SARS-CoV-2 presence in recreational seawater and evaluation of intestine permeability: experimental evidence of low impact on public health.

Introduction: Coastal seawater pollution poses a public health risk due to the potential ingestion of contaminated water during recreational activities. Wastewater-based epidemiology has revealed the abundant presence of SARS-CoV-2 in seawater emitted from wastewater outlets. The objective of this research was to investigate the impact of seawater on SARS-CoV-2 infectivity to assess the safety of recreational activities in seawater. Methods: Wild SARS-CoV-2 was collected from oral swabs of COVID-19 affected patients and incubated for up to 90 min using the following solutions: (a) standard physiological solution (control), (b) reconstructed seawater (3.5% NaCl), and (c) authentic seawater (3.8%). Samples were then exposed to two different host systems: (a) Vero E6 cells expressing the ACE2 SARS-CoV-2 receptor and (b) 3D multi-tissue organoids reconstructing the human intestine. The presence of intracellular virus inside the host systems was determined using plaque assay, quantitative real-time PCR (qPCR), and transmission electron microscopy. Results: Ultrastructural examination of Vero E6 cells revealed the presence of virus particles at the cell surface and in replicative compartments inside cells treated with seawater and/or reconstituted water only for samples incubated up to 2 min. After a 90-min incubation, the presence of the virus and its infectivity in Vero E6 cells was reduced by 90%. Ultrastructural analysis performed in 3D epi-intestinal tissue did not reveal intact viral particles or infection signs, despite the presence of viral nucleic acid detected by qPCR. Indeed, viral genes (Orf1ab and N) were found in the intestinal luminal epithelium but not in the enteric capillaries. These findings suggest that the intestinal tissue is not a preferential entry site for SARS-CoV-2 in the human body. Additionally, the presence of hypertonic saline solution did not increase the susceptibility of the intestinal epithelium to virus penetration; rather, it neutralized its infectivity. Conclusion: Our results indicate that engaging in recreational activities in a seawater environment does not pose a significant risk for COVID-19 infection, despite the possible presence of viral nucleic acid deriving from degraded and fragmented viruses.

Complement tunes glutamate release and supports synaptic impairments in an animal model of multiple sclerosis.

BACKGROUND AND PURPOSE: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post-immunization). EXPERIMENTAL APPROACH: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D-aspartate ([3H]D-ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. KEY RESULTS: In healthy mice, complement releases [3H]D-ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution-dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement-induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. CONCLUSION AND IMPLICATIONS: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS.

Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies.

Extracellular vesicles (EVs) have emerged as potential vehicles for targeted drug delivery and diagnostic applications. However, achieving consistent and reliable functionalization of EV membranes remains a challenge. Copper-catalyzed click chemistry, commonly used for EV surface modification, poses limitations due to cytotoxicity and interference with biological systems. To overcome these limitations, we developed a standardized method for functionalizing an EV membrane via copper-free click chemistry. EVs derived from plasma hold immense potential as diagnostic and therapeutic agents. However, the isolation and functionalization of EVs from such a complex biofluid represent considerable challenges. We compared three different EV isolation methods to obtain an EV suspension with an optimal purity/yield ratio, and we identified sucrose cushion ultracentrifugation (sUC) as the ideal protocol. We then optimized the reaction conditions to successfully functionalize the plasma-EV surface through a copper-free click chemistry strategy with a fluorescently labeled azide, used as a proof-of-principle molecule. Click-EVs maintained their identity, size, and, more importantly, capacity to be efficiently taken up by responder tumor cells. Moreover, once internalized, click EVs partially followed the endosomal recycling route. The optimized reaction conditions and characterization techniques presented in this study offer a foundation for future investigations and applications of functionalized EVs in drug delivery, diagnostics, and therapeutics.

Interactions between Glycine and Glutamate through Activation of Their Transporters in Hippocampal Nerve Terminals.

Evidence supports the pathophysiological relevance of crosstalk between the neurotransmitters Glycine and Glutamate and their close interactions; some reports even support the possibility of Glycine-Glutamate cotransmission in central nervous system (CNS) areas, including the hippocampus. Functional studies with isolated nerve terminals (synaptosomes) permit us to study transporter-mediated interactions between neurotransmitters that lead to the regulation of transmitter release. Our main aims here were: (i) to investigate release-regulating, transporter-mediated interactions between Glycine and Glutamate in hippocampal nerve terminals and (ii) to determine the coexistence of transporters for Glycine and Glutamate in these terminals. Purified synaptosomes, analyzed at the ultrastructural level via electron microscopy, were used as the experimental model. Mouse hippocampal synaptosomes were prelabeled with [3H]D-Aspartate or [3H]Glycine; the release of radiolabeled tracers was monitored with the superfusion technique. The main findings were that (i) exogenous Glycine stimulated [3H]D-Aspartate release, partly by activation of GlyT1 and in part, unusually, through GlyT2 transporters and that (ii) D-Aspartate stimulated [3H]glycine release by a process that was sensitive to Glutamate transporter blockers. Based on the features of the experimental model used, it is suggested that functional transporters for Glutamate and Glycine coexist in a small subset of hippocampal nerve terminals, a condition that may also be compatible with cotransmission; glycinergic and glutamatergic transporters exhibit different functions and mediate interactions between the neurotransmitters. It is hoped that increased information on Glutamate-Glycine interactions in different areas, including the hippocampus, will contribute to a better knowledge of drugs acting at "glycinergic" targets, currently under study in relation with different CNS pathologies.

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells.

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature.

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-ß-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-ß structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-ß structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

A quantitative biology approach correlates neuronal toxicity with the largest inclusions of TDP-43.

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Ultrastructural examination of lung "cryobiopsies" from a series of fatal COVID-19 cases hardly revealed infected cells.

Ultrastructural analysis of autopsy samples from COVID-19 patients usually suffers from significant structural impairment possibly caused by the rather long latency between death of the patient and an appropriate sample fixation. To improve structural preservation of the tissue, we obtained samples from ventilated patients using a trans-bronchial "cryobiopsy" within 30 min after their death and fixed them immediately for electron microscopy. Samples of six COVID-19 patients with a documented histopathology were systematically investigated by thin section electron microscopy. The different samples and areas inspected revealed the ultrastructural correlates of the different phases of diffuse alveolar damage, including detachment of the alveolar epithelium, hyperplasia of type 2 cells, exudates, and accumulation of extracellular material, such as the hyaline membranes and fibrin. Macrophages and neutrophilic granulocytes were regularly detected. Structural integrity of endothelium was intact in regions where the alveolar epithelium was already detached. Aggregates of erythrocytes, leukocytes with fibrin, and thrombocytes were not observed. Coronavirus particles were only found in and around very few cells in one of the six patient samples. The type and origin of these cells could not be assessed although the overall structural preservation of the samples allowed the identification of pulmonary cell types. Hence, the observed alveolar damage is not associated with virus presence or structural impairment due to ongoing replication at later stages of the disease in fatal cases, which implies that the lung damage in these patients is at least propagated by alternative mechanisms, perhaps, an inappropriate immune or stress response.

Neurodegenerative Disease-Associated TDP-43 Fragments Are Extracellularly Secreted with CASA Complex Proteins.

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs.

Ultrastructural imaging reveals vascular remodeling in migraine patients.

Migraine is a neurological disorder and one of the most common pain conditions worldwide. Despite its prevalence, the basic biology and underlying mechanisms contributing to the development of migraine are still poorly understood. It is still unclear, for instance, whether the vasculature, both extra and intracranial, plays a significant role in the generation of migraine pain. Neuroimaging data, indeed, have reported conflicting results on blood vessels abnormalities like vasodilation, while functional studies suggest that vessels dysfunction may extend beyond vasodilation. Here we combined light and electron microscopy imaging to investigate the fine structure of superficial temporal (STA) and occipital arteries (OA) from patients that underwent minimally invasive surgery for migraine. Using optical microscopy, we observed that both STA and OA vessels showed marked endothelial thickening and internal elastic lamina fragmentation. In the muscular layer, we found profound shape changes of vascular smooth muscle cells (VSMCs), abundant extracellular matrix, and the presence of clear extracellular vacuoles. The electron microscopy analysis confirmed putative VSMCs infiltrated within the intima layer and revealed a consistent shifting of VSMCs from contractile to a synthetically active phenotype. We also report the presence of (i) abundant extracellular vacuoles filled with fine granular material and membranes, (ii) multilamellar structures, (iii) endosome-like organelles, and (iv) bona fide extracellular vesicles in the matrix space surrounding synthetically active cells. As both the endothelial layer and VSMCs coordinate a variety of vascular functions, these results suggest that a significant vascular remodeling is occurring in STA and OA of migraine patients. Thus, this phenomenon may represent an important target for future investigation designed toward the development of new therapeutic approaches.

Role of extracellular vesicles from adipose tissue- and bone marrow-mesenchymal stromal cells in endothelial proliferation and chondrogenesis.

The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle- and small-sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC-EVs overexpressed pro-angiogenic factors in comparison to the BMSC-counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Simulated Microgravity Effects on Human Adenocarcinoma Alveolar Epithelial Cells: Characterization of Morphological, Functional, and Epigenetic Parameters.

BACKGROUND: In space, the reduction or loss of the gravity vector greatly affects the interaction between cells. Since the beginning of the space age, microgravity has been identified as an informative tool in biomedicine, including cancer research. The A549 cell line is a hypotriploid human alveolar basal epithelial cell line widely used as a model for lung adenocarcinoma. Microgravity has been reported to interfere with mitochondrial activity, energy metabolism, cell vitality and proliferation, chemosensitivity, invasion and morphology of cells and organelles in various biological systems. Concerning lung cancer, several studies have reported the ability of microgravity to modulate the carcinogenic and metastatic process. To investigate these processes, A549 cells were exposed to simulated microgravity (µG) for different time points. METHODS: We performed cell cycle and proliferation assays, ultrastructural analysis of mitochondria architecture, as well as a global analysis of miRNA modulated under µG conditions. RESULTS: The exposure of A549 cells to microgravity is accompanied by the generation of polynucleated cells, cell cycle imbalance, growth inhibition, and gross morphological abnormalities, the most evident are highly damaged mitochondria. Global miRNA analysis defined a pool of miRNAs associated with µG solicitation mainly involved in cell cycle regulation, apoptosis, and stress response. To our knowledge, this is the first global miRNA analysis of A549 exposed to microgravity reported. Despite these results, it is not possible to draw any conclusion concerning the ability of µG to interfere with the cancerogenic or the metastatic processes in A549 cells. CONCLUSIONS: Our results provide evidence that mitochondria are strongly sensitive to µG. We suggest that mitochondria damage might in turn trigger miRNA modulation related to cell cycle imbalance.

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells.

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration-approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30-100 nm) ERBB2- EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs.

Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells.

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

Liposomes Loaded with the Proteasome Inhibitor Z-Leucinyl-Leucinyl-Norleucinal are Effective in Inducing Apoptosis in Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is one of the main causes of cancer-related death in developed countries. Targeted therapies and conventional chemotherapeutics have been developed to help treat this type of aggressive cancer. Among these, the monoclonal antibodies cetuximab (Cxm) and panitumumab specifically target and inactivate the signaling of ERBB1 (EGF receptor), a key player in the development and progression of this cancer. Unfortunately, these antibodies are effective only on a small fraction of patients due to primary or secondary/acquired resistance. However, as ERBB1 cell surface expression is often maintained in resistant tumors, ERBB1 can be exploited as a target to deliver other drugs. Liposomes and immunoliposomes are under intensive investigation as pharmaceutical nanocarriers and can be functionalized with specific antibodies. In this study, we first investigated the anti-cancer activity of a cell permeable tripeptide, leucine-leucin-norleucinal (LLNle), an inhibitor of gamma-secretase and proteasome, in three different CRC cell lines that express ERBB1. We formulated LLNle-liposomes and Cxm-conjugated LLNle-loaded liposomes (LLNle-immunoliposomes) and evaluated their efficacy in inhibiting cell survival. Despite similar pro-apoptotic effects of free LLNle and LLNle-liposomes, immunoliposomes-LLNle were significantly less effective than their unconjugated counterparts. Indeed, immunoliposomes-LLNle were readily internalized and trafficked to lysosomes, where LLNle was likely trapped and/or inactivated. In conclusion, we demonstrated that LLNle was readily delivered to CRC cell lines by liposomes, but immunoliposomes-LLNle failed to show significant anti-cancer activity.

Exosomes From Astrocyte Processes: Signaling to Neurons.

It is widely recognized that extracellular vesicles subserve non-classical signal transmission in the central nervous system. Here we assess if the astrocyte processes, that are recognized to play crucial roles in intercellular communication at the synapses and in neuron-astrocyte networks, could convey messages through extracellular vesicles. Our findings indicate, for the first time that freshly isolated astrocyte processes prepared from adult rat cerebral cortex, can indeed participate to signal transmission in central nervous system by releasing exosomes that by volume transmission might target near or long-distance sites. It is noteworthy that the exosomes released from the astrocyte processes proved ability to selectively target neurons. The astrocyte-derived exosomes were proven positive for neuroglobin, a protein functioning as neuroprotectant against cell insult; the possibility that exosomes might transfer neuroglobin to neurons would add a mechanism to the potential astrocytic neuroprotectant activity. Notably, the exosomes released from the processes of astrocytes maintained markers, which prove their parental astrocytic origin. This potentially allows the assessment of the cellular origin of exosomes that might be recovered from body fluids.