A new cell labelling strategy to study the biogenesis of exosomes and their role in human melanoma progression driven by microenvironmental acidic pH

Gli esosomi sono nanovescicole di origine endosomiale, che rappresentano un mezzo importante di comunicazione cellula-cellula. Essendo coinvolti in diversi tipi di processi, sia fisiologici che patologici, sono diventati oggetto di sperimentazione clinica, nonostante molti meccanismi alla base della loro biogenesi rimangono tuttora sconosciuti. Quindi nel nostro laboratorio è stata messa a punto una metodica atta a produrre esosomi fluorescenti, in modo da poterne seguire la biogenesi nei diversi compartimenti intracellulari, il rilascio nell’ambiente extracellulare e la fusione con la membrana delle cellule riceventi. È stato quindi studiato, in un modello cellulare di melanoma umano, come questo precursore lipidico fluorescente è assorbito dalla cellula, accumulato all’interno del reticolo endoplasmatico, per poi diventare parte integrante della membrana degli esosomi. Queste nanovescicole fluorescenti sono state caratterizzate per dimensione, densità e presenza di marcatori proteici e successivamente è stata studiata con esperimenti di cinetica (5min – 24h) la secrezione. In seguito tale metodica è stata applicata allo studio della progressione del melanoma in condizioni di acidità microambientale. Nel melanoma è già noto che il pH microambientale è in grado di promuovere invasione e migrazione cellulare, e in questa tesi abbiamo analizzato il ruolo degli esosomi in tali processi. Quindi abbiamo studiato come cellule di melanoma umano, corrispondente a uno stadio intermedio, coltivate in ambiente acido producano una maggior quantità di esosomi, con un’aumentata capacità di trasferimento in cellule bersaglio. Inoltre queste vescicole presentano un profilo proteico che permette alle cellule riceventi di acquisire capacità migratorie e invasive. Infine, attraverso studi di meta-analisi ed ex vivo su biopsie di pazienti, è stato confermato che le molecole arricchite negli esosomi secreti in condizioni di acidità possano rappresentare dei marcatori della progressione del melanoma, convalidando quindi il valore diagnostico e prognostico degli esosomi.Exosomes, nanosized vesicles of endosomal origin, are worldwide recognized for their ability to transfer biological molecules, from cell to cell, crucial for both physiological and pathological processes. Hundred studies have been focused on exosome application also to clinics although biogenesis modalities are under investigation. Therefore, we set up a new effective fluorescent labelling strategy to trace exosome biogenesis and release with the aim to seek in human melanoma cell lines the impact that tumor progression may exert on their secretion and composition. Cells exposed to a fluorescent analogue of palmitic acid (Bodipy FL C16) were able to promptly synthetize fluorescent phospholipids, constituents of exosome membrane bilayer. Afterwards, we were able to follow exosome biogenesis from the intracellular sites of origin to cell secretion, chasing over time by direct cytofluorimetric analysis. To get insight into their function, we focused our studies on exosomes derived from melanoma cells maintained at low pH, which is a microenvironmental leverage for primary tumor to be transformed into widespread metastasis. When melanoma cells at specific intermediate stage were subjected to an acidic microenvironment, showed an increase in exosome release and transfer capability. Most importantly, when control melanoma cells were incubated with exosomes secreted in acidic medium acquired migratory and invasive capacities, demonstrating that exosomes carrying molecular payload can modify recipient cell program. Finally, meta-analysis and ex vivo studies confirmed the importance of acidic exosomes molecule content as marker of melanoma progression and so exosomes prognostic and diagnostic value

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT's toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3',5'-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Acidic and Hypoxic Microenvironment in Melanoma: Impact of Tumour Exosomes on Disease Progression

The mechanisms of melanoma progression have been extensively studied in the last decade, and despite the diagnostic and therapeutic advancements pursued, malignant melanoma still accounts for 60% of skin cancer deaths. Therefore, research efforts are required to better define the intercellular molecular steps underlying the melanoma development. In an attempt to represent the complexity of the tumour microenvironment (TME), here we analysed the studies on melanoma in acidic and hypoxic microenvironments and the interactions with stromal and immune cells. Within TME, acidity and hypoxia force melanoma cells to adapt and to evolve into a malignant phenotype, through the cooperation of the tumour-surrounding stromal cells and the escape from the immune surveillance. The role of tumour exosomes in the intercellular crosstalk has been generally addressed, but less studied in acidic and hypoxic conditions. Thus, this review aims to summarize the role of acidic and hypoxic microenvironment in melanoma biology, as well as the role played by melanoma-derived exosomes (Mexo) under these conditions. We also present a perspective on the characteristics of acidic and hypoxic exosomes to disclose molecules, to be further considered as promising biomarkers for an early detection of the disease. An update on the use of exosomes in melanoma diagnosis, prognosis and response to treatment will be also provided and discussed

Exosome-induced differentiation of neural stem progenitor cells

Cell-cell communication is a crucial event during neural development and differentiation. Extracellular membrane vesicles, including exosomes, have been recently discovered to play an important role in these events. Exosomes originate from multivescicular bodies, which may fuse with the plasma membrane and release exosomes in the extracellular space. Exosomes may contain proteins and nucleic acids and can either reach the blood flow and be transported all over the organism, or can fuse with the plasma membrane of other cells and release their content into the target cell. They have been demonstrated to exhert an influence on a large number of biological functions and to be implicated in the progression of several pathologies. They may also play an important role during tissue development, and we have been specifically interested to investigate their role in neural differentiation. Neural stem progenitor cells (NSPCs), obtained from E13,5 embyos, can be maintained in culture under proliferating conditions (i.e. in the presence of bFGF and EGF). Upon treatments with growth factors they may differentiate towards neuronal or glial phenotypes, or both. In this work we were interested in determining whether NSPCs can produce and secrete exosomes and if exosome content may exhert a developmental effect on proliferating and differentiated cells. The capability of these cells to produce and release exosomes was assessed by analyzing the presence of specific markers, such as CD63 and TSG101. Our results indicate that cultured NSPCs produce and secrete exosomes both under proliferating conditions as well as when they are cultured in differentiation medium (e.g. upon removal of EGF). Treatment of proliferating NSPCs with exosomes derived from differentiated cells triggers cell differentiation, as demonstrated by glial and neuronal marker expression; the expression of these markers is also enhanced when exosomes are added to differentiated NSPCs cultures. We also show that the effects of exosome treatment are dose dependent. Characterization of protein and nucleic acid content of NSPCs exosomes is currently under wa

Fmr1-KO mice failure to detect object novelty associates with a post-test decrease of structural and synaptic plasticity upstream of the hippocampus

Abstract Mice with deletion of the FMR1 gene show episodic memory impairments and exhibit dendritic spines and synaptic plasticity defects prevalently identified in non-training conditions. Based on evidence that synaptic changes associated with normal or abnormal memory emerge when mice are cognitively challenged, here we examine whether, and how, fragile entorhinal and hippocampal synapses are remodeled when mice succeed or fail to learn. We trained Fmr1 knockout (KO) and wild-type C57BL/6J (WT) mice in the novel object recognition (NOR) paradigm with 1 h or 24 h training-to-test intervals and then assessed whether varying the time between the presentation of similar and different objects modulates NOR performance and plasticity along the entorhinal cortex-hippocampus axis. At the 1 h-interval, KO mice failed to discriminate the novel object, showed a collapse of spines in the lateral entorhinal cortex (LEC), and of long-term potentiation (LTP) in the lateral perforant path (LPP), but a normal increase in hippocampal spines. At the 24 h, they exhibited intact NOR performance, typical LEC and hippocampal spines, and exaggerated LPP-LTP. Our findings reveal that the inability of mice to detect object novelty primarily stands in their impediment to elaborate, and convey to the hippocampus, sensory/perceptive object representations

Generation, quantification, and tracing of metabolically labeled fluorescent exosomes

Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY® fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells

DataSheet_1_Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.pdf

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.</p