The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma ...(DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that ...primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion.
A comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis.
In conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases.
The hypermethylation of CpG islands within gene promoter regions is an epigenetic phenomenon that is often, but not always, associated with the transcriptional silencing of downstream genes and ...contributes to carcinogenesis. We have determined the pattern of methylation of several genes involved in distinct biological pathways, including cell proliferation and apoptosis, in neuroblastoma and in the nonmalignant ganglioneuroma. The purpose of this work was to search for epigenetic signatures that could be associated with defined clinical and biological parameters and that, in prospective, could identify specific risk categories among the patients. We have analysed 31 malignant neuroblastoma with or without MYCN amplification and 13 benign ganglioneuroma and we have observed dramatic differences in the methylation pattern of five genes (CASP8, 14.3.3sigma, DeltaN-p73, RASSF1A and DCR2) between these tumors indicating that this phenomenon is not tissue-specific and can be considered as cancer-dependent. Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any ...gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth
and
. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.
Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. ...The coordinate methylation of many loci in cancer is defined as ‘CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose
The purpose of this study was to determine whether specific
HOXA
epigenetic signatures could differentiate glioma with distinct biological, pathological, and clinical characteristics.
Methods
...We evaluated
HOXA3
,
7
,
9,
and
10
methylation in 63 glioma samples by MassARRAY and pyrosequencing.
Result
s
We demonstrated the direct statistical correlation between the level of methylation of all
HOXA
genes examined and WHO grading. Moreover, in glioblastoma patients, higher level of
HOXA9
and
HOXA10
methylation significantly correlated with increased survival probability (
HOXA9
—HR: 0.36,
P
= 0.007;
HOXA10
—HR: 0.46,
P
= 0.045; combined
HOXA9
and
10
—HR 0.28,
P
= 0.004).
Conclusions
This study identifies
HOXA3
,
7
,
9,
and
10
as methylation targets mainly in high-grade glioma and hypermethylation of the
HOXA9
and
10
as prognostic factor in glioblastoma patients. Our data indicate that these epigenetic changes may be biomarkers of clinically different subgroups of glioma patients that could eventually benefit from personalized therapeutic strategies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Extraventricular neurocytoma (EVN) is an extremely rare tumor of neuroglial origin with a tendency toward ganglionic or glial differentiation. In the 2016 World Health Organization Classification, ...EVN was classified as a grade II tumor and described as a neoplasm with good outcome. However, the presence of cellular atypia is an important unfavorable prognostic factor. Here, we describe the first case of a patient with a congenital EVN localized in the brainstem. After a sub-total resection, his disease rapidly progressed despite several chemotherapies, including molecular targeting approaches. He died 13 months after diagnosis. In conclusion, we report an atypical case of EVN presenting an extremely aggressive behavior, despite the absence of cellular atypia. The brainstem origin and the age of the patient may have represented two important prognostic factors for our patient.
The aim of our study was to identify threshold levels of DNA methylation predictive of the outcome to better define the risk group of stage 4 neuroblastic tumor patients. Quantitative pyrosequencing ...analysis was applied to a training set of 50 stage 4, high risk patients and to a validation cohort of 72 consecutive patients. Stage 4 patients at lower risk and ganglioneuroma patients were included as control groups. Predictive thresholds of methylation were identified by ROC curve analysis. The prognostic end points of the study were the overall and progression‐free survival at 60 months. Data were analyzed with the Cox proportional hazard model. In a multivariate model the methylation threshold identified for the SFN gene (14.3.3σ) distinguished the patients presenting favorable outcome from those with progressing disease, independently from all known predictors (Training set: Overall Survival HR 8.53, p = 0.001; Validation set: HR 4.07, p = 0.008). The level of methylation in the tumors of high‐risk patients surviving more than 60 months was comparable to that of tumors derived from lower risk patients and to that of benign ganglioneuroma. Methylation above the threshold level was associated with reduced SFN expression in comparison with samples below the threshold. Quantitative methylation is a promising tool to predict survival in neuroblastic tumor patients. Our results lead to the hypothesis that a subset of patients considered at high risk—but displaying low levels of methylation—could be assigned at a lower risk group.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogenous tumours comprised of distinct cell sub-populations co-existing within the same tumour mass. We have shown that ...primary patient-derived sub-clones, as well as optical-barcoded sub-clones, function as an interconnected network conferring an aggressive phenotype. Here, we explored the role of exosomes in mediating PDHGG inter-clonal communication. A comprehensive characterization of 7 optical-barcoded single cell-derived clones obtained from two patient-derived cell lines (one DMGH3K27-altered and one diffuse high-grade paediatric-type glioma H3WT), confirmed extensive genomic and phenotypic heterogeneity. Live single-cell tracking in 3D migration and invasion assays demonstrated the key role of the inter-clonal crosstalk in driving a more aggressive phenotype. To determine the exosome role in this crosstalk, we first characterised them in terms of size, marker expression and cargo. Moreover, we demonstrated that exosomes were actively internalized by the sub-clones. Exosomal proteomic analysis showed differential protein contents implicated in the regulation of biological processes such as focal adhesion and extracellular matrix organization. The analysis of exosomal miRNome did not show differentially expressed miRNAs between sub-clones, however, specific and distinct exosomal miRNAs were found uniquely expressed by each sub-clone. The abrogation of the exosome biogenesis by GW4869 phospholipase inhibitor did not affect sub-clones viability, but significantly inhibited their motility, when cultured individually and more prominently in co-culture condition. Analysis of the exo-miRNAs uniquely expressed by the sub-clones highlighted a set of target genes regulating cell motility/invasion/migration. These target genes were differentially expressed when sub-clones were co-cultured compared to mono-culture. Moreover, the expression levels of these genes (e.g. CD44, PTRZ, GLI3, NTRK2) were significantly modulated upon GW4869 treatments. In conclusion, our study highlights the importance of the exosomes in the inter-clonal communication and suggests that interfering with the exosome biogenesis may be a valuable strategy to inhibit cell motility in PDHGG.
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