Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

Correction to: Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.

Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling.

Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

Targeted metabolomics detects a putatively diagnostic signature in plasma and dried blood spots from head and neck paraganglioma patients.

Head and neck paragangliomas (HNPGLs), rare chemoresistant tumors curable only with surgery, are strongly influenced by genetic predisposition, hence patients and relatives require lifetime follow-up with MRI and/or PET-CT because of de novo disease risk. This entails exposure to electromagnetic/ionizing radiation, costs, and organizational challenges, because patients and relatives are scattered far from reference centers. Simplified first-line screening strategies are needed. We employed flow injection analysis tandem mass spectrometry, as used in newborn metabolic screening, to compare the plasma metabolic profile of HNPGL patients (59 samples, 56 cases) and healthy controls (24 samples, 24 cases). Principal Component Analysis (PCA) and Partial Least Discriminant Analysis (PLS-DA) highlighted a distinctive HNPGL signature, likely reflecting the anaplerotic conversion of the TCA cycle to glutaminolysis and catabolism of branched amino acids, DNA damage and deoxyadenosine (dAdo) accumulation, impairment of fatty acid oxidation, switch towards the Warburg effect and proinflammatory lysophosphatidylcholines (LPCs) signaling. Statistical analysis of the metabolites that most impacted on PLS-DA was extended to 10 acoustic neuroma and 2 cholesteatoma patients, confirming significant differences relative to the HNPGL plasma metabolomic profile. The best confusion matrix from the ROC curve built on 2 metabolites, dAdo and C26:0-LPC, provided specificity of 94.29% and sensitivity of 89.29%, with positive and negative predictive values of 96.2% and 84.6%, respectively. Analysis of dAdo and C26:0-LPC levels in dried venous and capillary blood confirmed that dAdo, likely deriving from 2'-deoxy-ATP accumulated in HNPGL cells following endogenous genotoxic damage, efficiently discriminated HNPGL patients from healthy controls and acoustic neuroma/cholesteatoma patients on easily manageable dried blood spots.

Evolutionarily-Related Helicobacter pylori Genotypes and Gastric Intraepithelial Neoplasia in a High-Risk Area of Northern Italy.

Helicobacter pylori (Hp) is the major recognized risk factor for non-cardia gastric cancer (GC), but only a fraction of infected subjects develop GC, thus GC risk might reflect other genetic/environmental cofactors and/or differences in virulence among infectious Hp strains. Focusing on a high GC risk area of Northern Italy (Cremona, Lombardy) and using archived paraffin-embedded biopsies, we investigated the associations between the Hp vacA and cagA genotype variants and gastric intraepithelial neoplasia (GIN, 33 cases) versus non-neoplastic gastroduodenal lesions (NNGDLs, 37 cases). The glmM gene and the cagA and vacA (s and m) genotypes were determined by polymerase chain reaction (PCR) and sequencing. Hp was confirmed in 37/37 (100%) NNGDLs and detected in 9/33 GINs (27%), consistently with the well-known Hp loss in GC. CagA was detected in 4/9 Hp-positive GINs and in 29/37 NNGDLs. The vacA s1a and m1 subtypes were more common in GINs than in NNGDLs (6/7 vs. 12/34, p=0.014, for s1a; 7/7 vs. 18/34, p=0.020 for m1), with significant vacA s genotype-specific variance. The GIN-associated vacA s1a sequences clustered together, suggesting that aggressive Hp strains from a unique founder contribute to GC in the high-risk area studied.

A Developmental Perspective on Paragangliar Tumorigenesis.

In this review, we propose that paraganglioma is a fundamentally organized, albeit aberrant, tissue composed of neoplastic vascular and neural cell types that share a common origin from a multipotent mesenchymal-like stem/progenitor cell. This view is consistent with the pseudohypoxic footprint implicated in the molecular pathogenesis of the disease, is in harmony with the neural crest origin of the paraganglia, and is strongly supported by the physiological model of carotid body hyperplasia. Our immunomorphological and molecular studies of head and neck paragangliomas demonstrate in all cases relationships between the vascular and the neural tumor compartments, that share mesenchymal and immature vasculo-neural markers, conserved in derived cell cultures. This immature, multipotent phenotype is supported by constitutive amplification of NOTCH signaling genes and by loss of the microRNA-200s and -34s, which control NOTCH1, ZEB1, and PDGFRA in head and neck paraganglioma cells. Importantly, the neuroepithelial component is distinguished by extreme mitochondrial alterations, associated with collapse of the ΔΨm. Finally, our xenograft models of head and neck paraganglioma demonstrate that mesenchymal-like cells first give rise to a vasculo-angiogenic network, and then self-organize into neuroepithelial-like clusters, a process inhibited by treatment with imatinib.

Effects of dichloroacetate as single agent or in combination with GW6471 and metformin in paraganglioma cells.

Paragangliomas (PGLs) are infiltrating autonomic nervous system tumors that cause important morbidity. At present, surgery is the only effective therapeutic option for this rare tumor. Thus, new agents for PGL treatment should be identified. Using unique PGL cell models established in our laboratory, we evaluated the effect of dichloroacetate (DCA) as single agent or in a novel combination with other metabolic drugs, including GW6471 and metformin. DCA and metformin had not been tested before in PGL. DCA reduced PGL cell viability and growth through mechanisms involving reactivation of PDH complex leading to promotion of oxidative metabolism, with lowering of lactate and enhanced ROS production. This resulted in cell cycle inhibition and induction of apoptosis in PGL cells, as shown by flow cytometry and immunoblot analyses. Moreover, DCA drastically impaired clonogenic activity and migration of PGL cells. Also metformin reduced PGL cell viability as single agent and the combinations of DCA, GW6471 and metformin had strong effects on cell viability. Furthermore, combined treatments had drastic and synergistic effects on clonogenic ability. In conclusion, DCA, GW6471 and metformin as single agents and in combination appear to have promising antitumor effects in unique cell models of PGL.

Cytotoxic effect of a family of peroxisome proliferator-activated receptor antagonists in colorectal and pancreatic cancer cell lines.

Recent studies report an interesting role of peroxisome proliferator-activated receptor (PPAR) antagonists in different tumor models, being these compounds able to perturb metabolism and viability in cancer cells. In this work, the identification of a novel PPAR antagonist, showing inhibitory activity on PPARα and a weaker antagonism on PPARγ, is described. The activity of this compound and of a series of chemical analogues was investigated in selected tumor cell lines, expressing both PPARα and PPARγ. Data obtained show a dose-dependent cytotoxic effect of the novel PPAR antagonist in colorectal and pancreatic cancer models.

Interaction with culture medium components, cellular uptake and intracellular distribution of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts.

The mechanistic understanding of nanotoxicity requires the physico-chemical characterisation of nanoparticles (NP), and their comparative investigation relative to the corresponding ions and microparticles (MP). Following this approach, the authors studied the dissolution, interaction with medium components, bioavailability in culture medium, uptake and intracellular distribution of radiolabelled Co forms (CoNP, CoMP and Co(2+)) in Balb/3T3 mouse fibroblasts. Co(2+) first saturates the binding sites of molecules in the extracellular milieu (e.g., albumin and histidine) and on the cell surface. Only after saturation, Co(2+) is actively uptaken. CoNP, instead, are predicted to be internalised by endocytosis. Dissolution of Co particles allows the formation of Co compounds (CoNP-rel), whose mechanism of cellular internalisation is unknown. Co uptake (ranking CoMP > CoNP > Co(2+)) reached maximum at 4 h. Once inside the cell, CoNP spread into the cytosol and organelles. Consequently, massive amounts of Co ions and CoNP-rel can reach subcellular compartments normally unexposed to Co(2+). This could explain the fact that the nuclear and mitochondrial Co concentrations resulted significantly higher than those obtained with Co(2+).

Cytotoxicity and morphological transforming potential of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model.

We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co(2+)), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co(2+), but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co(2+). However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co(2+) released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co(2+) and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation.

Effects of palladium nanoparticles on the cytokine release from peripheral blood mononuclear cells of palladium-sensitized women.

OBJECTIVE: To investigate the effects of palladium (Pd) nanoparticles on cytokine release from peripheral blood mononuclear cells (PBMCs) of control or Pd-sensitized nonatopic women. METHODS: TNF-α, IL-5, IL-10 and IFN-γ release and/or expression from PBMCs incubated in presence of 5 to 10 nm Pd nanoparticles or Pd salt (potassium hexachloropalladate) were determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis. Transmission electronmicroscopy was performed. RESULTS: In lipopolysaccharide-stimulated PBMCs from controls, Pd salt inhibited IFN-γ and IL-10 release, whereas Pd nanoparticles enhanced IFN-γ release and inhibited TNF-α secretion. In lipopolysaccharide-stimulated PBMCs from Pd-sensitized women showing high IFN-γ release, Pd nanoparticles inhibited TNF-α release and Pd salt IL-10 release. TNF-α and IFN-γ release and messenger RNA expression were correlated. Transmission electronmicroscopy demonstrated uptake of nanoparticles in the endocytic compartment and activation of autophagy. CONCLUSIONS: Palladium ions and nanoparticles exert different effects in vitro on the expression and release of cytokines.