Colorectal cancer-derived small extracellular vesicles induce TGFß1-mediated epithelial to mesenchymal transition of hepatocytes.

BACKGROUND: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown. METHODS: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment. RESULTS: Our study shows for the first time that TGFß1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFß1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. CONCLUSIONS: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480.

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells.

Overview and Update on Extracellular Vesicles: Considerations on Exosomes and Their Application in Modern Medicine.

In recent years, there has been a rapid growth in the knowledge of cell-secreted extracellular vesicle functions. They are membrane enclosed and loaded with proteins, nucleic acids, lipids, and other biomolecules. After being released into the extracellular environment, some of these vesicles are delivered to recipient cells; consequently, the target cell may undergo physiological or pathological changes. Thus, extracellular vesicles as biological nano-carriers, have a pivotal role in facilitating long-distance intercellular communication. Understanding the mechanisms that mediate this communication process is important not only for basic science but also in medicine. Indeed, extracellular vesicles are currently seen with immense interest in nanomedicine and precision medicine for their potential use in diagnostic, prognostic, and therapeutic applications. This paper aims to summarize the latest advances in the study of the smallest subtype among extracellular vesicles, the exosomes. The article is divided into several sections, focusing on exosomes' nature, characteristics, and commonly used strategies and methodologies for their separation, characterization, and visualization. By searching an extended portion of the relevant literature, this work aims to give a quick outline of advances in exosomes' extensive nanomedical applications. Moreover, considerations that require further investigations before translating them to clinical applications are summarized.

Tumor-Derived Small Extracellular Vesicles Induce Pro-Inflammatory Cytokine Expression and PD-L1 Regulation in M0 Macrophages via IL-6/STAT3 and TLR4 Signaling Pathways.

Tumor-associated macrophages play a key role in promoting tumor progression by exerting an immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1). It is well known that tumor-derived small extracellular vesicles (SEVs) affect the tumor microenvironment, influencing TAM behavior. The present study aimed to examine the effect of SEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with SEVs derived from a colorectal cancer (CRC) cell line, SW480, and a multiple myeloma (MM) cell line, MM1.S. The expression of PD-L1, interleukin-6 (IL-6), and other inflammatory cytokines as well as of the underlying molecular mechanisms were evaluated. Our results indicate that SEVs can significantly upregulate the expressions of PD-L1 and IL-6 at both the mRNA and protein levels and can activate the STAT3 signaling pathway. Furthermore, we identified the TLR4/NF-kB pathway as a convergent mechanism for SEV-mediated PD-L1 expression. Overall, these preliminary data suggest that SEVs contribute to the formation of an immunosuppressive microenvironment.

A 3D­scaffold of PLLA induces the morphological differentiation and migration of primary astrocytes and promotes the production of extracellular vesicles.

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly­L­lactic acid (PLLA) via thermally­induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co­culture systems.

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.

Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis.

Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death.

Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing.Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment.

Ultrastructural aspects of naturally occurring wound in the tunic of two ascidians: Ciona intestinalis and Styela plicata (Tunicata).

Efficient wound healing is essential for all animals from insects to mammals. Ciona intestinalis and Styela plicata are solitary ascidians belonging to urochordates, a subphylum that occupies a key phylogenetic position as it includes the closest relative to vertebrates. Urochordate first physical barrier against invaders is the tunic, an extracellular matrix that is constantly exposed to all kinds of insults. Thus, when damage occurs, an innate immune response is triggered to eliminate impaired tissue and potentially pathogenic microbes, and restore tissue functionality. Ultrastructural aspects of the tunic in the wound healing process of two ascidians are described. In the injured areas, we evidenced thinning of the tunic and areas of low fibre density, dense intratunic bacterial and protozoan population, and inflammatory aspects such as the increase in tunic cells, their aggregates, and phagocytosis. This is the first report on tunic physical wounding occurring in the natural habitat.

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae).

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.

Screening of subtelomeric rearrangements in autistic disorder: identification of a partial trisomy of 13q34 in a patient bearing a 13q;21p translocation.

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the triplicate region does not exceed 300 kb about, that is, the distance from telomere to the first normally inherited marker. In addition, gene dosage analysis performed on the derivative chromosome 21, did not reveal loss of the most telomeric protein-encoding genes on 21p. The potential relationship between a postulated increased expression of genes on 13q34 and the complex phenotype in this trisomic patient is discussed.

Comparative study of T84 and T84SF human colon carcinoma cells: in vitro and in vivo ultrastructural and functional characterization of cell culture and metastasis.

To better understand the relationship between tumor heterogeneity, differentiation, and metastasis, suitable experimental models permitting in vitro and in vivo studies are necessary. A new variant cell line (T84SF) exhibiting an altered phenotype was recently selected from a colon cancer cell line (T84) by repetitive plating on TNF-alpha treated human endothelial cells and subsequent selection for adherent cells. The matched pair of cell lines provides a useful system to investigate the extravasation step of the metastatic cascade. Since analysis of morphological differences can be instructive to the understanding of metastatic potential of tumor cells, we compared the ultrastructural and functional phenotype of T84 and T84SF cells in vitro and in vivo. The reported ultrastructural features evidence differences between the two cell lines; selected cells showed a marked pleomorphism of cell size and nuclei, shape, and greater surface complexity. These morphological differences were also coupled with biochemical data showing a distinct tyrosine phosphorylation-based signaling, an altered localization of beta-catenin, MAPK, and AKT activation, as well as an increased expression in T84SF cells of Bcl-X(L), a major regulator of apoptosis. Therefore, these cell lines represent a step forward in the development of appropriate models in vitro and in vivo to investigate colon cancer progression.

Aspects of cell production in mantle tissue of Ciona intestinalis L. (Tunicata, Ascidiacea).

Renewal of cell population is needed in the tunic of ascidians, as the tunic cells are involved in many biological functions. Tunic cells are thought to arrive by migrating across the mantle epithelium into the tunic from the blood lacunae or the mesenchymal space. Electron microscope observations show that the mantle epithelium of Ciona intestinalis shares some proliferative characteristics, releasing cells into the tunic and thus providing an increase renewal of tunical cells in restricted zones of adult animals.