Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma

BACKGROUND: The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31. METHODS: Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients. RESULTS: We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%-97%) compared to patients with higher values (40%, 95%CI, 12%-67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio. CONCLUSION: CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.We thank Professor Franco Locatelli for critical reading this paper and for his suggestions. We would also like to thank the children ’ s parents, who gave their informed consent for publication and “Il cuore grande di Flavio ”Onlus. Dr. Marta Colletti is a post-doctoral fellow of the Umberto Veronesi Founda- tion. To Valentina Polcini for proofreading.S

Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas

Background: 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. Results: 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. Conclusions: 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

Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2–9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow–liver–thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal—in combination with appropriate tools for systemic clearance of persistent HIV infection—greatly increases opportunities for HIV eradication

An Update on Circular RNA in Pediatric Cancers

Circular RNAs (circRNAs) are a class of single-stranded closed noncoding RNA molecules which are formed as a result of reverse splicing of mRNAs. Despite their relative abundance, only recently there appeared an increased interest in the understanding of their regulatory importance. Among their most relevant characteristics are high stability, abundance and evolutionary conservation among species. CircRNAs are implicated in several cellular functions, ranging from miRNA and protein sponges to transcriptional modulation and splicing. Additionally, circRNAs’ aberrant expression in pathological conditions is bringing to light their possible use as diagnostic and prognostic biomarkers. Their use as indicator molecules of pathological changes is also supported by their peculiar covalent closed cyclic structure which bestows resistance to RNases. Their regulatory role in cancer pathogenesis and metastasis is supported by studies involving human tumors that have investigated different expression profiles of these molecules. As endogenous competitive RNA, circRNAs can regulate tumor proliferation and invasion and they arouse great consideration as potential therapeutic biomarkers and targets for cancer. In this review, we describe the most recent findings on circRNAs in the most common pediatric solid cancers (such as brain tumors, neuroblastomas, and sarcomas) and in more rare ones (such as Wilms tumors, hepatoblastomas, and retinoblastomas)

Recent Advancements on the Use of Exosomes as Drug Carriers for the Treatment of Glioblastoma

Glioblastoma (GBM) is the most common and aggressive cancer of the brain. Presently, GBM patients have a poor prognosis, and therapy primarily aims to extend the life expectancy of affected patients. The current treatment of GBM in adult cases and high-grade gliomas in the pediatric population involves a multimodal approach that includes surgical resection followed by simultaneous chemo/radiotherapy. Exosomes are nanoparticles that transport proteins and nucleic acids and play a crucial role in mediating intercellular communication. Recent evidence suggests that these microvesicles may be used as biological carriers and offer significant advantages in targeted therapy. Due to their inherent cell-targeting properties, circulation stability, and biocompatibility, exosomes are emerging as promising new carriers for drugs and biotherapeutics. Furthermore, these nanovesicles are a repository of potential diagnostic and prognostic markers. In this review, we focus on the therapeutic potentials of exosomes in nano-delivery and describe the latest evidence of their use as a therapeutic tool in GBM

MicroRNAs in neuroblastoma: Biomarkers with therapeutic potential

Background: Neuroblastoma is the most common extracranial solid tumor in infancy. The majority of children have a disseminated disease at diagnosis with bone marrow as the most common site of metastasis. Although several prognostic factors have been defined (i.e. age, stage, histology, recurrent genetic anomalies), the identification of non-invasive biomarkers for disease follow-up and therapy monitoring is indeed still a clinical need. Aberrant regulation of microRNAs (miRNAs) expression has been implicated in several malignancies. Objectives: In this mini-review, we describe the recent findings about miRNAs in neuroblastoma, both in the tumor and circulation, with particular focus on those involved in tumor progression and drug resistance. Furthermore, we will discuss the use of specific miRNAs as potential therapeutic tools in this tumor. Results: Several miRNAs have been identified to be down- or up-regulated in primary tumors and have been associated with MYCN amplification, differentiation, dissemination and chemoresistance. Little evidence is available in the literature about circulating miRNAs which are of particular interest as potential biomarkers for liquid biopsy. Conclusion: Identification of body-fluid markers for non-invasive diagnosis, risk tratification, treatment monitoring and tumor follow-up, is gaining growing interest, especially in the pediatric field. miRNAs are suitable candidates as biomarkers in neuroblastoma but further investigations are needed to expand knowledge regarding their role in this malignancy to design specific approaches of miRNAs-mediated therapies

Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity

Proteomics of Aqueous Humor as a Source of Disease Biomarkers in Retinoblastoma

Aqueous humor (AH) can be easily and safely used to evaluate disease-specific biomarkers in ocular disease. The aim of this study was to identify specific proteins biomarkers in the AH of retinoblastoma (RB) patients at various stages of the disease. We analyzed the proteome of 53 AH samples using high-resolution mass spectrometry. We grouped the samples according to active vitreous seeding (Group 1), active aqueous seeding (Group 2), naive RB (group 3), inactive RB (group 4), and congenital cataracts as the control (Group 5). We found a total of 889 proteins in all samples. Comparative parametric analyses among the different groups revealed three additional proteins expressed in the RB groups that were not expressed in the control group. These were histone H2B type 2-E (HISTH2B2E), InaD-like protein (PATJ), and ubiquitin conjugating enzyme E2 V1 (UBE2V1). Upon processing the data of our study with the OpenTarget Tool software, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and CD44 were more highly expressed in the RB groups. Our results provide a proteome database regarding AH related to RB disease that may be used as a source of biomarkers. Further prospective studies should validate our finding in a large cohort of RB patients