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Abstract 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.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.

Summary: Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. : Borghesan et al. show that the soluble fraction and small extracellular vesicles (sEVs) mediate paracrine senescence. RNA sequencing and loxP reporter systems confirm sEV-mediated paracrine senescence, while preventing sEV release averts senescence. Mass spectrometry and functional analysis show that the IFN protein, IFITM3, is partially responsible for this phenotype. Keywords: exosomes, small extracellular vesicles, EV, paracrine senescence, OIS, DDIS, aging, interferon, IFITM3, fragilis

Summary: Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr−/− mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr−/− mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr−/− mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Long noncoding RNAs (lncRNAs) are regulatory molecules which have been traditionally considered as “non-coding”. Strikingly, recent evidence has demonstrated that many non-coding regions, including lncRNAs, do in fact contain small-open reading frames that code for small proteins that have been called microproteins. Only a few of them have been characterized so far, but they display key functions in a wide variety of cellular processes. Here, we show that TUNAR lncRNA encodes an evolutionarily conserved microprotein expressed in the nervous system that we have named pTUNAR. pTUNAR deficiency in mouse embryonic stem cells improves their differentiation potential towards neural lineage both in vitro and in vivo. Conversely, pTUNAR overexpression impairs neuronal differentiation by reduced neurite formation in different model systems. At the subcellular level, pTUNAR is a transmembrane protein that localizes in the endoplasmic reticulum and interacts with the calcium transporter SERCA2. pTUNAR overexpression reduces cytoplasmatic calcium, consistent with a possible role of pTUNAR as an activator of SERCA2. Altogether, our results suggest that our newly discovered microprotein has an important role in neural differentiation and neurite formation through the regulation of intracellular calcium. From a more general point of view, our results provide a proof of concept of the role of lncRNAs-encoded microproteins in neural differentiation.

Pre-metastatic niches provide favorable conditions for tumor cells to disseminate, home to and grow in otherwise unfamiliar and distal microenvironments. Tumor-derived extracellular vesicles are now recognized as carriers of key messengers secreted by primary tumors, signals that induce the formation of pre-metastatic niches. Recent evidence suggests that tumor cells can disseminate from the very earliest stages of primary tumor development. However, once they reach distal sites, tumor cells can persist in a dormant state for long periods of time until their growth is reactivated and they produce metastatic lesions. In this new scenario, the question arises as to whether extracellular vesicles could influence the formation of these metastatic niches with dormant tumor cells? (here defined as “sleepy niches”). If so, what are the molecular mechanisms involved? In this perspective-review article, we discuss the possible influence of extracellular vesicles in early metastatic dissemination and whether they might play a role in tumor cell dormancy. In addition, we comment whether extracellular vesicle-mediated signals may be involved in tumor cell awakening, considering the possibility that extracellular vesicles might serve as biomarkers to detect early metastasis and/or minimal residual disease (MRD) monitoring.

The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging.