Novel mediator in anaphylaxis: decreased levels of miR-375-3p in serum and within extracellular vesicles of patients.

Introduction: Anaphylaxis is among the most severe manifestations of allergic disorders, but its molecular basis remains largely unknown and reliable diagnostic markers are not currently available. MicroRNAs (miRNAs) regulate several pathophysiological processes and have been proposed as non-invasive biomarkers. Therefore, this study aims to evaluate their involvement in anaphylactic reaction and their value as biomarkers. Methods: Acute (anaphylaxis) and baseline (control) serum samples from 67 patients with anaphylaxis were studied. Among them, 35 were adults with drug-induced anaphylaxis, 13 adults with food-induced anaphylaxis and 19 children with food-induced anaphylaxis. The circulating serum miRNAs profile was characterized by next-generation sequencing (NGS). For this purpose, acute and baseline samples from 5 adults with drug-induced anaphylaxis were used. RNA was extracted, retrotranscribed, sequenced and the readings obtained were mapped to the human database miRBase_20. In addition, a system biology analysis (SBA) was performed with its target genes and revealed pathways related to anaphylactic mediators signaling. Moreover, functional and molecular endothelial permeability assays were conducted with miR-375-3p-transfected cells in response to cAMP. Results: A total of 334 miRNAs were identified, of which 21 were significant differentially expressed between both phases. Extracellular vesicles (EVs) were characterized by Western blot, electron microscopy and NanoSight. A decrease of miR-375-3p levels was determined by qPCR in both serum and EVs of patients with anaphylaxis (****p<.0001). Precisely, the decrease of miR-375-3p correlated with the increase of two inflammatory cytokines: monocyte chemoattractant protein-1 (MCP-1) and granulocyte macrophage colony-stimulating factor (GM-CSF). On the other hand, functional and molecular data obtained showed that miR-375-3p partially blocked the endothelial barrier maintenance and stabilization by disassembly of cell-cell junctions exhibiting low Rac1-Cdc42 levels. Discussion: These findings demonstrate a differential serum profile of circulating miRNAs in patients with anaphylaxis and exhibit the miR-375-3p modulation in serum and EVs during drug- and food-mediated anaphylactic reactions. Furthermore, the in silico and in vitro studies show a negative role for miR-375-3p/Rac1-Cdc42 in the endothelial barrier stability.

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.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

Establishment and Characterization of a Cell Line (S-RMS1) Derived from an Infantile Spindle Cell Rhabdomyosarcoma with SRF-NCOA2 Fusion Transcript.

Background: Spindle cell rhabdomyosarcoma (S-RMS) is a rare tumor that was previously considered as an uncommon variant of embryonal RMS (ERMS) and recently reclassified as a distinct RMS subtype with NCOA2, NCOA1, and VGLL2 fusion genes. In this study, we established a cell line (S-RMS1) derived from a four-month-old boy with infantile spindle cell RMS harboring SRF-NCOA2 gene fusion. Methods: Morphological and molecular characteristics of S-RMS1 were analyzed and compared with two RMS cell lines, RH30 and RD18. Whole genome sequencing of S-RMS1 and clinical exome sequencing of genomic DNA were performed. Results: S-RMS1 showed cells small in size, with a fibroblast-like morphology and positivity for MyoD-1, myogenin, desmin, and smooth muscle actin. The population doubling time was 3.7 days. Whole genome sequencing demonstrated that S-RMS1 retained the same genetic profile of the tumor at diagnosis. A Western blot analysis showed downregulation of AKT-p and YAP-p while RT-qPCR showed upregulation of endoglin and GATA6 as well as downregulation of TGFßR1 and Mef2C transcripts. Conclusion: This is the first report of the establishment of a cell line from an infantile spindle cell RMS with SRF-NCOA2 gene fusion. S-RMS1 should represent a useful tool for the molecular characterization of this rare and almost unknown tumor.

Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

A Chart Review on the Feasibility and Safety of the Vincristine Irinotecan Pazopanib (VIPaz) Association in Children and Adolescents With Resistant or Relapsed Sarcomas.

Background: Pediatric patients with relapsed or refractory sarcomas have poor outcome and need novel therapies that provide disease control while maintaining an acceptable quality of life. The safety of vincristine, irinotecan, and pazopanib (VIPaz) association has not yet been published in this population. Methods: A chart review was conducted in children and adolescents with relapsed or refractory bone and soft tissue sarcomas who received VIPaz in our institution. Results: One hundred sixty-six patients with a diagnosis of soft or bone sarcoma were admitted to our hospital in the period between March 2015 and August 2018, 30 were relapsed or resistant. Seventeen out of 30 resistant or relapsed patients (median age, 14 years) received 114 VIPaz cycles (median six cycles per patient, range 1-17). Sixteen courses (15%) resulted in gastrointestinal toxicity with Grade two diarrhea; 35 courses (30%) resulted in Grade ≥3 neutropenia. One patient presented Grade two hypothyroidism after nine courses, and another one had Grade two hyperbilirubinemia after 12 courses. Two and five patients required a 25% dose reduction of irinotecan (because of diarrhea) and pazopanib (because of neutropenia four and hyperbilirubinemia 1), respectively. No patient experienced heart failure, hypertension, nor posterior reversible encephalopathy syndrome. Pneumothorax was not reported in any case even in lung metastatic patients. After two and four VIPaz cycles, we observed one complete response (CR), five partial responses (PRs), seven stable diseases (SDs), and four progressive diseases (PDs). With a median follow-up of 15 months (range 3-32), five out of 17 (29%) patients were alive, and four patients were in continuous CR after 12 VIPaz cycles. Conclusions: The VIPaz regimen might be a safe option in children and adolescents with relapsed or refractory sarcomas otherwise unable to be enrolled in other clinical trials; on the other hand, the efficacy of pazopanib observed cannot be sustained from the current study.

Proteomic Profiling of Retinoblastoma-Derived Exosomes Reveals Potential Biomarkers of Vitreous Seeding.

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids.

Circulating Biomarkers for Tumor Angiogenesis: Where Are We?

BACKGROUND: In recent years, several anti-angiogenic drugs have been developed and their addition to standard treatment has been associated with clinical benefits. However, the response to anti-angiogenic therapy is characterized by considerable variability. In this context, the development of dynamic non-invasive biomarkers would be helpful to elucidate the emergence of anti-angiogenic resistance as well as to correctly address the treatment. OBJECTIVES: The purpose of this review is to describe current reports on circulating diagnostic and prognostic biomarkers related to angiogenesis. We further discuss how this non-invasive strategy could improve the monitoring of tumor treatment and help clinical strategy. RESULTS: We discuss the latest evidence in the literature regarding circulating anti-angiogenic markers. Besides growth factor proteins, different circulating miRNAs could exert a pro- or anti-angiogenic activity so as to represent suitable candidates for a non-invasive strategy. Recent reports indicate that tumor-derived exosomes, which are small membrane vesicles abundant in biological fluids, also have an impact on vascular remodeling. CONCLUSION: Numerous circulating biomarkers related to angiogenesis have been recently identified. Their use will allow identifying patients who are more likely to benefit from a specific anti-angiogenic treatment, as well as detecting those who will develop resistance and/or adverse effects. Nonetheless, further studies are required to elucidate the role of these biomarkers in clinical settings.

Exosomal MiRNAs in Pediatric Cancers.

MicroRNAs (miRNAs) have generated great attention in oncology as they play a fundamental role in the regulation of gene expression and their aberrant expression is present in almost all types of tumors including pediatric ones. The discovery that miRNAs can be transported by exosomes, which are vesicles of 40-120 nm involved in cellular communication, that are produced by different cell types, and that are present in different biological fluids, has opened the possibility of using exosomal miRNAs as biomarkers. The possibility to diagnose and monitor the progression and response to drugs through molecules that can be easily isolated from biological fluids represents a particularly important aspect in the pediatric context where invasive techniques are often used. In recent years, the idea of liquid biopsy as well as studies on the possible role of exosomal miRNAs as biomarkers have developed greatly. In this review, we report an overview of all the evidences acquired in recent years on the identification of exosomal microRNAs with biomarker potential in pediatric cancers. We discuss the following herein: neuroblastoma, hepatoblastoma, sarcomas (osteosarcoma, Ewing's sarcoma and rhabdoid tumors, and non-rhabdomyosarcoma soft tissue sarcoma), brain tumors, lymphomas, and leukemias.

Expression profiles of exosomal miRNAs isolated from plasma of patients with desmoplastic small round cell tumor.

AIM: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive mesenchymal tumor, lacking biomarkers for diagnosis, treatment stratification and prognosis. We investigated the exosomal miRNA profile in plasma samples collected from DSRCT patients, evaluating their potential as circulating biomarkers for this tumor. PATIENTS & METHODS: We isolated exosomes from plasma of three DSRCT adolescents and four age-matched healthy controls; expression of circulating miRNAs was quantified by qPCR. RESULTS: We identified 55 miRNAs significantly modulated compared with healthy controls. Among these miRNAs, 14 were highly dysregulated in at least one patient and 5 were expressed in all patients. CONCLUSION: To our knowledge, this is the first report describing exosomal miRNAs as promising biomarkers to characterize disease status in DSRCT patients.

Congenital Rhabdomyosarcoma: a different clinical presentation in two cases.

BACKGROUND: Rhabdomyosarcoma (RMS), one of the most common soft tissue sarcomas of childhood, is very rare in the neonatal period (0.4-2% of cases). In order to gain a deeper understanding of this disease at such age, patient and tumor features, as well as treatment modality and outcome need to be reported. CASE PRESENTATION: We describe two cases with congenital RMS treated at Bambino Gesù Children's Hospital between 2000 and 2016. They represent only 2.24% of all RMS patients diagnosed during that period in our Institution; this data is in agreement with the incidence reported in the literature. They reflect the two different clinical forms in which the disease may manifest itself. One patient, with the alveolar subtype (positive for specific PAX3-FOXO1 fusion transcript) and disseminated disease, had a fatal outcome with central nervous system (CNS) progression despite conventional and high dose chemotherapy. The other child, with the localized embryonal subtype, was treated successfully with conservative surgery and conventional chemotherapy, including prolonged maintenance therapy. He is disease free at 7 years of follow-up. CONCLUSIONS: RMS can also be diagnosed during the neonatal period. Given the young age, disease management is often challenging, and especially for the alveolar subtype, the outcome is dismal despite intensified multimodality therapy. In fact, it characteristically manifests with multiple subcutaneous nodules and progression most commonly occurs in the CNS (Rodriguez-Galindo et al., Cancer 92(6):1613-20, 2001). In this context, CNS prophylaxis could play a role in preventing leptomeningeal dissemination, and molecular studies can allow a deeper tumor characterization, treatment stratification and identification of new potential therapeutic targets.

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.

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker.

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population.

BACKGROUND: The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). METHODS: Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. RESULTS: MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. CONCLUSIONS: Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

HGF Modulates Actin Cytoskeleton Remodeling and Contraction in Testicular Myoid Cells.

The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-ß activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII-VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-ß have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in vitro in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1) the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2) the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-ß, the secretion of this factor was also evaluated. We found that both uPA and TGF-ß activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-ß activation and myoid cell contraction are abrogated by uPA or Met inhibitor administration.

Expression and functional activity of PACAP and its receptors on cumulus cells: effects on oocyte maturation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation.