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Simple Summary Lung cancer is the leading cause of cancer-related death worldwide as patients are burdened with incredibly poor prognosis. Low survival rates are primarily attributed to lack of early detection and, therefore, timely therapeutic interventions. Late diagnosis is essentially caused by absent and non-specific symptoms, and compounded by inadequate diagnostic tools. We show here that a lung cancer biomarker, based on a simple blood test, might provide promising advantages for diagnostic assessment. Small extracellular vesicles (sEVs) are miniscule messengers that carry cancer biomarkers and are easily detected in the blood. We identify that the abundance of a specific micro-RNA, miR-3182, in these sEVs can be detected in the blood of lung cancer patients but not in controls with benign lung conditions. This demonstrates the potential use of miR-3182 as a biomarker for lung cancer diagnosis. Abstract With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.

BackgroundMesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton’s jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer’s disease-linked amyloid beta oligomers (AβOs).MethodsWe isolated and characterized EVs released by human Wharton’s jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AβOs.ResultshMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AβOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AβOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole.ConclusionshMSC-EVs protected hippocampal neurons from damage induced by AβOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer’s disease.

Breast cancer (BC) is a heterogeneous disease composed of multiple subtypes with different molecular characteristics and clinical outcomes. The metastatic process in BC depends on the transcription factors (TFs) related to epithelial-mesenchymal transition (EMT), including the master regulator Twist1. However, its role beyond EMT in BC subtypes remains unclear. Our study aimed to investigate the role of Twist1, beyond EMT, in the molecular subtypes of BC. In patients, we observed the overexpression of TWIST1 in the HER2+ group. The silencing of TWIST1 in HER2+ BC cells resulted in the upregulation of 138 genes and the downregulation of 174 genes compared to control cells in a microarray assay. In silico analysis revealed correlations between Twist1 and important biological processes such as the Th17-mediated immune response, suggesting that Twist1 could be relevant for IL-17 signaling in HER2+ BC. IL-17 signaling was then examined, and it was shown that TWIST1 knockdown caused the downregulation of leading members of IL-17 signaling pathway. Taken together, our findings suggest that Twist1 plays a role on IL-17 signaling in HER2+ BC.

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

While tumour cells are classically known to communicate via direct cell-to-cell contact and the secretion of soluble protein-based factors such as cytokines and growth factors, alternative novel mechanisms that promote tumour progression have recently emerged. Now, new critical components of the secretome thought to be involved in tumour progression are exosomes, small vesicles of endocytic origin that carry a variety of bioactive molecules, including proteins, lipids, RNA, as well as DNA molecules. Cancer cell-derived exosomes have been shown to participate in crucial steps of metastatic spread of a primary tumour, ranging from oncogenic reprogramming of malignant cells to formation of pre-metastatic niches. These effects are achieved through the mediation of intercellular cross-talk and subsequent modification of both local and distant microenvironments in an autocrine and paracrine fashion. Here, we summarise the recent findings that implicate this non-canonical signalling within the tumour as a critical driver of metastatic disease progression, and discuss how understanding the molecular mechanisms involved in exosome-mediated metastasis is of great value for the development of new therapeutic strategies to prevent cancer progression.

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow–derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer–derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816–27. ©2016 AACR.

Fine-mapping of breast cancer GWAS regions has identified 195 high confidence signals containing more than 5,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements and thus may confer the risk by altering gene expression in cis. We analyzed allelic expression imbalance (AEI) of genes surrounding known breast cancer signals, using normal breast and breast tumor transcriptome data and imputed genotypes. Fourteen genes, including NTN4, were identified whose expression was associated with CCV genotype. We showed that CCVs at this signal were located within an enhancer that physically interacts with the NTN4 promoter. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. Here, we present the most comprehensive AEI analysis of breast cancer CCVs resulting in identification of new candidate risk genes.

Hypoxia and necrosis are fundamental features of glioma, and their emergence is critical for the rapid biological progression of this fatal tumor. The presence of vaso-occlusive thrombus is higher in grade IV tumors [glioblastoma multiforme (GBM)] compared with lower grade tumors, suggesting that the procoagulant properties of the tumor contribute to its aggressive behavior, as well as the establishment of tumor hypoxia and necrosis. Tissue factor (TF), the primary cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Phosphatase and tensin homolog (PTEN) loss and hypoxia are two common alterations observed in glioma that may be responsible for TF upregulation. In the present study, ST1 and P7 rat glioma lines, with different levels of aggressiveness, were comparatively analyzed with the aim of identifying differences in procoagulant mechanisms. The results indicated that P7 cells display potent procoagulant activity compared with ST1 cells. Flow cytometric analysis showed less pronounced levels of TF in ST1 cells compared with P7 cells. Notably, P7 cells supported factor X (FX) activation via factor VIIa, whereas no significant FXa generation was observed in ST1 cells. Furthermore, the exposure of phosphatidylserine on the surface of P7 and ST1 cells was investigated. The results supported the assembly of prothrombinase complexes, accounting for the production of thrombin. Furthermore, reverse transcription-quantitative polymerase chain reaction showed that CoCl2 (known to induce a hypoxic-like stress) led to an upregulation of TF levels in P7 and ST1 cells. Therefore, increased TF expression in P7 cells was accompanied by increased TF procoagulant activity. In addition, hypoxia increased the shedding of procoagulant TF-bearing microvesicles in both cell lines. Finally, hypoxic stress induced by treatment with CoCl2 upregulated the expression of the PAR1 receptor in both P7 and ST1 cells. In addition to PAR1, P7, but not ST1 cells, expressed higher levels of PAR2 under hypoxic stress. Thus, modulating these molecular interactions may provide additional insights for the development of more efficient therapeutic strategies against aggressive glioma.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling. CA extern

Cancer-derived exosomes have been recognised as important mediators of cell-cell communication in the tumour microenvironment, thereby playing crucial pro-tumourigenic roles. Exosomes are found in different biological fluids obtained from cancer patients, suggesting that these vesicles can leave the primary cancer lesion, circulate systemically and distribute to other distant tissues. Recently, circulating cancer-derived exosomes have been reported to contribute to the guidance of metastatic dissemination to specific sites of secondary tumour outgrowth, a phenomenon termed organotropism. Clinical research has also provided evidence that exosomes can be used as novel delivery vehicles for therapeutic molecules, as well as serve as biomarkers for predicting cancer patient outcomes. In this chapter, we review the mechanisms that influence the fate of tumour-derived exosomes in vivo. We also consider the use of novel technical approaches that in the future will permit assessment of cancer-derived exosomes biodistribution in a physiologically precise manner, thereby furthering our current understanding of the systemic effects of these vesicles.

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell-of-origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway-enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial-mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell-derived exosomes reflect the phenotype of the cells-of-origin.

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57/BL6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies.

Human β-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and β2–β3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure–function relation of the hBD6–CCR2 interaction, which is a promising target for the design of novel anticancer agents.

Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.

BackgroundRhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.Methodology/principal findingsHere we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.Conclusions/significanceTaken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

SummaryShedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte- derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation- related protumoural responses.

Exposure of phosphatidylserine (PS) on cellular membranes and membrane-derived microvesicles stimulates a number of anti-inflammatory responses involved in malignant processes. Herein we show that B16F10 cells, a highly metastatic melanoma cell line, produce large quantities of PS-containing microvesicles in vitro. Tumor microvesicles increased TGF-beta(1) production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, microvesicles induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Altogether, this is the first demonstration that tumor-derived microvesicles favor the establishment of melanoma metastasis in a PS-dependent manner, possibly by down-regulating the host's inflammatory and/or anti-tumoral immune responses.

Background: It is largely accepted that cancer patients have a high incidence of thrombosis which is strictly related to prognosis of cancer. Aim: We prospectively evaluated 81 consecutively lung adenocarcinoma patients in order to identify prothrombotic factors and thrombosis incidence. Methods: Patients were submitted to Doppler examination of arms and limbs every three months or in case of thrombosis suspicion. Additional image evaluations were made in case of inconclusive diagnosis. Levels of C protein, platelets, and fibrinogen, factor VIII and IX, D-dimmer, antitrombin and apoptotic microparticles were evaluated each six months. Samples were also tested for lupus anticoagulant (LA) antibodies and levels of IL-1, IL-6 and Tumor Necrosis Factor (TNF). A group of 30 healthy blood donors was used as control. Results: From the studied patients 21% had thrombosis, 38% had fibrinogen above 500 mg/dl, 42% had FVIII activity above 200%, 63% had increased D-dimmer, 60% had more than 1x106 microparticles/ml serum, and none of them have decreased levels of C protein or antitrombin. A small fraction of patients had increase of platelet counts. At immunological evaluation 52% of patients had LA detection and 63% showed increased leves of IL-6. Patients with at least one of the variables thrombosis, high levels of IL-6 (p Conclusion: There is a high incidence of thrombosis in lung adenocarcinoma patients, on which FVIII and LA significantly influenced and microparticles only showed a tendency. However, presence of LA, thrombosis or increased levels of IL-6 or FVIII showed directly interfered with patient’s survival.

Background Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. Results We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region. Conclusions Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.