments. Identifying peripheral nerve tumors can be tricky and are often misdiagnosed as other cancers. To avoid this, we have developed a detailed protocol using histology, immunohistochemistry, and grading to accurately identify and grade these tumors.This improves the reliability of future research studies. Our work provides a framework to investigate and validate genetically engineered mouse model derived tumors to ensure they properly mimic the human disease. We will use current genetically engineered mouse models to understand tumor microenvironment and the factors essential to tumor transformation.What causes plexiform neurofibromas to progress to malignant peripheral tumors.

Verificando a Identidade de Células MPNST de Passagem Precoce e Aloenxerto de Células Tumorais de Passagem Precoce para Demonstrar Tumorigenicidade

Para começar, coloque lamínulas estéreis nos poços de placas de cultura de tecidos de seis poços e despeje um volume adequado de Poli-L-lisina e solução laminada nos poços para cobrir a lamínula. Em seguida, sele a placa com filme plástico para reduzir a evaporação e incube a quatro graus Celsius durante a noite. Na manhã seguinte, lave as tampas três vezes com PBS estéril.Adicione dois mililitros de suspensão celular e meio de crescimento à placa 10.000 células em cada poço contendo a lamínula. Após lavar as lamínulas com PBS, fixar as células com uma solução de paraformaldeído a 4% e PBS por 18 minutos. Enxaguar as lamínulas três vezes com PBS por cinco minutos antes da imunomarcação.Incubar as lamínulas imunocoradas e lavadas em um a cinco microgramas de corante Hoechst por 10 minutos. Em seguida, lave as tampas com PBS por mais cinco minutos. Monte as lâminas usando uma proporção igual de PBS e mistura de glicerol e capture as imagens usando um microscópio fluorescente.Tratar as células com uma solução de dissociação celular não enzimática durante 30 segundos a um minuto. Em seguida, adicione cinco mililitros de DMEM para cada mililitro do reagente de dissociação celular não enzimática. Conte células usando um hemocitômetro.Centrifugar as células no 5G por cinco minutos. Ressuspender as células em uma concentração de uma a duas vezes 10 das sextas células por 100 microlitros de DMEM. Coloque o animal de bruços e esterilize o local da injeção com etanol 70%.Injetar uma a duas vezes 10 das sextas células por via subcutânea no flanco direito. Coloque o rato sozinho numa gaiola sem roupa de cama e deixe-o recuperar. Para evitar a hipotermia, coloque uma parte da gaiola sobre a almofada de aquecimento.Imagens de baixa e alta potência de células MPNST P0 GGF beta 3 de passagem precoce são mostradas. As células mostraram-se tumorogênicas, formando colônias em ágar mole e enxertos em camundongos imunodeficientes.

Research

JoVE Journal

Cancer Research

Verifying the Identity of Early-Passage MPNST Cells and Allograft of Early-Passage Tumor Cells to Demonstrate Tumorigenicity

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that ...

Pesquisa do câncer

Identifying Neurofibromas and Malignant Peripheral Nerve Sheath Tumors (MPNSTs) in P0-GGF&#946;3 Mice

Identificação de neurofibromas e tumores malignos da bainha do nervo periférico (MPNSTs) em camundongos P 0-GGFβ3

Preparing Hematoxylin and Eosin-Stained Sections of Grossly Visible Tumors

Preparação de seções coradas com hematoxilina e eosina de tumores grosseiramente visíveis

Identifying Potential Plexiform Neurofibromas (PNs) and Malignant Peripheral Nerve Sheath Tumors (MPNSTs) by Performing Special Stains to Confirm Diagnoses

Identificação de potenciais neurofibromas plexiformes (NPs) e tumores malignos da bainha nervosa periférica (MPNSTs) através da realização de colorações especiais para confirmar diagnósticos