Angiogenic content of microparticles in patients with diabetes and coronary artery disease predicts networks of endothelial dysfunction.

BACKGROUND: Elevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD). METHODS: A total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs' apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array. RESULTS: Levels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-ß1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-ß1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes. CONCLUSION: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction.

Akt-Activated Endothelium Increases Cancer Cell Proliferation and Resistance to Treatment in Ovarian Cancer Cell Organoids.

Ovarian cancer (OC) is a heterogeneous disease characterized by its late diagnosis (FIGO stages III and IV) and the importance of abdominal metastases often observed at diagnosis. Detached ovarian cancer cells (OCCs) float in ascites and form multicellular spheroids. Here, we developed endothelial cell (EC)-based 3D spheroids to better represent in vivo conditions. When co-cultured in 3D conditions, ECs and OCCs formed organized tumor angiospheres with a core of ECs surrounded by proliferating OCCs. We established that Akt and Notch3/Jagged1 pathways played a role in angiosphere formation and peritoneum invasion. In patients' ascites we found angiosphere-like structures and demonstrated in patients' specimens that tumoral EC displayed Akt activation, which supports the importance of Akt activation in ECs in OC. Additionally, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCC-EC crosstalk relating to OCC proliferation and during peritoneal invasion. Our results support the use of multicellular spheroids to better model tumoral and stromal interaction. Such models could help decipher the complex pathways playing critical roles in metastasis spread and predict tumor response to chemotherapy or anti-angiogenic treatment.

Angiocrine endothelium: from physiology to cancer.

The concept of cancer as a cell-autonomous disease has been challenged by the wealth of knowledge gathered in the past decades on the importance of tumor microenvironment (TM) in cancer progression and metastasis. The significance of endothelial cells (ECs) in this scenario was initially attributed to their role in vasculogenesis and angiogenesis that is critical for tumor initiation and growth. Nevertheless, the identification of endothelial-derived angiocrine factors illustrated an alternative non-angiogenic function of ECs contributing to both physiological and pathological tissue development. Gene expression profiling studies have demonstrated distinctive expression patterns in tumor-associated endothelial cells that imply a bilateral crosstalk between tumor and its endothelium. Recently, some of the molecular determinants of this reciprocal interaction have been identified which are considered as potential targets for developing novel anti-angiocrine therapeutic strategies.

Pathogenesis of chronic rhinosinusitis with nasal polyps: role of IL-6 in airway epithelial cell dysfunction.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by an alteration in airway epithelial cell functions including barrier function, wound repair mechanisms, mucociliary clearance. The mechanisms leading to epithelial cell dysfunction in nasal polyps (NPs) remain poorly understood. Our hypothesis was that among the inflammatory cytokines involved in NPs, IL-6 could alter epithelial repair mechanisms and mucociliary clearance. The aim of this study was to evaluate the in vitro effects of IL-6 on epithelial repair mechanisms in a wound repair model and on ciliary beating in primary cultures of Human Nasal Epithelial Cells (HNEC). METHODS: Primary cultures of HNEC taken from 38 patients during surgical procedures for CRSwNP were used in an in vitro model of wound healing. Effects of increasing concentrations of IL-6 (1 ng/mL, 10 ng/mL, and 100 ng/mL) and other ILs (IL-5, IL-9, IL-10) on wound closure kinetics were compared to cultures without IL-modulation. After wound closure, the differentiation process was characterized under basal conditions and after IL supplementation using cytokeratin-14, MUC5AC, and ßIV tubulin as immunomarkers of basal, mucus, and ciliated cells, respectively. The ciliated edges of primary cultures were analyzed on IL-6 modulation by digital high-speed video-microscopy to measure: ciliary beating frequency (CBF), ciliary length, relative ciliary density, metachronal wavelength and the ciliary beating efficiency index. RESULTS: Our results showed that: (i) IL-6 accelerated airway wound repair in vitro, with a dose-response effect whereas no effect was observed after other ILs-stimulation. After 24 h, 79% of wounded wells with IL6-100 were fully repaired, vs 46% in the IL6-10 group, 28% in the IL6-1 group and 15% in the control group; (ii) specific migration analyses of closed wound at late repair stage (Day 12) showed IL-6 had the highest migration compared with other ILs (iii) The study of the IL-6 effect on ciliary function showed that CBF and metachronal wave increased but without significant modifications of ciliary density, length of cilia and efficiency index. CONCLUSION: The up-regulated epithelial cell proliferation observed in polyps could be induced by IL-6 in the case of prior epithelial damage. IL-6 could be a major cytokine in NP physiopathology.

A de novo synonymous variant in EFTUD2 disrupts normal splicing and causes mandibulofacial dysostosis with microcephaly: case report.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is a rare autosomal dominant genetic disease characterized by intellectual and growth retardations, as well as major microcephaly, induced by missense and splice site variants or microdeletions in the EFTUD2 gene. CASE PRESENTATION: Here, we investigate the case of a young girl with symptoms of MFDM and a normal karyotype. Whole-exome sequencing of the family was performed to identify genetic alterations responsible for this phenotype. We identified a de novo synonymous variant in the EFTUD2 gene. We demonstrated that this synonymous variant disrupts the donor splice-site in intron 9 resulting in the skipping of exon 9 and a frameshift that leads to a premature stop codon. CONCLUSIONS: We present the first case of MFDM caused by a synonymous variant disrupting the donor splice site, leading to exon skipping.

Hyperthermic intraperitoneal chemotherapy (HIPEC): Should we look closer at the microenvironment?

The age of cancer as an isolated single-cell concept is now behind us. It is now established that epithelial ovarian cancer, like other cancers, interacts with the healthy bystander cells to influence them and takes advantage of their nutritional, immunological, disseminating and other capacities. This interaction has become a therapeutic target, as shown by the numerous studies on this subject. Intraperitoneal chemo-hyperthermia has been part of the therapeutic armamentarium for some time yet its efficiency in ovarian cancer has only been recently proven in a randomized controlled trial. However, its therapeutic performance is not revolutionary and epithelial ovarian cancer maintains a high mortality. In this review, we studied the impact of HIPEC on the microenvironment and vice versa to determine whether it could be the key to this lukewarm efficacy. We began by exploring the modalities of HIPEC and establishing the reasons that make this treatment topical. Then, we examined its impact on each element of the tumor environment to obtain a global view of the resistance mechanisms at work in HIPEC.

En bloc greenlight laser enucleation of prostate (GreenLEP): about the first hundred cases.

PURPOSE: To report the functional outcomes, perioperative morbidity and surgical learning curve key points using "en bloc" greenlight enucleation of prostate (EB-GreenLEP) for patients with refractory lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH). METHODS: Between December, 2015 and May, 2018, all consecutive patients with refractory LUTS due to BPH in our institution were included and underwent EB-GreenLEP by a single surgeon. Perioperative data, complications and functional outcomes at 1-, 6- and 12-month follow-ups were collected and retrospectively analyzed. RESULTS: One hundred patients were included whose median age was 69 years. The median prostate volume (PV) was 84 mL and median enucleated PV was 45.5 mL. Mean irrigation, catheterization and hospitalization times were 1.3, 1.4 and 1.6 days, respectively. Average follow-up was 9.3 months. A single high-grade Clavien-Dindo complication occurred. No urinary retention was reported. Two conversions to conventional resection of the prostate were noted. Three patients had postoperative urinary incontinence at 6 months, only one at 1 year (1%). At 1, 6 and 12 months, there was a significant improvement in IPSS score, QoL and Qmax. Enucleation and energy efficiency ratios were shorter after the 30th procedure. We demonstrated a linear correlation between enucleation time and PV (r = 0.53, p < 0.0001). CONCLUSION: Our study shows that the mid-term functional results of EB-GreenLEP are comparable to other laser sources for the endoscopic enucleation of the prostate but with a shorter learning curve. We showed that, with (a) low rates of complications and a short hospital stay, EB-GreenLEP can manage medium-size glands (60-90 mL).

Skin trophicity improvement by mechanotherapy for lipofilling-based breast reconstruction postradiation therapy.

BACKGROUND: Post-mastectomy irradiation severely impairs skin trophicity resulting in poor prosthetic implant outcome. Autologous fat grafting improves skin quality allowing minimally invasive approach with prosthetic reconstruction. Here, we report our pilot experience of preoperative mechanotherapy to optimize lipofilling and subsequent prosthetic reconstruction outcome. METHODS: We retrospectively included 65 women that had breast reconstruction using autologous fat grafting and implant placement from 2012 to 2018 benefiting or not from mechanotherapy before the reconstructive procedure. Demographic and surgical outcomes were recorded. RESULTS: The volume of fat injected was significantly superior in the mechanotherapy group compared with the controls for the first and second lipofilling (259.3 mL vs 150.6 mL and 251.8 mL vs 154 mL, respectively). Sixteen patients among controls required a pre-expansion prosthesis compared with none in the endermology group. The prosthesis volume was smaller in the endermology group. Six patients in the endermology group had a reconstruction without prosthesis. The aesthetic score evaluated by patients was 4.8 with no statistically significant difference between the two groups. CONCLUSION: Preoperative skin mechanotherapy and postoperative skin mechanotherapy increase skin compliance. It is associated with a higher volume of fat injection and lower prosthesis volume. If confirmed in a prospective study, endermology could become a standard in patients' preparation for lipofilling-based reconstruction.

Akt-activated endothelium promotes ovarian cancer proliferation through notch activation.

BACKGROUND: One main challenge in ovarian cancer rests on the presence of a relapse and an important metastatic disease, despite extensive surgical debulking and chemotherapy. The difficulty in containing metastatic cancer is partly due to the heterotypic interaction of tumor and its microenvironment. In this context, evidence suggests that endothelial cells (EC) play an important role in ovarian tumor growth and chemoresistance. Here, we studied the role of tumor endothelium on ovarian cancer cells (OCCs). METHODS: We evaluated the effect of activated endothelial cells on ovarian cancer cell proliferation and resistance to chemotherapy and investigated the survival pathways activated by endothelial co-culture. RESULTS: The co-culture between OCCs and E4+ECs, induced an increase of OCCs proliferation both in vitro and in vivo. This co-culture induced an increase of Notch receptors expression on OCC surface and an increase of Jagged 1 expression on E4+ECs surface and activation of survival pathways leading to chemoresistance by E4+ECs. CONCLUSION: The targeting of aberrant NOTCH signaling could constitute a strategy to disrupt the pro-tumoral endothelial niche.

Direct and indirect P-glycoprotein transfers in MCF7 breast cancer cells.

The efflux protein P-glycoprotein (P-gp) is over expressed in many cancer cells and has a known capacity to confer multi-drug resistance to cytotoxic therapies. We provide a mathematical model for the direct cell-to-cell transfer of proteins between cells and the indirect transfer between cells and the surrounding liquid. After a mathematical analysis of the model, we construct an adapted numerical scheme and give some numerical simulations. We observe that we obtain a better fit with the experimental data when we take into account the indirect transfer of the protein released in a dish. This quantity, usually neglected by the experimenters, seems to influence the results.

CCL2/CCL5 secreted by the stroma induce IL-6/PYK2 dependent chemoresistance in ovarian cancer.

BACKGROUND: Minimal residual disease is the main issue of advanced ovarian cancer treatment. According to the literature and previous results, we hypothesized that Mesenchymal Stromal Cells (MSC) could support this minimal residual disease by protecting ovarian cancer cells (OCC) from chemotherapy. In vitro study confirmed that MSC could induce OCC chemoresistance without contact using transwell setting. Further experiments showed that this induced chemoresistance was dependent on IL-6 OCC stimulation. METHODS: We combined meticulous in vitro profiling and tumor xenograft models to study the role of IL-6 in MSC/OCC intereactions. RESULTS: We demonstrated that Tocilizumab® (anti-IL-6R therapy) in association with chemotherapy significantly reduced the peritoneal carcinosis index (PCI) than chemotherapy alone in mice xenografted with OCCs+MSCs. Further experiments showed that CCL2 and CCL5 are released by MSC in transwell co-culture and induce OCCs IL-6 secretion and chemoresistance. Finally, we found that IL-6 induced chemoresistance was dependent on PYK2 phosphorylation. CONCLUSIONS: These findings highlight the potential key role of the stroma in protecting minimal residual disease from chemotherapy, thus favoring recurrences. Future clinical trials targeting stroma could use anti-IL-6 therapy in association with chemotherapy.

Surgical peritoneal stress creates a pro-metastatic niche promoting resistance to apoptosis via IL-8.

BACKGROUND: The mainstay of treatment of advanced ovarian cancer (AOC) involves chemotherapy, and debulking surgery. However, despite optimal surgical procedure and adjuvant chemotherapy, 60% of patients with AOC will relapse within 5 years. Most recurrences occur in the peritoneal cavity, suggesting the existence of occult sanctuaries where ovarian cancer cells (OCC) are protected. In murine models, surgical stress favors tumor growth; however, it has never been established that surgery may affect OCC sensitivity to subsequent chemotherapy. In this study, we investigated how the surgical stress could affect the chemosensitivity of OCC. METHODS: To avoid bias due to tumor burden in peritoneal cavity and duration of surgery, we used peritoneal biopsies from patients without a malignancy at precise time points. During laparotomies, peritoneal biopsies at the incision site were performed at the time of incision (H0 sample) and 1 h after initiation of surgery (H1 sample). We evaluated the chemoresistance to Taxol (0-20 µM) induced by H0 or H1 incubation (24 h) in two ovarian cancer cell lines OVCAR3 and SKOV3 and a primary cancer cell lines derived in our laboratory. RESULTS: Our results indicate that stressed peritoneum overexpressed cytokines, resulting in OCC increased resistance to therapy. Among these cytokines, IL8 was responsible for the resistance to apoptosis through the AKT pathway activation. Chemoresistance in OCC persists through the establishment of an autocrine IL8 loop. Finally, in a cohort of 32 patients, we showed an impact of IL8 tumoral overexpression on chemosensitivity and survival outcomes with a significant association to earlier recurrence. CONCLUSIONS: Our study demonstrated that precision surgery where targeted treatment would be used in combination with surgery is essential to obtain better tumor control.

Halfway between 2D and Animal Models: Are 3D Cultures the Ideal Tool to Study Cancer-Microenvironment Interactions?

An area that has come to be of tremendous interest in tumor research in the last decade is the role of the microenvironment in the biology of neoplastic diseases. The tumor microenvironment (TME) comprises various cells that are collectively important for normal tissue homeostasis as well as tumor progression or regression. Seminal studies have demonstrated the role of the dialogue between cancer cells (at many sites) and the cellular component of the microenvironment in tumor progression, metastasis, and resistance to treatment. Using an appropriate system of microenvironment and tumor culture is the first step towards a better understanding of the complex interaction between cancer cells and their surroundings. Three-dimensional (3D) models have been widely described recently. However, while it is claimed that they can bridge the gap between in vitro and in vivo, it is sometimes hard to decipher their advantage or limitation compared to classical two-dimensional (2D) cultures, especially given the broad number of techniques used. We present here a comprehensive review of the different 3D methods developed recently, and, secondly, we discuss the pros and cons of 3D culture compared to 2D when studying interactions between cancer cells and their microenvironment.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Structural and functional analysis of tunneling nanotubes (TnTs) using gCW STED and gconfocal approaches.

BACKGROUND INFORMATION: Tunneling nanotubes (TnTs) are thin plasma membrane bridges mediating transfers of materials and signals between cells. Heterogeneity of heterocellular and homocellular TnTs is largely described but ultrafine imaging of these light-sensitive floating nanometric structures represents a real challenge in microscopy. We propose here imaging strategies designed to dissect structural and dynamic aspects of TnT formation and function in fixed or living PC12 cells. RESULTS: Through time-gated Continuous Wave STimulated Emission Depletion (gCW STED) nanoscopy associated with deconvolution, we provided nanoscale details of membrane and cytoskeleton organisations in two subtypes of TnTs, namely type 1 TnT (TnT1) and type 2 TnT (TnT2). In fixed PC12 cells, TnT1 (length, several tens of micrometres; diameter, 100-650 nm) exhibited a large trumpet-shaped origin, a clear cytosolic tunnel and different bud-shaped connections from closed-ended to open-ended tips. TnT1 contained both actin and tubulin. TnT2 (length, max 20 µm, diameter, 70-200 nm) only contained actin without clear cytosolic tunnel. In living PC12 cells, we observed through gCW STED additional details, unrevealed so far, including a filament spindle emerging from an organising centre at the origin of TnT1 and branched or bulbous attachments of TnT2. However, the power of depletion laser in STED nanoscopy was deleterious for TnTs and prolonged time-lapse experiments were almost prohibited. By circumventing the hazard of photoxicity, we were able to monitor dynamics of bud-shaped tips and intercellular transfer of wheat germ agglutinin labelled cellular elements through time-gated confocal microscopy. CONCLUSIONS: Our work identified new structural characteristics of two subtypes of TnTs in PC12 cells as well as dynamics of formation and transfer through complementary imaging methods combined with image processing. Therefore, we could achieve maximum lateral resolution and sample preservation during acquisitions to reveal new insights into TnT studies. SIGNIFICANCE: Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication.

Metabolic signatures differentiate ovarian from colon cancer cell lines.

BACKGROUND: In this era of precision medicine, the deep and comprehensive characterization of tumor phenotypes will lead to therapeutic strategies beyond classical factors such as primary sites or anatomical staging. Recently, "-omics" approached have enlightened our knowledge of tumor biology. Such approaches have been extensively implemented in order to provide biomarkers for monitoring of the disease as well as to improve readouts of therapeutic impact. The application of metabolomics to the study of cancer is especially beneficial, since it reflects the biochemical consequences of many cancer type-specific pathophysiological processes. Here, we characterize metabolic profiles of colon and ovarian cancer cell lines to provide broader insight into differentiating metabolic processes for prospective drug development and clinical screening. METHODS: We applied non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography and gas chromatography for the metabolic phenotyping of four cancer cell lines: two from colon cancer (HCT15, HCT116) and two from ovarian cancer (OVCAR3, SKOV3). We used the MetaP server for statistical data analysis. RESULTS: A total of 225 metabolites were detected in all four cell lines; 67 of these molecules significantly discriminated colon cancer from ovarian cancer cells. Metabolic signatures revealed in our study suggest elevated tricarboxylic acid cycle and lipid metabolism in ovarian cancer cell lines, as well as increased ß-oxidation and urea cycle metabolism in colon cancer cell lines. CONCLUSIONS: Our study provides a panel of distinct metabolic fingerprints between colon and ovarian cancer cell lines. These may serve as potential drug targets, and now can be evaluated further in primary cells, biofluids, and tissue samples for biomarker purposes.

Breast cancer cells promote a notch-dependent mesenchymal phenotype in endothelial cells participating to a pro-tumoral niche.

BACKGROUND: Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness. METHODS: Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition. RESULTS: Our results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECs(Mes)) in vitro. The ECs(Mes) acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECs(Mes) were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECs(Mes) were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFß and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFß down-regulated Smad1/5 phosphorylation and Jag1(KD) tumor cells were unable to initiate the process. CONCLUSIONS: Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFß pathways in the initiation of mesenchymal phenotype may propose new stromal targets.

SDF-1alpha concentration dependent modulation of RhoA and Rac1 modifies breast cancer and stromal cells interaction.

BACKGROUND: The interaction of SDF-1alpha with its receptor CXCR4 plays a role in the occurrence of distant metastasis in many solid tumors. This interaction increases migration from primary sites as well as homing at distant sites. METHODS: Here we investigated how SDF-1α could modulate both migration and adhesion of cancer cells through the modulation of RhoGTPases. RESULTS: We show that different concentrations of SDF-1α modulate the balance of adhesion and migration in cancer cells. Increased migration was obtained at 50 and 100 ng/ml of SDF-1α; however migration was reduced at 200 ng/ml. The adhesion between breast cancer cells and BMHC was significantly increased by SDF-1α treatment at 200 ng/ml and reduced using a blocking monoclonal antibody against CXCR4. We showed that at low SDF-1α concentration, RhoA was activated and overexpressed, while at high concentration Rac1 was promoting SDF-1α mediating-cell adhesion. CONCLUSION: We conclude that SDF-1α concentration modulates migration and adhesion of breast cancer cells, by controlling expression and activation of RhoGTPases.

Epigenetics and Cardiovascular Disease in Diabetes.

Type 2 diabetes has become a major health issue worldwide. Chronic hyperglycemia induces a low-grade inflammation that, on top of other mechanisms, leads to endothelial dysfunction. Mounting evidence suggests that DNA methylation, post-translational modifications of histones, and long non-coding RNAs play an important role in the initiation, maintenance, and progression of both macro- and micro-vascular complications of diabetes. Long-term exposure to hyperglycemia induces epigenetic changes that could become irreversible, a phenomenon known as the 'metabolic memory.' Whether epigenetic-based therapies could be used to slow or limit the progression of cardiovascular disease remains unclear. While non-coding RNAs are currently investigated as potential biomarkers that predict diabetic cardiovascular disease incidence and progression, their therapeutic role is only hypothetical. In this review, we highlight the latest findings in experimental and clinical studies relevant to epigenetics and cardiovascular disease in diabetes.

Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.