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.

CONNECTOR, fitting and clustering of longitudinal data to reveal a new risk stratification system.

MOTIVATION: The transition from evaluating a single time point to examining the entire dynamic evolution of a system is possible only in the presence of the proper framework. The strong variability of dynamic evolution makes the definition of an explanatory procedure for data fitting and clustering challenging. RESULTS: We developed CONNECTOR, a data-driven framework able to analyze and inspect longitudinal data in a straightforward and revealing way. When used to analyze tumor growth kinetics over time in 1599 patient-derived xenograft growth curves from ovarian and colorectal cancers, CONNECTOR allowed the aggregation of time-series data through an unsupervised approach in informative clusters. We give a new perspective of mechanism interpretation, specifically, we define novel model aggregations and we identify unanticipated molecular associations with response to clinically approved therapies. AVAILABILITY AND IMPLEMENTATION: CONNECTOR is freely available under GNU GPL license at https://qbioturin.github.io/connector and https://doi.org/10.17504/protocols.io.8epv56e74g1b/v1.

Systematic review of long term follow-up and transitional care in adolescents and adults with esophageal atresia - why is transitional care mandatory?

PURPOSE:  to review recent literature concerning long-term health issues and transitional care in esophageal atresia (EA) patients. PubMed, Scopus, Embase and Web of Science databases were screened for studies regarding EA patients aged more than or equal to 11 years, published between August 2014 and June 2022. Sixteen studies involving 830 patients were analyzed. Mean age was 27.4 years (range 11-63). EA subtype distribution was: type C (48.8%), A (9.5%), D (1.9%), E (0.5%) and B (0.2%). 55% underwent primary repair, 34.3% delayed repair, 10.5% esophageal substitution. Mean follow-up was 27.2 years (range 11-63). Long-term sequelae were: gastro-esophageal reflux (41.4%), dysphagia (27.6%), esophagitis (12.4%), Barrett esophagus (8.1%), anastomotic stricture (4.8%); persistent cough (8.7%), recurrent infections (4.3%) and chronic respiratory diseases (5.5%). Musculo-skeletal deformities were present in 36 out of 74 reported cases. Reduced weight and height were detected in 13.3% and 6% cases, respectively. Impaired quality of life was reported in 9% of patients; 9.6% had diagnosis or raised risk of mental disorders. 10.3% of adult patients had no care provider. Meta-analysis was conducted on 816 patients. Estimated prevalences are: GERD 42.4%, dysphagia 57.8%, Barrett esophagus 12.4%, respiratory diseases 33.3%, neurological sequelae 11.7%, underweight 19.6%. Heterogeneity was substantial (> 50%).   Conclusion: EA patients must continue follow-up beyond childhood, with a defined transitional-care path by a highly specialized multidisciplinary team due to the multiple long-term sequelae. WHAT IS KNOWN: • Survival rates of esophageal atresia patients is now more than 90% thanks to the improvements in surgical techniques and intensive care, therefore patients' needs throughout adolescence and adulthood must be taken into account. WHAT IS NEW: • This review, by summarizing recent literature concerning long term sequelae of esophageal atresia, may contribute to raise awareness on the importance of defining standardized protocols of transitional and adulthood care for esophageal atresia patients.

Temperature-Dependent Re-alignment of the Short Multifunctional Peptide BP100 in Membranes Revealed by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

BP100 is a cationic undecamer peptide with antimicrobial and cell-penetrating activities. The orientation of this amphiphilic α-helix in lipid bilayers was examined under numerous conditions using solid-state 19 F, 15 N and 2 H NMR. At high temperatures in saturated phosphatidylcholine lipids, BP100 lies flat on the membrane surface, as expected. Upon lowering the temperature towards the lipid phase transition, the helix is found to flip into an upright transmembrane orientation. In thin bilayers, this inserted state was stable at low peptide concentration, but thicker membranes required higher peptide concentrations. In the presence of lysolipids, the inserted state prevailed even at high temperature. Molecular dynamics simulations suggest that BP100 monomer insertion can be stabilized by snorkeling lysine side chains. These results demonstrate that even a very short helix like BP100 can span (and thereby penetrate through) a cellular membrane under suitable conditions.

Regulation of CD45 phosphatase by oncogenic ALK in anaplastic large cell lymphoma.

Anaplastic Large Cell Lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma frequently driven by the chimeric tyrosine kinase NPM-ALK, generated by the t (2,5)(p23;q35) translocation. While ALK+ ALCL belongs to mature T cell lymphomas, loss of T cell identity is observed in the majority of ALCL secondary to a transcriptional and epigenetic repressive program induced by oncogenic NPM-ALK. While inhibiting the expression of T cell molecules, NPM-ALK activates surrogate TCR signaling by directly inducing pathways downstream the TCR. CD45 is a tyrosine phosphatase that plays a central role in T cell activation by controlling the TCR signaling and regulating the cytokine responses through the JAK/STAT pathway and exists in different isoforms depending on the stage of T-cell maturation, activation and differentiation. ALK+ ALCL cells mainly express the isoform CD45RO in keeping with their mature/memory T cell phenotype. Because of its regulatory effect on the JAK/STAT pathway that is essential for ALK+ ALCL, we investigated whether CD45 expression was affected by oncogenic ALK. We found that most ALK+ ALCL cell lines express the CD45RO isoform with modest CD45RA expression and that NPM-ALK regulated the expression of these CD45 isoforms. Regulation of CD45 expression was dependent on ALK kinase activity as CD45RO expression was increased when NPM-ALK kinase activity was inhibited by treatment with ALK tyrosine kinase inhibitors (TKIs). Silencing ALK expression through shRNA or degradation of ALK by the PROTAC TL13-112 caused upregulation of CD45RO both at mRNA and protein levels with minimal changes on CD45RA, overall indicating that oncogenic ALK downregulates the expression of CD45. CD45 repression was mediated by STAT3 as demonstrated by ChIP-seq data on ALCL cells treated with the ALK-TKI crizotinib or cells treated with a STAT3 degrader. Next, we found that knocking-out CD45 with the CRISPR/Cas9 system resulted in increased resistance to ALK TKI treatment and CD45 was down-regulated in ALCL cells that developed resistance in vitro to ALK TKIs. Overall, these data suggest that CD45 expression is regulated by ALK via STAT3 and acts as a rheostat of ALK oncogenic signaling and resistance to TKI treatment in ALCL.

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.

Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma.

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

A Rare Case of Renal Sarcoidosis.

Sarcoidosis is a multisystem granulomatous disorder characterized by non-caseating granulomas in multiple organs. It most commonly involves lungs and it is very rare to find isolated cases affecting other organ systems with no associated pulmonary findings. We hereby present a case of a young 30-year-old male who was referred to the hospital by his primary medical doctor due to right eye pain secondary to iritis and acute kidney injury (AKI). His initial laboratory studies revealed anemia, AKI, mild hypercalcemia, and the urinary analysis revealed proteinuria. Imaging studies were negative and a kidney biopsy was performed and showed results from the biopsy that revealed diffuse tubulointerstitial disease with early fibrosis, widespread moderate inflammation, multifocal tubulitis, and focal aggregate of epithelioid cells suggestive of granuloma consistent with sarcoidosis. The patient was treated with prednisone. Renal involvement of sarcoidosis is extremely rare (around 0.7%). It has a wide spectrum of presentation including abnormal calcium metabolism, nephrolithiasis, nephrocalcinosis, and acute tubulointerstitial nephritis with or without granulomas. This is a unique case as it shows renal sarcoidosis without coexisting pulmonary finding of hilar lymphadenopathy on chest X-ray. There are very few reported cases of renal sarcoidosis in the literature and this case can add to the pool of those cases. It also emphasizes the need for urgent renal biopsy in the settings of AKI associated with mild to moderate proteinuria. Lack of availability of comprehensive research on the disease may lead to misdiagnosis and delay in treatment.

Application of the Euro Clonality next-generation sequencing-based marker screening approach to detect immunoglobulin heavy chain rearrangements in mantle cell lymphoma patients: first data from the Fondazione Italiana Linfomi MCL0208 trial.

Minimal residual disease (MRD) determined by classic polymerase chain reaction (PCR) methods is a powerful outcome predictor in mantle cell lymphoma (MCL). Nevertheless, some technical pitfalls can reduce the rate of of molecular markers. Therefore, we applied the EuroClonality-NGS IGH (next-generation sequencing immunoglobulin heavy chain) method (previously published in acute lymphoblastic leukaemia) to 20 MCL patients enrolled in an Italian phase III trial sponsored by Fondazione Italiana Linfomi. Results from this preliminary investigation show that EuroClonality-NGS IGH method is feasible in the MCL context, detecting a molecular IGH target in 19/20 investigated cases, allowing MRD monitoring also in those patients lacking a molecular marker for classical screening approaches.

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.

Inhaled high molecular weight hyaluronan ameliorates respiratory failure in acute COPD exacerbation: a pilot study.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) carry significant morbidity and mortality. AECOPD treatment remains limited. High molecular weight hyaluronan (HMW-HA) is a glycosaminoglycan sugar, which is a physiological constituent of the lung extracellular matrix and has notable anti-inflammatory and hydrating properties. RESEARCH QUESTION: We hypothesized that inhaled HMW-HA will improve outcomes in AECOPD. METHODS: We conducted a single center, randomized, placebo-controlled, double-blind study to investigate the effect of inhaled HMW-HA in patients with severe AECOPD necessitating non-invasive positive-pressure ventilation (NIPPV). Primary endpoint was time until liberation from NIPPV. RESULTS: Out of 44 screened patients, 41 were included in the study (21 for placebo and 20 for HMW-HA). Patients treated with HMW-HA had significantly shorter duration of NIPPV. HMW-HA treated patients also had lower measured peak airway pressures on the ventilator and lower systemic inflammation markers after liberation from NIPPV. In vitro testing showed that HMW-HA significantly improved mucociliary transport in air-liquid interface cultures of primary bronchial cells from COPD patients and healthy primary cells exposed to cigarette smoke extract. INTERPRETATION: Inhaled HMW-HA shortens the duration of respiratory failure and need for non-invasive ventilation in patients with AECOPD. Beneficial effects of HMW-HA on mucociliary clearance and inflammation may account for some of the effects (NCT02674880, www.clinicaltrials.gov ).

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft.

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches.

A Case of Rheumatoid Arthritis-Associated Interstitial Lung Disease - An Unfortunate Complication of Untreated Rheumatoid Arthritis!

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a rare extraarticular manifestation of the systemic autoimmune disease rheumatoid arthritis. RA-ILD is one of the leading causes of morbidity and mortality in patients with rheumatoid arthritis. It is more commonly seen in patients with risk factors, which include male sex, severe rheumatoid arthritis, and smoking. The presentation depends on the extent of the underlying pathology. Patients can remain asymptomatic for extended periods of time and progressively develop symptoms such as shortness of breath on exertion and a nonproductive cough. The diagnosis is made using a combination of clinical presentation, pulmonary function testing, and imaging. It is difficult for a practitioner to distinguish the presentation of interstitial lung disease from other causes and RA-ILD. However, it is extremely important to differentiate them due to the differences in management. The treatment usually involves a multidisciplinary team guided approach towards treating the underlying rheumatoid arthritis. Asymptomatic patients are managed by observation, while symptomatic patients are managed with glucocorticoids and immunosuppressants. Accurate and early treatment can lead to an improvement in a patient's symptoms and significantly improve their quality of life. We present an interesting case of a female with a long-standing history of rheumatoid arthritis not on any treatment presenting to the ED with exertional shortness of breath, dry cough, and abdominal distension.

Euglycemic Diabetic Ketoacidosis With Sodium-Glucose Cotransporter-2 Inhibitor Use Post-Bariatric Surgery: A Brief Review of the Literature.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are antihyperglycemic drugs that are currently being recommended as second-line therapy for patients with diabetes mellitus. SGLT-2 Inhibitors function by inhibiting renal cotransporters, which reduces the reabsorption of glucose in the kidney, ultimately decreasing the concentration of glucose in the body. They have gained popularity in recent years due to their protective effects on the heart and kidneys - both organ systems that diabetes mellitus has shown to have a deleterious effect on. However, despite their growing fame, they have been found to increase the risk of euglycemic diabetic ketoacidosis (DKA). Euglycemic DKA is particularly dangerous as there is a chance that it can be missed by clinicians due to glucose levels generally being less than 200 mg/dL. There is an increasing body of literature detailing cases of euglycemic DKA after bariatric surgery. We present a brief review of the literature regarding this important side effect of SGLT-2 inhibitors seen in patients after bariatric surgery.

Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

PIK3R1W624R Is an Actionable Mutation in High Grade Serous Ovarian Carcinoma.

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis.

Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA-target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424-5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton.

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.