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

Valutazione patologica della progressione di PN-MPNST nel modello murino 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.

Autore in primo piano: Modelli murini geneticamente modificati e caratterizzazione patologica dei tumori associati alla neurofibromatosi di tipo 1

Identificazione, diagnosi e classificazione dei tumori maligni della guaina dei nervi periferici in modelli murini geneticamente modificati

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.

Verifica dell'identità delle cellule MPNST di passaggio precoce e dell'allotrapianto di cellule tumorali di passaggio precoce per dimostrare la tumorigenicità

Per iniziare, posizionare i vetrini coprioggetti sterili nei pozzetti delle piastre di coltura tissutale a sei pozzetti e versare un volume adeguato di poli-L-lisina e soluzione laminata nei pozzetti per coprire il vetrino coprioggetto. Quindi, sigilla la piastra con pellicola trasparente per ridurre l'evaporazione e incuba a quattro gradi Celsius per una notte. La mattina successiva, lavare i vetrini coprioggetti tre volte con PBS sterile.Aggiungere due millilitri di sospensione cellulare e terreno di coltura alla piastra 10.000 cellule in ciascun pozzetto contenente il vetrino coprioggetto. Dopo aver lavato i vetrini coprioggetti con PBS, fissare le celle con una soluzione di paraformaldeide al 4% e PBS per 18 minuti. Sciacquare i vetrini coprioggetti tre volte con PBS per cinque minuti prima dell'immunocolorazione.Incubare i vetrini coprioggetti immunocolorati e lavati in uno o cinque microgrammi di colorante Hoechst per 10 minuti. Quindi lavare i vetrini coprioggetti con PBS per altri cinque minuti. Montare i vetrini utilizzando un rapporto uguale di PBS e miscela di glicerolo e acquisire le immagini utilizzando un microscopio a fluorescenza.Trattare le cellule con una soluzione di dissociazione cellulare non enzimatica per 30 secondi a un minuto. Quindi aggiungere cinque millilitri di DMEM per ogni millilitro del reagente di dissociazione cellulare non enzimatica. Conta le cellule usando un emocitometro.Centrifugare le celle a 5G per cinque minuti. Risospendere le cellule ad una concentrazione di una o due volte 10 delle sesta cellule per 100 microlitri di DMEM. Posizionare l'animale a pancia in giù e sterilizzare il sito di iniezione con etanolo al 70%.Iniettare una o due volte 10 delle sesta cellule per via sottocutanea nel fianco destro. Metti il topo da solo in una gabbia senza lettiera e lascialo riprendere. Per prevenire l'ipotermia, posizionare una parte della gabbia sul termoforo.Vengono mostrate immagini a bassa e alta potenza di cellule MPNST P0 GGF beta 3 di passaggio iniziale. Si è scoperto che le cellule sono tumoregeniche, formando colonie in agar molle e innesti in topi immunodeficienti.

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 ...

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

Identificazione di neurofibromi e tumori maligni della guaina dei nervi periferici (MPNST) nei topi P 0-GGFβ3

Preparing Hematoxylin and Eosin-Stained Sections of Grossly Visible Tumors

Preparazione di sezioni colorate con ematossilina ed eosina di tumori grossolanamente visibili

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

Identificazione di potenziali neurofibromi plessiformi (PN) e tumori maligni della guaina dei nervi periferici (MPNST) mediante l'esecuzione di colorazioni speciali per confermare le diagnosi