Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

Teneurins: Role in Cancer and Potential Role as Diagnostic Biomarkers and Targets for Therapy.

Teneurins have been identified in vertebrates as four different genes (TENM1-4), coding for membrane proteins that are mainly involved in embryonic and neuronal development. Genetic studies have correlated them with various diseases, including developmental problems, neurological disorders and congenital general anosmia. There is some evidence to suggest their possible involvement in cancer initiation and progression, and drug resistance. Indeed, mutations, chromosomal alterations and the deregulation of teneurins expression have been associated with several tumor types and patient survival. However, the role of teneurins in cancer-related regulatory networks is not fully understood, as both a tumor-suppressor role and pro-tumoral functions have been proposed, depending on tumor histotype. Here, we summarize and discuss the literature data on teneurins expression and their potential role in different tumor types, while highlighting the possibility of using teneurins as novel molecular diagnostic and prognostic biomarkers and as targets for cancer treatments, such as immunotherapy, in some tumors.

Sparsely Connected Autoencoders: A Multi-Purpose Tool for Single Cell omics Analysis.

BACKGROUND: Biological processes are based on complex networks of cells and molecules. Single cell multi-omics is a new tool aiming to provide new incites in the complex network of events controlling the functionality of the cell. METHODS: Since single cell technologies provide many sample measurements, they are the ideal environment for the application of Deep Learning and Machine Learning approaches. An autoencoder is composed of an encoder and a decoder sub-model. An autoencoder is a very powerful tool in data compression and noise removal. However, the decoder model remains a black box from which is impossible to depict the contribution of the single input elements. We have recently developed a new class of autoencoders, called Sparsely Connected Autoencoders (SCA), which have the advantage of providing a controlled association among the input layer and the decoder module. This new architecture has the benefit that the decoder model is not a black box anymore and can be used to depict new biologically interesting features from single cell data. RESULTS: Here, we show that SCA hidden layer can grab new information usually hidden in single cell data, like providing clustering on meta-features difficult, i.e. transcription factors expression, or not technically not possible, i.e. miRNA expression, to depict in single cell RNAseq data. Furthermore, SCA representation of cell clusters has the advantage of simulating a conventional bulk RNAseq, which is a data transformation allowing the identification of similarity among independent experiments. CONCLUSIONS: In our opinion, SCA represents the bioinformatics version of a universal "Swiss-knife" for the extraction of hidden knowledgeable features from single cell omics data.

MET Exon 14 Skipping: A Case Study for the Detection of Genetic Variants in Cancer Driver Genes by Deep Learning.

BACKGROUND: Disruption of alternative splicing (AS) is frequently observed in cancer and might represent an important signature for tumor progression and therapy. Exon skipping (ES) represents one of the most frequent AS events, and in non-small cell lung cancer (NSCLC) MET exon 14 skipping was shown to be targetable. METHODS: We constructed neural networks (NN/CNN) specifically designed to detect MET exon 14 skipping events using RNAseq data. Furthermore, for discovery purposes we also developed a sparsely connected autoencoder to identify uncharacterized MET isoforms. RESULTS: The neural networks had a Met exon 14 skipping detection rate greater than 94% when tested on a manually curated set of 690 TCGA bronchus and lung samples. When globally applied to 2605 TCGA samples, we observed that the majority of false positives was characterized by a blurry coverage of exon 14, but interestingly they share a common coverage peak in the second intron and we speculate that this event could be the transcription signature of a LINE1 (Long Interspersed Nuclear Element 1)-MET (Mesenchymal Epithelial Transition receptor tyrosine kinase) fusion. CONCLUSIONS: Taken together, our results indicate that neural networks can be an effective tool to provide a quick classification of pathological transcription events, and sparsely connected autoencoders could represent the basis for the development of an effective discovery tool.

Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases.

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.

SeqBox: RNAseq/ChIPseq reproducible analysis on a consumer game computer.

Summary: Short reads sequencing technology has been used for more than a decade now. However, the analysis of RNAseq and ChIPseq data is still computational demanding and the simple access to raw data does not guarantee results reproducibility between laboratories. To address these two aspects, we developed SeqBox, a cheap, efficient and reproducible RNAseq/ChIPseq hardware/software solution based on NUC6I7KYK mini-PC (an Intel consumer game computer with a fast processor and a high performance SSD disk), and Docker container platform. In SeqBox the analysis of RNAseq and ChIPseq data is supported by a friendly GUI. This allows access to fast and reproducible analysis also to scientists with/without scripting experience. Availability and implementation: Docker container images, docker4seq package and the GUI are available at http://www.bioinformatica.unito.it/reproducibile.bioinformatics.html. Contact: beccuti@di.unito.it. Supplementary information: Supplementary data are available at Bioinformatics online.

Reproducible bioinformatics project: a community for reproducible bioinformatics analysis pipelines.

BACKGROUND: Reproducibility of a research is a key element in the modern science and it is mandatory for any industrial application. It represents the ability of replicating an experiment independently by the location and the operator. Therefore, a study can be considered reproducible only if all used data are available and the exploited computational analysis workflow is clearly described. However, today for reproducing a complex bioinformatics analysis, the raw data and the list of tools used in the workflow could be not enough to guarantee the reproducibility of the results obtained. Indeed, different releases of the same tools and/or of the system libraries (exploited by such tools) might lead to sneaky reproducibility issues. RESULTS: To address this challenge, we established the Reproducible Bioinformatics Project (RBP), which is a non-profit and open-source project, whose aim is to provide a schema and an infrastructure, based on docker images and R package, to provide reproducible results in Bioinformatics. One or more Docker images are then defined for a workflow (typically one for each task), while the workflow implementation is handled via R-functions embedded in a package available at github repository. Thus, a bioinformatician participating to the project has firstly to integrate her/his workflow modules into Docker image(s) exploiting an Ubuntu docker image developed ad hoc by RPB to make easier this task. Secondly, the workflow implementation must be realized in R according to an R-skeleton function made available by RPB to guarantee homogeneity and reusability among different RPB functions. Moreover she/he has to provide the R vignette explaining the package functionality together with an example dataset which can be used to improve the user confidence in the workflow utilization. CONCLUSIONS: Reproducible Bioinformatics Project provides a general schema and an infrastructure to distribute robust and reproducible workflows. Thus, it guarantees to final users the ability to repeat consistently any analysis independently by the used UNIX-like architecture.

Chimeric DNA Vaccines: An Effective Way to Overcome Immune Tolerance.

The fact that cancer immunotherapy is considered to be a safe and successful weapon for use in combination with surgery, radiation, and chemotherapy treatments means that it has recently been chosen as Breakthrough of the Year 2013 by Science editors. Anticancer vaccines have been extensively tested, in this field, both in preclinical cancer models and in the clinic. However, tumor-associated antigens (TAAs) are often self-tolerated molecules and cancer patients suffer from strong immunosuppressive effects, meaning that the triggering of an effective anti-tumor immune response is difficult. One possible means to overcome immunological tolerance to self-TAAs is of course the use of vaccines that code for xenogeneic proteins. However, a low-affinity antibody response against the self-homologous protein expressed by cancer cells is generally induced by xenovaccination. This issue becomes extremely limiting when working with tumors in which the contribution of the humoral rather than the cellular immune response is required if tumor growth is to be hampered. A possible way to avoid this problem is to use hybrid vaccines which code for chimeric proteins that include both homologous and xenogeneic moieties. In fact, a superior protective anti-tumor immune response against ErbB2+ transplantable and autochthonous mammary tumors was observed over plasmids that coded for the fully rat or fully human proteins when hybrid plasmids that coded for chimeric rat/human ErbB2 protein were tested in ErbB2 transgenic mice. In principle, these findings may become the basis for a new rational means of designing effective vaccines against TAAs.

Strengths and Weaknesses of Pre-Clinical Models for Human Melanoma Treatment: Dawn of Dogs' Revolution for Immunotherapy.

Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed. The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models. For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated. The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized. However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics. In this panorama, the concept of comparative oncology acquires a priceless value. The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.

Recombinant human lactoferrin induces human and mouse dendritic cell maturation via Toll-like receptors 2 and 4.

Lactoferrin, a key component of innate immunity, is a cationic monomeric 80-kDa glycoprotein of the transferrin superfamily. Recombinant human lactoferrin, known as talactoferrin (TLF), induces a distinct functional maturation program in human dendritic cells (DCs) derived from peripheral blood monocytes. However, the receptors and molecular mechanisms involved in this induction have not been fully determined. By exploiting genome-wide transcription profiling of immature DCs, TNF-α- and IL-1ß-matured DCs (m-DCs), and TLF-matured DCs (TLF-DCs), we have detected a set of transcripts specific for m-DCs and one specific for TLF-DCs. Functional network reconstruction highlighted, as expected, the association of m-DC maturation with IL-1ß, TNF-α, and NF-κB, whereas TLF-DC maturation was associated with ERK and NF-κB. This involvement of ERK and NF-κB transduction factors suggests direct involvement of Toll-like receptors (TLRs) in TLF-induced maturation. We have used MyD88 inhibition and siRNA silencing TLRs on human DCs and mouse TLR-2-knockout cells, to show that TLF triggers the maturation of both human and mouse DCs through TLR-2 and TLR-4.

Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers. RESULTS: We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-ras(LA1)/p53(R172HΔg) and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-ras(LA1)/p53(R172HΔg) tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-ras(LA1)/p53(R172HΔg) mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive. CONCLUSIONS: Our study provided a complete transcription overview of the K-ras(LA1)/p53(R172HΔg) mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

miR-135b coordinates progression of ErbB2-driven mammary carcinomas through suppression of MID1 and MTCH2.

In an attempt to reveal deregulated miRNAs associated with the progression of carcinomas developed in BALB-neuT transgenic mice, we found increased expression of miR-135b during malignancy. Relevantly, we observed that miR-135b is up-regulated in basal or normal-like human breast cancers, and it correlates with patient survival and early metastatization. Therefore, we investigated its biological functions by modulating its expression (up- or down-regulation) in mammary tumor cells. Although no effect was observed on proliferation in cell culture and in orthotopically injected mice, miR-135b was able to control cancer cell stemness in a mammosphere assay, anchorage-independent growth in vitro, and lung cancer cell dissemination in mice after tail vein injections. Focusing on the miR-135b molecular mechanism, we observed that miR-135b controls malignancy via its direct targets, midline 1 (MID1) and mitochondrial carrier homolog 2 (MTCH2), as proved by biochemical and functional rescuing/phenocopying experiments. Consistently, an anti-correlation between miR-135b and MID1 or MTCH2 was found in human primary tumor samples. In conclusion, our research led us to the identification of miR-135b and its targets, MID1 and MTCH2, as relevant coordinators of mammary gland tumor progression.

The noninflammatory role of high mobility group box 1/Toll-like receptor 2 axis in the self-renewal of mammary cancer stem cells.

Cancer stem cells (CSCs) are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, the identification of molecules involved in CSC self-renewal is a necessary step toward more effective therapies. To this aim, through the transcription profiling of the murine ErbB2(+) tumor cell line TUBO vs. derived CSC-enriched mammospheres, Toll-like receptor 2 (TLR2) was identified as 2-fold overexpressed in CSCs, as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA-MB-231 (50%), HCC1806 (60%), and MCF7 (50%) cells. In CSC, TLR2 was activated by endogenous high-mobility-group box 1 (HMGB1), inducing IκBα phosphorylation, IL-6 and TGFß secretion, and, consequently, STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2-fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively, these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self-renewal, tumorigenesis, and metastatic ability. These findings, while providing evidence against the controversial use of TLR2 agonists in antitumor therapy, lay out new paths toward the design of anticancer treatments.

A single cell RNAseq benchmark experiment embedding "controlled" cancer heterogeneity.

Single-cell RNA sequencing (scRNA-seq) has emerged as a vital tool in tumour research, enabling the exploration of molecular complexities at the individual cell level. It offers new technical possibilities for advancing tumour research with the potential to yield significant breakthroughs. However, deciphering meaningful insights from scRNA-seq data poses challenges, particularly in cell annotation and tumour subpopulation identification. Efficient algorithms are therefore needed to unravel the intricate biological processes of cancer. To address these challenges, benchmarking datasets are essential to validate bioinformatics methodologies for analysing single-cell omics in oncology. Here, we present a 10XGenomics scRNA-seq experiment, providing a controlled heterogeneous environment using lung cancer cell lines characterised by the expression of seven different driver genes (EGFR, ALK, MET, ERBB2, KRAS, BRAF, ROS1), leading to partially overlapping functional pathways. Our dataset provides a comprehensive framework for the development and validation of methodologies for analysing cancer heterogeneity by means of scRNA-seq.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening.

Background: Personalized disease models are crucial for assessing the specific response of diseased cells to drugs, particularly novel biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells. Methods: EVs were isolated from kidney progenitor cells (nKPCs) derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients' urine and a line of Alport patient podocytes were characterized and used to assess albumin permeability in response to various drugs or to nKPC-EVs. RNA sequencing was conducted to identify commonly modulated pathways. Results: Podocytes appeared unresponsive to pharmacological treatments, except for a podocyte line demonstrating responsiveness, in alignment with the patient's clinical response at 48 months. At variance, treatment with the nKPC-EVs was able to significantly reduce permeability in all the steroid-resistant patients-derived podocytes as well as in the line of Alport-derived podocytes. RNA sequencing of nKPC-EV-treated podocytes revealed the common upregulation of two genes (small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2)) involved in the SUMOylation pathway, a process recently demonstrated to play a role in slit diaphragm stabilization. Gene ontology analysis on podocyte expression profile highlighted cell-to-cell adhesion as the primary upregulated biological activity in treated podocytes. Conclusions: nKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocyte dysfunction. Furthermore, our findings suggest the possibility of establishing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

Additional copies of 1q negatively impact the outcome of multiple myeloma patients and induce transcriptomic deregulation in malignant plasma cells.

Additional copies of chromosome 1 long arm (1q) are frequently found in multiple myeloma (MM) and predict high-risk disease. Available data suggest a different outcome and biology of patients with amplification (Amp1q, ≥4 copies of 1q) vs. gain (Gain1q, 3 copies of 1q) of 1q. We evaluated the impact of Amp1q/Gain1q on the outcome of newly diagnosed MM patients enrolled in the FORTE trial (NCT02203643). Among 400 patients with available 1q data, 52 (13%) had Amp1q and 129 (32%) Gain1q. After a median follow-up of 62 months, median progression-free survival (PFS) was 21.2 months in the Amp1q group, 54.9 months in Gain1q, and not reached (NR) in Normal 1q. PFS was significantly hampered by the presence of Amp1q (HR 3.34 vs. Normal 1q, P < 0.0001; HR 1.99 vs. Gain1q, P = 0.0008). Patients with Gain1q had also a significantly shorter PFS compared with Normal 1q (HR 1.68, P = 0.0031). Concomitant poor prognostic factors or the failure to achieve MRD negativity predicted a median PFS < 12 months in Amp1q patients. Carfilzomib-lenalidomide-dexamethasone plus autologous stem cell transplantation treatment improved the adverse effect of Gain1q but not Amp1q. Transcriptomic data showed that additional 1q copies were associated with deregulation in apoptosis signaling, p38 MAPK signaling, and Myc-related genes.

Functional and pharmacodynamic evaluation of metronomic cyclophosphamide and docetaxel regimen in castration-resistant prostate cancer.

AIM: The aim of our study was to investigate the association of docetaxel and metronomic cyclophosphamide (CYC) in castration-resistant prostate cancer (CRPC). MATERIALS & METHODS: CRPC xenografts were established with PC3 cells. Mice were treated with a combination of CYC (50 mg/kg/day) and docetaxel (10-30 mg/kg/week) or with docetaxel alone. Docetaxel plasma levels were analyzed in patients receiving the drug alone or combined with CYC. RESULTS: Metronomic CYC is an effective adjuvant in blocking tumor growth in vivo, with comparable efficacy and less toxic effects compared with docetaxel treatment. CYC acts by downregulating cell proliferation and inducing apoptosis thorough upregulation of p21 and inhibition of angiogenesis. Finally, CYC increases docetaxel plasma levels in patients. CONCLUSION: Metronomic CYC exerts anti-tumoral effects in an in vivo model of prostate cancer and in patients with CRPC, and also increases the bioavailability of docetaxel. These results explain the favorable toxicity and activity profiles observed in patients treated with this regimen.

Tissue RNA Integrity in Visium Spatial Protocol (Fresh Frozen Samples).

The transcriptome of a tissue can be acquired both by single-cell RNAseq (scRNA-seq) and by spatial transcriptomics (ST). The dissociation step, which is mandatory in scRNA-seq methods, might lead to the loss of fragile cells and of spatial information, thus limiting the acquisition of the tissue cellular organization. Spatial transcriptomics methods moderate the above-mentioned issues and provide single-cell transcripts detection over an intact fresh frozen tissue section. Visium platform, commercialized from 10× Genomics, provides a whole transcriptome spatial transcriptomics platform, which does not require dedicated instruments, other than those available in any pathology laboratory. In spatial transcriptomics, proper tissue handling is mandatory to preserve the morphological quality of the tissue sections and the integrity of mRNA transcripts. Proper tissue handling is critical for downstream library preparation and sequencing performance. In this chapter, we describe the most critical steps of Visium protocol on fresh frozen tissues and we provide indications on how to interpret the data obtained from the quality control analysis recommended during the workflow.

Circulating hsa-miR-5096 predicts 18F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors.

Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. 18F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with 18F-FDG-PET/CT status, higher risk and lower response to PRRT. Methods: Whole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between 18F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models. Results: While no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with 18F-FDG-PET/CT in PanNETs (p-value:<0.005). Statistical analysis has shown that, hsa-miR-5096 can predict 6-month PFS (p-value:<0.001) and 12-month Overall Survival upon PRRT treatment (p-value:<0.05), as well as identify 18F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:<0.005). In addition, hsa-miR-5096 inversely correlated with both SSTR2 expression in PanNET tissue and with the 68Gallium-DOTATOC captation values (p-value:<0.05), and accordingly it was able to decrease SSTR2 when ectopically expressed in PanNET cells (p-value:<0.01). Conclusions: hsa-miR-5096 well performs as a biomarker for 18F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.

RNAseq Analysis of Livers from Pigs Treated with Testosterone and Nandrolone Esters: Selection and Field Validation of Transcriptional Biomarkers.

The use of anabolic-androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon®) and other nine with nandrolone esters (Myodine®). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.