Nerve Sheath Tumor Cell Lines Research Articles

Establishment and characterization of a recurrent malignant peripheral nerve sheath tumor cell line: RsNF.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive and recurrent soft tissue sarcoma. It most commonly occurs secondary to neurofibromatosis type I, and it has a 5-year survival rate of only 8-13%. To better study the tumor heterogeneity of MPNST and to develop diverse treatment options, more tumor-derived cell lines are needed to obtain richer biological information. Here, we established a primary cell line of relapsed MPNST RsNF cells derived from a patient diagnosed with NF1 and detected the presence of NF1 mutations and SUZ12 somatic mutations through whole-exome sequencing(WES). Through tumor molecular marker targeted sequencing and single-cell transcriptome sequencing, it was found that chromosome 7 copy number variation (CNV) was gained in this cell line, and ZNF804B, EGFR, etc., were overexpressed on chromosome 7. Therefore, RsNF cells can be used as a useful tool in NF1-associated MPNST genomic amplification studies and to develop new therapeutic strategies.

Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.

Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.

Assessment of oncolytic HSV efficacy following increased entry-receptor expression in malignant peripheral nerve sheath tumor cell lines.

Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.

Aborted Autophagy and Nonapoptotic Death Induced by Farnesyl Transferase Inhibitor and Lovastatin

Exposure of the human malignant peripheral nerve sheath tumor cell lines STS-26T, ST88-14, and NF90-8 to nanomolar concentrations of both lovastatin and farnesyl transferase inhibitor (FTI)-1 but not to either drug alone induced cell death. ST88-14 and NF90-8 cells underwent apoptosis, yet dying STS-26T cells did not. FTI-1 cotreatment induced a strong and sustained autophagic response as indicated by analyses of microtubule-associated protein-1 light chain 3 (LC3)-II accumulation in STS-26T cultures. Extensive colocalization of LC3-positive punctate spots was observed with both lysosome-associated membrane protein (LAMP)-1 and LAMP-2 (markers of late endosomes/lysosomes) in solvent or FTI-1 or lovastatin-treated STS-26T cultures but very little colocalization in lovastatin/FTI-1-cotreated cultures. The absence of colocalization in the cotreatment protocol correlated with loss of LAMP-2 expression. Autophagic flux studies indicated that lovastatin/FTI-1 cotreatment inhibited the completion of the autophagic program. In contrast, rapamycin induced an autophagic response that was associated with cytostasis but maintenance of viability. These studies indicate that cotreatment of STS-26T cells with lovastatin and FTI-1 induces an abortive autophagic program and nonapoptotic cell death.

SOX9 expression increases with malignant potential in tumors from patients with neurofibromatosis 1 and is not correlated to desert hedgehog

We investigated the expression of SOX9, a transcription factor linked to the hedgehog pathways, and desert hedgehog, previously shown to be expressed in a malignant peripheral nerve sheath tumor cell line, in neurofibromatosis 1 tumors (neurofibromas and malignant peripheral nerve sheath tumor), and in a group of sporadic spindle cell sarcomas. We found that SOX9 was expressed in Schwann cells from neurofibromatosis 1 tumors, and was significantly up-regulated in malignant peripheral nerve sheath tumor versus neurofibromas at the protein level (P < .0001) and in malignant peripheral nerve sheath tumor and plexiform neurofibroma as compared to diffuse tumors at the mRNA level (P = .002 and P = .009, respectively). SOX9, however, was not a specific feature of malignant peripheral nerve sheath tumor because a significant proportion of spindle cell sarcomas unrelated to neurofibromatosis 1 were also positive. Interestingly, within neurofibromas, SOX9 expression correlated to the histology mostly found in plexiform areas, whereas it was negative or weak in diffuse neurofibroma (P < .0001). In sporadic sarcomas, the proportion of positive cells was actually heterogeneous among cases. Desert hedgehog expression was mostly found in sarcomas (P = .0002), but diffuse staining was only seen in non-neurofibromatosis 1 tumors, whereas malignant peripheral nerve sheath tumor displayed only focal expression. Finally, we found no significant correlation between the expression of SOX9 and desert hedgehog, and neither SOX9 nor desert hedgehog expression was correlated to the histoprognostic grade in sarcomas. Altogether, these results indicate that, although not a specific feature of neurofibromatosis 1 tumors, SOX9 may play a role in the development of malignant peripheral nerve sheath tumor in patients with neurofibromatosis 1 and does not appear to be linked to autocrine stimulation by the desert hedgehog signaling pathway.

Establishment and characterization of a novel human malignant peripheral nerve sheath tumor cell line, FMS-1, that overexpresses epidermal growth factor receptor and cyclooxygenase-2

Malignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma. We established a new human MPNST cell line (designated FMS-1) from MPNST of the right brachial plexus of a 69-year-old woman with NF1. The cell line has been maintained for >24 months with >100 passages. FMS-1 cells showed a fibrosarcoma-like or epithelioid pattern in the heterotransplanted tumor, compared with a fascicular growth pattern of short-spindle tumor cells in the primary tumor. Immunophenotypically, FMS-1 cells showed almost the same characteristics as the primary tumor. Cytogenetic and molecular analyses revealed a deletion in exons 5-8 of the p53 gene. Epidermal growth factor receptor (EGFR) and cyclooxygenase (COX)-2 were expressed in FMS-1 cells. To improve the highly aggressive course and poor prognosis and establish new therapeutic methods, molecular genetic and biological characterizations of MPNST are required. Thus, FMS-1 cells might be useful for investigating biological behaviors and developing new molecular-targeting antitumor drugs for MPNST expressing EGFR or COX-2.

Suppression of proliferation of two independent NF1 malignant peripheral nerve sheath tumor cell lines by the pan-ErbB inhibitor CI-1033

Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain malignancies, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable, and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases, and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2, and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.

Neuronal Differentiation of Synovial Sarcoma and Its Therapeutic Application

Synovial sarcoma is a rare sarcoma of unknown histologic origin. We previously reported the gene expression profile of synovial sarcoma was closely related to that of malignant peripheral nerve sheath tumors, and the fibroblast growth factor (FGF) signal was one of the main growth signals in synovial sarcoma. Here we further demonstrate the neural origin of synovial sarcoma using primary tumors and cell lines. The expression of neural tissue-related genes was confirmed in synovial sarcoma tumor tissues, but the expression of some genes was absent in synovial sarcoma cell lines. Treatment of synovial sarcoma cell lines with BMP4 or FGF2 enhanced or restored the expression of neural tissue-related genes and induced a neuron-like morphology with positive Tuj-1 expression. Treatment with all-trans-retinoic acid also induced the expression of neural tissue-related genes in association with growth inhibition, which was not observed in other cell lines except a malignant peripheral nerve sheath tumor cell line. A growth-inhibitory effect of all-trans-retinoic acid was also observed for xenografted tumors in athymic mice. The simultaneous treatment with FGF signal inhibitors enhanced the growth-inhibitory effect of all-trans-retinoic acid, suggesting the combination of growth signaling inhibition and differentiation induction could be a potential molecular target for treating synovial sarcoma.

Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication

Oncolytic herpes simplex viruses (oHSVs) are promising anticancer therapeutics. We sought to characterize the functional genomic response of human cancer cells to oHSV infection using G207, an oHSV previously evaluated in a phase I trial. Five human malignant peripheral nerve sheath tumor cell lines, with differing sensitivity to oHSV, were infected with G207 for 6 h. Functional genomic analysis of virus-infected cells demonstrated large clusters of downregulated cellular mRNAs and smaller clusters of those upregulated, including 21 genes commonly upregulated in all five lines. Of these, 7 are known to be HSV-1 induced and 14 represent novel virus-regulated genes. Gene ontology analysis revealed that a majority of G207-upregulated genes are involved in Janus kinase/signal transducer and activator of transcription signaling, transcriptional regulation, nucleic acid metabolism, protein synthesis and apoptosis. Ingenuity networks highlighted nodes for AP-1 subunits and interferon signaling via STAT1, suppressor of cytokine signaling-1 (SOCS1), SOCS3 and RANTES. As biological confirmation, we found that virus-mediated upregulation of SOCS1 correlated with sensitivity to G207 and that depletion of SOCS1 impaired virus replication by >10-fold. Further characterization of roles provided by oHSV-induced cellular genes during virus replication may be utilized to predict oncolytic efficacy and to provide rational strategies for designing next-generation oncolytic viruses.

Establishment of three malignant peripheral nerve sheath tumor cell lines, FU-SFT8611, 8710 and 9817: Conventional and molecular cytogenetic characterization

Malignant peripheral nerve sheath tumor (MPNST) is a rare malignant tumor, for which only a few cultured cell lines are available to date. In the present study, we established three new MPNST cell lines, FU-SFT8611, FU-SFT8710 and FU-SFT9817, from a 40-year-old Japanese man without neurofibromatosis 1 (NF1), a 43-year-old Japanese woman with NF1, and a 61-year-old Japanese woman without NF1, respectively. These cell lines were in culture for more than 3 years in vitro, and exhibit complex karyotypes lacking a common characteristic pattern. Two of the cell lines, FU-SFT8611 and FU-SFT9817, were tumorigenic in nude mice, with the resultant tumors showing an immunohistochemical phenotype similar to the original tumors. Comparative genomic hybridization analysis revealed that chromosomal imbalances were very similar between the original tumors and the established cell lines, but no consistent imbalances among the three cell lines as reported so far. As for chromosomal imbalances that have been reported to be associated with poor survival, FU-SFT8611 exhibited a gain of chromosome 17q22-qter, and FU-SFT9817 showed a loss of 17q22-qter, while gains of 17q24 and 7p15-p21 were observed in none of the cell lines. These newly established cell lines provide a valuable resource for biological and pathological investigations into new treatment regimes for MPNST.

Newly established Askin tumor cell line and overexpression of focal adhesion kinase in Ewing sarcoma family of tumors cell lines

Askin tumor is a malignant small round cell tumor that originates from the thoracopulmonary region and is a member of Ewing sarcoma family of tumors (ESFT). Only a few Askin tumor cell lines have been established. An Askin tumor cell line, designated MP-ASKIN-SA, was established from the left thoracic tumor of a 13-year-old Japanese boy. ESFT is known to have a high rate of distant metastases at diagnosis. The genes controlling the spread of ESFT cells, however, have not been elucidated. G-banding chromosome analysis revealed that the MP-ASKIN-SA cell line has complex chromosomal abnormalities including trisomy 8. The EWS/FLI1 chimeric transcript and c-myc overexpression were revealed by the reverse transcriptase-polymerase chain reaction and Northern blot analysis. Furthermore, we investigated the expression of the focal adhesion kinase ( FAK) gene in the ESFT cell lines using Northern blot analysis. In addition to the MP-ASKIN-SA cell line, six Ewing sarcoma cell lines, one peripheral nerve sheath tumor cell line, and two Askin tumor cell lines were analyzed. All ESFT cell lines, including MP-ASKIN-SA, expressed five- to twenty-eight-fold–increased values of FAK, as compared with fibroblasts obtained from the bone marrow of a healthy volunteer. These results raise the possibility that the overexpression of c-myc and FAK are involved in the poor prognosis of ESFT.