Proteomic profiling of tumour tissue-derived extracellular vesicles in colon cancer.

Colon cancer is one of the most commonly occurring tumours among both women and men, and over the past decades the incidence has been on the rise. As such, the need for biomarker identification as well as an understanding of the underlying disease mechanism has never been greater. Extracellular vesicles are integral mediators of cell-to-cell communication and offer a unique opportunity to study the machinery that drives disease progression, and they also function as vectors for potential biomarkers. Tumour tissue and healthy mucosal tissue from the colons of ten patients were used to isolate tissue-resident EVs that were subsequently subjected to global quantitative proteomic analysis through LC-MS/MS. In total, more than 2000 proteins were identified, with most of the common EV markers being among them. Bioinformatics revealed a clear underrepresentation of proteins involved in energy production and cellular adhesion in tumour EVs, while proteins involved in protein biosynthesis were overrepresented. Additionally, 53 membrane proteins were found to be significantly upregulated in tumour EVs. Among them were several proteins with enzymatic functions that degrade the extracellular matrix, and three of these, Fibroblast activating factor (FAP), Cell surface hyaluronidase (CEMIP2), as well as Ephrin receptor B3 (EPHB3), were validated and found to be consistent with the global quantitative results. These stark differences in the proteomes between healthy and cancerous tissue emphasise the importance of the interstitial vesicle secretome as a major player of disease development.

Three-dimensional reconstruction of interstitial extracellular vesicles in human liver as determined by electron tomography.

Extracellular vesicles (EVs) are lipid bilayer nanoparticles involved in cell-cell communication that are released into the extracellular space by all cell types. The cargo of EVs includes proteins, lipids, nucleic acids, and metabolites reflecting their cell of origin. EVs have recently been isolated directly from solid tissues, and this may provide insights into how EVs mediate communication between cells in vivo. Even though EVs have been isolated from tissues, their point of origin when they are in the interstitial space has been uncertain. In this study, we performed three-dimensional (3D) reconstruction using transmission electron tomography of metastatic and normal liver tissues with a focus on the presence of EVs in the interstitium. After chemical fixation of the samples and subsequent embedding of tissue pieces in resin, ultrathin slices (300 nm) were cut and imaged on a 120 ekV transmission electron microscopy as a tilt series (a series of subsequent images tilted at different angles). These were then computationally illustrated in a 3D manner to reconstruct the imaged tissue volume. We identified the cells delimiting the interstitial space in both types of tissues, and small distinct spherical structures with a diameter of 30-200 nm were identified between the cells. These round structures appeared to be more abundant in metastatic tissue compared to normal tissue. We suggest that the observed spherical structures in the interstitium of the metastatic and non-metastatic liver represent EVs. This work thus provides the first 3D visualization of EVs in human tissue.

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.

Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations.

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanoma.

Characterization of surface markers on extracellular vesicles isolated from lymphatic exudate from patients with breast cancer.

BACKGROUND: Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified. METHODS: Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit. RESULTS: Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors. CONCLUSIONS: Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.

Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy.

Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti-tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro-inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow-derived dendritic cells. Co-immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma-bearing mice through Th-1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti-PD-1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti-tumour responses using immunotherapeutic bacterial SyBV.

Isolation and characterization of extracellular vesicle subpopulations from tissues.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.

Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations is only beginning to be explored. Since EVs have been implicated in tumour microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Interestingly, two mitochondrial inner membrane proteins MT-CO2 (encoded by the mitochondrial genome) and COX6c (encoded by the nuclear genome) were highly prevalent in the plasma of melanoma patients, as well as in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs contains active mitochondrial enzymes. In summary, tumour tissues are enriched in EVs with mitochondrial membrane proteins and these mitochondrial membrane proteins can be detected in plasma and are increased in melanoma, ovarian cancer as well as breast cancer.

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia.

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA.

Role of WT1-ZNF224 interaction in the expression of apoptosis-regulating genes.

The transcription factor Wilms' tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. Physical interactions of other cellular proteins with WT1 are known to modulate its function. We previously identified the Krüppel-like zinc-finger protein, ZNF224, as a novel human WT1-associating protein that enhances the transcriptional activation of the human vitamin D receptor promoter by WT1. Here, we have analyzed the effects of WT1-ZNF224 interaction on the expression of apoptosis-regulating genes in the chronic myelogenous leukemia (CML) K562 cell line. The results demonstrated that ZNF224 acts in fine tuning of WT1-dependent control of gene expression, acting as a co-activator of WT1 in the regulation of proapoptotic genes and suppressing WT1 mediated transactivation of antiapoptotitc genes. Moreover, the DNA damaging drug cytosine arabinoside (ara-C) induces expression of ZNF224 in K562 cells and this induction enhances cell apoptotic response to ara-C. These findings suggest that ZNF224 can be a mediator of DNA damage-induced apoptosis in leukemia cells.