IntroductionFerroptosis is a promising new target for ovarian cancer (OVCA) treatment. However, some OVCA cell types resist the induction of ferroptosis by limiting the intracellular accumulation of the labile iron pool (LIP).MethodsHEY, COV318 and PEO4 were treated with erastin and assessed for cell viability by using PI flow cytometry assays. Erastin-affected iron metabolism was analysed by using FerroOrange assay, Western Blot (WB) analysis of ferritin heavy chain (FtH), transferrin receptor (CD71), and ferroportin (FPN). Mitochondrial reactive oxygen species (mitROS) and lipid peroxidation were quantified via MitoSOX and BODIPY-C11 flow cytometry assays, respectively. Exosomes (EVs) were collected from cell culture media through ultracentrifugation and then enumerated and analyzed by Nanoparticale Tracking Analysis (NTA) and transmission electron microscopy (TEM). CD63 protein expression in EVs was measured through WB by using CD9 as a loading control. Loss-of-function assays for FtH and CD63 were performed by using siRNA-mediated transient transfection.ResultsWe demonstrate that erastin treatment (8 µM, 8 h) is accompanied by the release of iron-rich ferritin via EV pathway in COV318 and PEO4 OVCA cells, thus failing to exert cytotoxic effects. Mechanistically, erastin causes the upregulation of CD63, a tetraspanin involved in forming multivesicular bodies (MVBs) and EVs, and the increase of MBVs assessed by transmission electron microscopy. Consistent with these findings, EV isolation followed by nanoparticle tracking analysis revealed a significant increase in EVs/cell in erastin-treated COV318 and PEO4 cells. Notably, EVs harvested from these cells contained CD63 and FtH, a major iron-storage protein. Inhibition of EV biogenesis with GW4869 prevented FtH release and restored LIP accumulation, lipid peroxidation, and ferroptosis sensitivity in COV318 and PEO4 cells.DiscussionOverall, our results indicate that OVCA cells can utilize CD63+ EVs to secrete iron-rich ferritin as a mechanism to evade erastin-induced ferroptosis. These findings suggest that combining erastin with EV inhibitors could offer promising strategy for overcoming ferroptosis resistance in OVCA.

Cerebral malaria (CM), caused by infection with Plasmodium falciparum, is characterized by brain inflammation. Type I IFN inflammatory response underlies CM pathogenesis leading to brain endothelium dysfunction with the loss of blood-brain-barrier (BBB). We identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that activates type I IFN pathway and CXCL10 expression governing brain leukocyte infiltration and CM lethality in mice. Brain endothelial-specific IFNβ-reporter and STING1 KO mice unveiled a critical role of brain endothelial cells (BECs) in fuelling type I IFN-driven inflammation in the brain during malaria infection. We show that extracellular particles (EPs) derived from Plasmodium-infected erythrocytes and containing digestive vacuoles trigger a STING1-dependent IFNβ response in BECs. This response increases with heme content in digestive vacuoles, which directly activates BECs via STING1. Together, our findings uncovered that heme-stimulated endothelial STING1 drives type I IFN response and subsequent brain pathology.

(1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients.

(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second-leading cause of cancer deaths by 2030. Imaging techniques are the standard for monitoring the therapy response in PDAC, but these techniques have considerable limits, including delayed disease progression detection and difficulty in distinguishing benign from malignant lesions. Extracellular vesicle (EV) liquid biopsy is an emerging diagnosis modality. Nonetheless, the majority of research for EV-based diagnosis relies on point analyses of EVs at specified times, while longitudinal EV population studies before and during therapeutic interventions remain largely unexplored. (2) Methods: We analyzed plasma EV protein composition at diagnosis and throughout PDAC therapy. (3) Results: We found that IgG is linked with the diagnosis of PDAC and the patient’s response to therapy, and that the IgG+ EV population increases with disease progression and reduces with treatment response. Importantly, this covers PDAC patients devoid of the standard PDAC seric marker CA19.9 expression. We also observed that IgG is bound to EVs via the tumor antigen MAGE B1, and that this is independent of the patient’s inflammatory condition and IgG seric levels. (4) Conclusions: We here propose that a population analysis of IgG+ EVs in PDAC plasma represents a novel method to supplement the monitoring of the PDAC treatment response.

Diffuse large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma characterized by a heterogeneous behavior and in need of more accurate biological characterization monitoring and prognostic tools. Extracellular vesicles are secreted by all cell types and are currently established to some extent as representatives of the cell of origin. The present study characterized and evaluated the diagnostic and prognostic potential of plasma extracellular vesicles (EVs) proteome in DLBCL by using state-of-the-art mass spectrometry. The EV proteome is strongly affected by DLBCL status, with multiple proteins uniquely identified in the plasma of DLBCL. A proof-of-concept classifier resulted in highly accurate classification with a sensitivity and specificity of 1 when tested on the holdout test data set. On the other hand, no proteins were identified to correlate with non-germinal center B-cell like (non-GCB) or GCB subtypes to a significant degree after correction for multiple testing. However, functional analysis suggested that antigen binding is regulated when comparing non-GCB and GCB. Survival analysis based on protein quantitative values and clinical parameters identified multiple EV proteins as significantly correlated to survival. In conclusion, the plasma extracellular vesicle proteome identifies DLBCL cancer patients from healthy donors and contains potential EV protein markers for prediction of survival.

Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnβ1 transcription in the brain of mice infected with Plasmodium berghei ( Pba ). This STING1/IFNβ-mediated response increases brain CXCL10 governing the extent of brain leucocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFNβ-reporter and STING1-deficient Pba -infected mice, which are significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba -infected erythrocytes activated STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNβ in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin triggering brain endothelial inflammatory responses via STING1/IFNβ/CXCL10 axis crucial to CM pathogenesis and lethality. Significance CM results from loss of blood-brain endothelial barrier function caused by unrestrained inflammatory response in the natural course of infection by Plasmodium parasites. However, the role of brain endothelium in triggering inflammatory mechanisms is still undetermined. We found that the innate immune sensor STING1 is crucial for production of IFNβ in brain endothelial cells in Plasmodium -infected mice. This in turn stimulates CXCL10-mediated recruitment of leukocytes and subsequent brain inflammation and tissue damage. We identified within extracellular particles released from Plasmodium -infected erythrocytes, a fraction containing products of hemoglobin degradation, namely heme, which we show can bind STING1. Our results unravel a new angle of CM pathogenesis: heme contained in particles triggers the STING/IFNβ/CXCL10 axis in brain endothelial cells.

Through lateral transfer, extra-cellular vesicles (EVs) transport their DNA, miRNA, mRNA and proteins such as enzymes mediating drug resistance, transporters as well as growth factors to neighboring cells. By virtue of this horizontal transfer, EVs potentially regulate cell growth, migration, angiogenesis and metastasis and increase tissue permeability in cancer. Furthermore, EVs regulate immune factors and allow the tumor cells to evade immune recognition and cell death. To explore if the proteomes of exosomes support functional transfer of cancer hallmarks, in this meta-analysis, we compared EVs and whole cell proteomes from the NCI-60 human tumor cell line panel. We observed a subgroup of proteins in each cancer hallmark signature as highly abundant and consistently expressed in EVs from all cell lines. Among these were oncoproteins frequently targeted in cancer therapies whose presence on EVs could potentially render therapies less effective by serving as decoys.

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.

Acellular bronchoalveolar lavage (BAL) proteomics can partially separate lung cancer from non-lung cancer patients based on principal component analysis and multivariate analysis. Furthermore, the variance in the proteomics data sets is correlated mainly with lung cancer status and, to a lesser extent, smoking status and gender. Despite these advances BAL small and large extracellular vehicles (EVs) proteomes reveal aberrant protein expression in paracrine signaling mechanisms in cancer initiation and progression. We consequently present a case-control study of 24 bronchoalveolar lavage extracellular vesicle samples which were analyzed by state-of-the-art liquid chromatography-mass spectrometry (LC-MS). We obtained evidence that BAL EVs proteome complexity correlated with lung cancer stage 4 and mortality within two years´ follow-up (p value = 0.006). The potential therapeutic target DNMT3B complex is significantly up-regulated in tumor tissue and BAL EVs. The computational analysis of the immune and fibroblast cell markers in EVs suggests that patients who deceased within the follow-up period display higher marker expression indicative of innate immune and fibroblast cells (four out of five cases). This study provides insights into the proteome content of BAL EVs and their correlation to clinical outcomes.

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

Pancreatic cancers (PC) are highly metastatic with poor prognosis, mainly due to delayed detection. We previously showed that PC-derived extracellular vesicles (EVs) act on macrophages residing in the liver, eliciting extracellular matrix remodeling in this organ and marked hepatic accumulation of CD11b+ bone marrow (BM) cells, which support PC liver metastasis. We here show that PC-EVs also bind to CD11b+ BM cells and induce the expansion of this cell population. Transcriptomic characterization of these cells shows that PC-EVs upregulate IgG and IgA genes, which have been linked to the presence of monocytes/macrophages in tumor microenvironments. We also report here the transcriptional downregulation of genes linked to monocyte/macrophage activation, trafficking, and expression of inflammatory molecules. Together, these results show for the first time the existence of a PC–BM communication axis mediated by EVs with a potential role in PC tumor microenvironments.

Tumors are not isolated entities, but complex systemic networks involving cell-cell communication between transformed and non-transformed cells. The milieu created by tumor-associated cells may either support or halt tumor progression. In addition to cell-cell contact, cells communicate through secreted factors via a highly complex system involving characteristics such as ligand concentration, receptor expression and integration of diverse signaling pathways. Of these, extracellular vesicles, such as exosomes, are emerging as novel cell-cell communication mediators in physiological and pathological scenarios. Exosomes, membrane vesicles of endocytic origin released by all cells (both healthy and diseased), ranging in size from 30 to 150nm, transport all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and perform intercellular transfer of components, locally and systemically. By acting not only in tumor cells, but also in tumor-associated cells such as fibroblasts, endothelium, leukocytes and progenitor cells, tumor- and non-tumor cells-derived exosomes have emerged as new players in tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and immunologic remodeling. In addition, due to their property of carrying molecules from their cell of origin to the peripheral circulation, exosomes have been increasingly studied as sources of tumor biomarkers in liquid biopsies. Here we review the current literature on the participation of exosomes in the communication between tumor and tumor-associated cells, highlighting the role of this process in the setup of tumor microenvironments that modulate tumor initiation and metastasis.

Oncologic diseases do not behave as isolated entities. Instead, they are based on complex systemic networks involving cell-cell communication between cancerous and healthy cells of the host, which may either facilitate or prevent cancer progression. In addition to cell-cell contacts, cells communicate through secreted factors in a process modulated by ligand concentration, receptor availability and synergy amongst several signaling circuits. Of these secreted factors, exosomes, 30-150 nm membrane vesicles of endocytic origin released by virtually all cells, have emerged as important cell-cell communication players both in physiological and pathological scenarios by being carriers of all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and performing intercellular transfer of components, locally and systemically. By acting both in tumor and non-tumor cells, such as fibroblasts, leukocytes, endothelial and progenitor cells, tumor-and non-tumor cells-derived exosomes can modulate tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and the immune system. In this Review, we summarize the main findings of the literature on the roles of exosomes in mediating interactions between tumor and tumor-associated cells. We also discuss how the molecular composition analysis of circulating exosomes in clinical settings has emerged as an attractive non-invasive source of liquid biopsies for early diagnosis, prognosis and follow-up of patients with oncologic diseases. (c) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

The protective effect of sunscreens has been extensively evaluated in vivo as a measure of erythema induced in human skin and is expressed as Sun Protection Factor (SPF). In vitro alternatives that use human cells might overcome the limitations of testing on human beings. Here is proposed a broad and accurate in vitro approach for evaluating the efficacy of commercial sunscreens even under environmental conditions. This Cell dosimeter allowed the determination of Sun Protection Factor for DNA (DNA-SPF), using specific DNA repair enzymes and antibodies, and Sun Protection Factor for Lethal Damage (LD-SPF), by measuring cell viability and apoptosis induced after the irradiation of human cells. The use of xeroderma pigmentosum (XP) cells, which are deficient in DNA repair, rendered this assay more sensitive. The results revealed significant protection against the effects elicited by UVB radiation; however, there was no efficient protection from DNA lesions and cell death induced by UVA radiation or natural sunlight. This work demonstrates the environmental application of this biodosimeter for measuring UV-induced biological damage to human cells and supports the need for better evaluation of the UVA protection efficacy conferred by commercial sunscreens, in terms of induction of DNA lesions and cell death.

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

Human cells are constantly exposed to DNA damage. Without repair, damage can result in genetic instability and eventually cancer. The strong association between the lack of DNA damage repair, mutations and cancer is dramatically demonstrated by a number of cancer-prone human syndromes, such as xeroderma pigmentosum (XP), ataxia-telangiectasia (AT) and Fanconi anemia (FA). This review focuses on the historical discoveries related with these three diseases and describes their impact on the understanding of DNA repair mechanisms and the causes of human cancer. As deficiencies in DNA repair are also often related with progeria symptoms, unrepaired damage and aging are somehow related. Several other pathologies associated with DNA repair defects, genetic instability and increased cancer risk are also discussed. In fact, studies with cells from these many syndromes have helped in understanding important levels of protection against cancer and aging, although little help has actually been conferred to the patients in terms of therapy. Finally, the recent advances in combined basic and translational research on DNA repair and chemotherapy are presented.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1–132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68–258, encompassing part of the RWD and the C-terminal “ancient domain”; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68–258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

ABSTRACT Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei , the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei , an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes.