Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1β, experiments were also performed in an OSC cell line, which does not express HNF1β. Metabolic profiles, GSH quantification, HNF1β, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1β knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1β regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1β as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H2S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

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Background: Colorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.Methods: Selected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.Results: Except for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A threegene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway. Conclusions:Combined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.

Bone marrow contains endothelial progenitor cells (EPCs) that, upon pro-angiogenic stimuli, migrate and differentiate into endothelial cells (ECs) and contribute to re-endothelialization and neo-vascularization. There are currently no reliable markers to characterize EPCs, leading to their inaccurate identification. In the past, we showed that, in a panel of tumors, some cells on the vessel wall co-expressed CD14 (monocytic marker) and CD31 (EC marker), indicating a putative differentiation route of monocytes into ECs. Herein, we disclosed monocytes as potential EPCs, using in vitro and in vivo models, and also addressed the cancer context. Monocytes acquired the capacity to express ECs markers and were able to be incorporated into blood vessels, contributing to cancer progression, by being incorporated in tumor neo-vasculature. Reactive oxygen species (ROS) push monocytes to EC differentiation, and this phenotype is reverted by cysteine (a scavenger and precursor of glutathione), which indicates that angiogenesis is controlled by the interplay between the oxidative stress and the scavenging capacity of the tumor microenvironment.Over the last decade, different studies reported EPCs as essential in restoring injured vessels. EPCs belong to a subset of cells, arising from hematopoietic stem cells in bone marrow; upon pro-angiogenic stimuli, they proliferate, migrate, and differentiate into endothelial cells (ECs) [4][5][6]. Some reports addressing EPCs and disease, such as systemic sclerosis, showed contradictory and discrepant results about EPCs mobilization and differentiation; in part, because there is a lack of a precise panel of cell surface markers used for the characterization of this subset of cells [4][5][6][7][8][9][10]. In mouse embryonic vascular endothelium, erythro-myeloid progenitors (EMPs) can differentiate into ECs [11] and in a mouse model of carotid injury, monocytes (CD14 + cells) are capable of improving re-endothelialization [12]. In vivo and in vitro targeting of Tie2-monocytes decreases angiogenesis by abrogating EC proliferation [13][14][15] and an in vivo CCR2 (chemokine (C-C motif) receptor 2) knockout impairs monocytes recruitment and VEGFA (also named VEGF, vascular endothelial growth factor) expression, accompanied by a reduction in the angiogenesis rate [16]. The release of cytokines and pro-angiogenic factors (e.g., VEGFA, VEGFC, and VEGFD, TNFα (tumor necrosis factor α), IL-8 (interleukin-8), and FGF-2 (fibroblast growth factor-2), and extracellular matrix (ECM) modifying proteins (e.g., metalloproteinase-9 (MMP-9)) by macrophages enhances the tissue's ability to support capillary sprouting and vascular density [17,18]. The precise mechanism by which monocytes influence angiogenesis in tissue development, homeostasis, and diseases is not fully understood. However, different studies, have shown that under in vitro pro-angiogenic pressure, blood mononuclear cells can acquire endothelial markers and morphology [19][20][21]. In addition, in a previous study, we showed that some ECs sim...

Ovarian carcinoma (OvC) remains a major therapeutic challenge due to its propensity to develop resistance after an initial response to chemotherapy. Interactions of tumour cells with the surrounding microenvironment play a role in tumour survival, invasion capacity and drug resistance. Cancer models that retain tissue architecture and tumour microenvironment components are therefore essential to understand drug response and resistance mechanisms. Herein, our goal was to develop a long-term OvC patient-derived explant (OvC-PDE) culture strategy in which architecture and cell type heterogeneity of the original tumour would be retained. Samples from 25 patients with distinct OvC types and one with a benign tumour, were cultured for 30 days in agitation-based culture systems with 100% success rate. OvC-PDE cultures retained the original tumour architecture and main cellular components: epithelial cells, fibroblasts and immune cells. Epithelial cells kept their original levels of proliferation and apoptosis. Moreover, the major extracellular components, such as collagen-I and -IV, were retained in explants. OvC-PDE cultures were exposed to standard-of-care chemotherapeutics agents for 2 weeks, attesting the ability of the platform for drug assays employing cyclic drug exposure regimens. We established an OvC-PDE dynamic culture in which tumour architecture and cell type heterogeneity were preserved for the different OvC types, replicating features of the original tumour and compatible with long-term drug exposure for drug efficacy and resistance studies.

Initial fast transport was not associated with systemic inflammation and atherosclerosis. In a population with preserved RRF and absence of baseline serious co-morbidity, it was not predictive of worse prognosis. Other determinants of early peritoneal fast transport deserve investigation.

Steal syndrome is a feared complication of dialysis vascular access in a population becoming older and frailer. The aim of this study was to determine the predictor factors of steal syndrome. All proximal arteriovenous fistulas (AVFs), patent at day 30, inserted between January 2008 and December 2009 were studied. Data on age, gender, diabetes mellitus (DM) status, presence of coronary or peripheral artery disease, date of initiation of renal replacement therapy, date of access construction, localization, type of anastomosis, previous interventions, and outcome for AVF and patients were analyzed. There were 324 AVFs placed into 309 individual patients. The mean age was 66.7 ± 15.3 years, and the majority (53.7%) of the patients was male. Mean follow-up of all 324 fistulas was 18.6 ± 8.5 months. During follow-up, steal syndrome occurred in 26 (8%) of the AVFs. Univariate analysis revealed correlations between steal syndrome and DM (P = 0.002), brachiomedian fistulas (P = 0.016), and side-to-side (STS) anastomosis (P = 0.003). However, in multivariate analysis, the presence of DM, STS anastomosis, and female gender were found to be the independent risk factors. The strongest predictive factor was DM (odds ratio: 6.7; 95% confidence interval: 2.5-17.9). Being diabetic is the factor most predictive of having steal syndrome. In diabetic women, with a proximal access, it seems preferable to construct fistulas with end-to-side anastomosis to minimize the risk.