Comparative excitation-emission dependence of the FV /FM ratio in model green algae and cyanobacterial strains.

The emission spectra collected under conditions of open (F0 ) and closed (FM ) photosystem II (PSII) reaction centres are close-to-independent from the excitation wavelength in Chlamydomonas reinhardtii and Chlorella sorokiniana, whereas a pronounced dependence is observed in Synechocystis sp. PCC6803 and Synechococcus PCC7942, instead. The differences in band-shape between the F0 and FM emission are limited in green algae, giving rise only to a minor trough in the FV /FM spectrum in the 705-720 nm range, irrespectively of the excitation. More substantial variations are observed in cyanobacteria, resulting in marked dependencies of the measured FV /FM ratios on both the excitation and the detection wavelengths. In cyanobacteria, the maximal FV /FM values (0.5-0.7), observed monitoring at approximately 684 nm and exciting Chl a preferentially, are comparable to those of green algae; however, FV /FM decreases sharply below approximately 660 nm. Furthermore, in the red emission tail, the trough in the FV /FM spectrum is more pronounced in cyanobacteria with respect to green algae, corresponding to FV /FM values of 0.25-0.4 in this spectral region. Upon direct phycobilisomes excitation (i.e. >520 nm), the FV /FM value detected at 684 nm decreases to 0.3-0.5 and is close-to-negligible (approximately 0.1) below 660 nm. At the same time, the FV spectra are, in all species investigated, almost independent on the excitation wavelength. It is concluded that the excitation/emission dependencies of the FV /FM ratio arise from overlapped contributions from the three independent emissions of PSI, PSII and a fraction of energetically uncoupled external antenna, excited in different proportions depending on the respective optical cross-section and fluorescence yield.

Non-endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4' ß-carotene oxygenase (CrtW) and 3,3' ß-carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD-212 under the control of a temperature-inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non-endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of ß-carotene (ß-car). When both Brevundimonas sp. SD-212 genes were coexpressed, significant amounts of non-endogenous Asx were obtained accompanied by a strong decrease in ß-car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added-value compounds.

Osteogenic commitment and differentiation of human mesenchymal stem cells by low-intensity pulsed ultrasound stimulation.

Low-intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm2 LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7, 14, 21 days). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21 days CD73+/CD90+: 6%; OCT-3/4+/NANOG+/SOX2+: 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Structure-activity relationship for the solid state emission of a new family of "push-pull"π-extended chromophores.

We report the design, synthesis, molecular optical properties, and solid state emissive behaviour of a series of novel compounds, which, similar to the archetypal AIE luminogen tetraphenylethene, are formed of a central olefin stator and decorated with either three or four rotors. These rotors, being either electron-rich substituted benzenes, or electron-withdrawing functional groups (esters, ketones, cyano groups) confer a "push-pull" character to the overall molecular structure. Building on both new and already published contributions, a comprehensive picture of the properties and the potential of these compounds is provided.

Inhibition of PID1/NYGGF4/PCLI1 gene expression highlights its role in the early events of the cell cycle in NIH3T3 fibroblasts.

The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.

Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521.

The study of protein-protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labor-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labeling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel protein-protein interactions. A cleavable cross-linker, dithiobis[succinimidyl propionate] (DSP), is utilized to stabilize protein complexes before immunoprecipitation. Protein complex detachment from the antibody is achieved by limited proteolysis. Finally, protein quantitation is performed via (18)O labeling. The workflow has been optimized concerning (i) DSP concentration and (ii) incubation times for limited proteolysis, using the stem cell-associated transcription cofactor ZNF521 as a model target. The interaction of ZNF521 with the core components of the nuclear remodelling and histone deacetylase (NuRD) complex, already reported in the literature, was confirmed. Additionally, interactions with newly discovered molecular partners of potentially relevant functional role, such as ZNF423, Spt16, Spt5, were discovered and validated by Western blotting.

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody.

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti-EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab-sensitive and -resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose-6-phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab-resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia-inducible factor 1-associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti-EGFR therapy in colorectal cancer.

Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

Anti-Oxidation Agents to Prevent Dye Degradation in Organic-Based Host-Guest Systems Suitable for Luminescent Solar Concentrators.

Luminescent solar concentrators (LSCs) have been extensively studied as they offer a practical solution to increase the efficiency of silicon-based photovoltaics (PVs). In this context, the use of natural and organic luminescent materials is desirable in order to obtain sustainable and environmentally friendly devices. Moreover, solution-processable organic host-guest systems based on Foerster Resonant Energy Transfer (FRET) processes offer the possibility to exploit a low-cost technique to obtain an efficient energy downshift from the UV-visible to red or deep red emissions in order to concentrate the radiation in the area of maximum efficiency of the PV device. Nevertheless, organic materials are subjected to photodegradation that reduces their optical properties when exposed to UV light and oxygen. In this work, we incorporated two different antioxidant molecules (i.e., octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (Octa) and L-ascorbic acid (L-Asc)) in a three-dye host-guest system and studied the corresponding optical properties after prolonged irradiation times in air. It was found that the presence of the antioxidants, especially L-Asc, slowed the system's photodegradation down whilst at the same time retaining high emission efficiencies and without interfering with the cascade Resonant Energy Transfer processes among the dyes inserted in the nanochannels of the host.

Special Issue: Innovations in Semiconducting Block Copolymers.

The current Special Issue entitled "Innovations in Semiconducting Block Copolymers" aims to discuss cutting-edge research regarding the synthesis, characterization and application of semiconducting block copolymers, with a special focus on the realization of novel and innovative nanostructured materials for the production of advanced devices suitable in different fields, ranging from sensors applications to optic photovoltaics [...].

Proteomic signatures in thapsigargin-treated hepatoma cells.

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca(2+)-store depletion within the ER and simultaneously increases Ca(2+) level in the cytosol. Perturbation of Ca(2+) homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway.

Controlled swapping of nanocomposite surface wettability by multilayer photopolymerization.

Single-layered photopolymerized nanocomposite films of polystyrene and TiO(2) nanorods change their wetting characteristics from hydrophobic to hydrophilic when deposited on substrates with decreasing hydrophilicity. Interestingly, the addition of a second photopolymerized layer causes a swapping in the wettability, so that the final samples result converted from hydrophobic to hydrophilic or vice versa. The wettability characteristics continue to be swapped as the number of photopolymerized layers increases. In fact, odd-layered samples show the same wetting behavior as single-layered ones, while even-layered samples have the same surface characteristics as double-layered ones. Analytical surface studies demonstrate that all samples, independently of the number of layers, have similar low roughness, and that the wettability swap is due to the different concentration of the nanocomposites constituents on the samples surface. Particularly, the different interactions between the hydrophilic TiO(2) nanorods and the underlying layer lead to different amounts of nanorods exposed on the nanocomposites surface. Moreover, due to the unique property of TiO(2) to reversibly increase its wettability upon UV irradiation and subsequent storage, the wetting characteristics of the multilayered nanocomposites can be tuned in a reversible manner. In this way, a combination of substrate, number of photopolymerized layers, and external UV light stimulus can be used in order to precisely control the surface wettability properties of nanocomposite films, opening the way to a vast number of potential applications in microfluidics, protein assays, and cell growth.

Light-controlled directional liquid drop movement on TiO2 nanorods-based nanocomposite photopatterns.

Patterned polymeric coatings enriched with colloidal TiO(2) nanorods and prepared by photopolymerization are found to exhibit a remarkable increase in their water wettability when irradiated with UV laser light. The effect can be completely reversed using successive storage in vacuum and dark ambient environment. By exploiting the enhancement of the nanocomposites hydrophilicity upon UV irradiation, we prepare wettability gradients along the surfaces by irradiating adjacent surface areas with increasing time. The gradients are carefully designed to achieve directional movement of water drops along them, taking into account the hysteresis effect that opposes the movement as well as the change in the shape of the drop during its motion. The accomplishment of surface paths for liquid flow, along which the hydrophilicity gradually increases, opens the way to a vast number of potential applications in microfluidics.

Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis.

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

SWATH-MS based quantitative proteomics analysis reveals that curcumin alters the metabolic enzyme profile of CML cells by affecting the activity of miR-22/IPO7/HIF-1α axis.

BACKGROUND: Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by expression of the chimeric BCR-ABL tyrosine kinase oncogene, resulting from the t(9;22) chromosomal translocation. Imatinib (gleevec, STI-571) is a selective inhibitor of BCR-ABL activity highly effective in the treatment of CML. However, even though almost all CML patients respond to treatment with imatinib or third generation inhibitors, these drugs are not curative and need to be taken indefinitely or until patients become resistant. Therefore, to get a definitive eradication of leukemic cells, it is necessary to find novel therapeutic combinations, for achieving greater efficacy and fewer side effects. Curcumin is an Indian spice with several therapeutic properties: anti-oxidant, analgesic, anti-inflammatory, antiseptic and anti-cancer. In cancer disease, it acts by blocking cell transformation, proliferation, and invasion and by inducing cell apoptosis. METHODS: In the present study, the effect of a sub-toxic dose of curcumin on K562 cells was evaluated by using the technique of Sequential Window Activation of All Theoretical Mass Spectra (SWATH-MS). Bioinformatic analysis of proteomic data was performed to highlight the pathways mostly affected by the treatment. The involvement of Hypoxia inducible factor 1 α (HIF-1α) was assayed by evaluating its activation status and the modulation of importin 7 (IPO7) and miR-22 was assessed by quantitative PCR and western blot analysis. Finally, K562 cells transfected with miR-22 inhibitor were used to confirm the ability of curcumin to elicit miR-22 expression. RESULTS: Our findings revealed that the most relevant effect induced by curcumin was a consistent decrease of several proteins involved in glucose metabolism, most of which were HIF-1α targets, concomitant with the up-regulation of functional and structural mitochondrial proteins. The mechanism by which curcumin affects metabolic enzyme profile was associated with the reduction of HIF-1α activity, due to the miR-22-mediated down-regulation of IPO7 expression. Finally, the ability of curcumin to enhance in vitro the efficiency of imatinib was reported. CONCLUSIONS: In summary, our data indicates that the miR-22/IPO7/HIF-1α axis may be considered as a novel molecular target of curcumin adding new insights to better define therapeutic activity and anticancer properties of this natural compound. The MS proteomic data have been deposited to the ProteomeXchange with identifier .

Label-free quantitative proteomic profiling of colon cancer cells identifies acetyl-CoA carboxylase alpha as antitumor target of Citrus limon-derived nanovesicles.

We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SIGNIFICANCE: This study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.

The phospholipase DDHD1 as a new target in colorectal cancer therapy.

BACKGROUND: Our previous study demonstrates that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability, and that this effect is associated with the downregulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on the contribution of DDHD1 in neurological disorders, there is no information on its role in cancer. This study investigates the role of DDHD1 in colon cancer. METHODS: DDHD1 siRNAs and an overexpression vector were transfected into colorectal cancer and normal cells to downregulate or upregulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics using SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. RESULTS: The results indicate that DDHD1 supports colon cancer cell proliferation and survival, since its downregulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumors, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Additionally, we found that functional categories, significantly affected by DDHD1 silencing, were specifically related to cancer phenotype and for the first time associated to DDHD1 activity. CONCLUSIONS: In conclusion, this study provides the first evidence confirming the role of DDHD1 in cancer, providing a possibility to define a new target to design more effective therapies for colon cancer patients.

Poly-histidine grafting leading to fishbone-like architectures.

A small series of Morita-Baylis-Hillman adduct (MBHA) derivatives was synthesized and made to react with imidazole, N-acetylhistidine, and N-acetylhexahistidine as models of poly-histidine derivatives. Intriguingly, the reaction of MBHA derivatives 1a and b with imidazole in acetonitrile-phosphate buffered saline (PBS) gave the imidazolium salt biadducts 3a and b as the main reaction products. These results were confirmed by experiments performed with N-acetylhistidine and 1b and suggested the possible occurrence of these structures in the products of poly-histidine labeling with MBHA derivatives 1a and b. These compounds were then transformed into the corresponding water-soluble derivatives 1c-e by introducing oligo(ethylene glycol) chains and their reactivity was evaluated in preliminary experiments with imidazole and then with N-acetylhexahistidine in PBS. The structure of polymeric materials Ac-His-6-MBHA-1d and Ac-His-6-MBHA-1e obtained using ten-fold excesses of compounds 1d and e was investigated using mass spectrometry, NMR spectroscopy, and photophysical studies, which suggested the presence of biadduct residues in both polymeric materials. These results provide the basis for the preparation of fishbone-like polymer brushes, the characterization of their properties, and the exploration of their potential applications in different fields of science such as in vivo fluorogenic labeling, fluorescence microscopy, protein PEGylation, up to the production of smart materials and biosensors.

Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity.

The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.