Implicaciones funcionales del pericito en la respuesta inmune y en angiogénesis: identificación de potenciales marcadores predictivos de respuesta a terapias antiangiogénicas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 4 de Diciembre de 2013

Basement membrane-rich Organoids with functional human blood vessels are permissive niches for human breast cancer metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention

The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling

SARS-CoV-2 Infection in Multiple Sclerosis

To understand COVID-19 characteristics in people with multiple sclerosis (MS) and identify high-risk individuals due to their immunocompromised state resulting from the use of disease-modifying treatments. Retrospective and multicenter registry in patients with MS with suspected or confirmed COVID-19 diagnosis and available disease course (mild = ambulatory; severe = hospitalization; and critical = intensive care unit/death). Cases were analyzed for associations between MS characteristics and COVID-19 course and for identifying risk factors for a fatal outcome. Of the 326 patients analyzed, 120 were cases confirmed by real-time PCR, 34 by a serologic test, and 205 were suspected. Sixty-nine patients (21.3%) developed severe infection, 10 (3%) critical, and 7 (2.1%) died. Ambulatory patients were higher in relapsing MS forms, treated with injectables and oral first-line agents, whereas more severe cases were observed in patients on pulsed immunosuppressors and critical cases among patients with no therapy. Severe and critical infections were more likely to affect older males with comorbidities, with progressive MS forms, a longer disease course, and higher disability. Fifteen of 33 patients treated with rituximab were hospitalized. Four deceased patients have progressive MS, 5 were not receiving MS therapy, and 2 were treated (natalizumab and rituximab). Multivariate analysis showed age (OR 1.09, 95% CI, 1.04-1.17) as the only independent risk factor for a fatal outcome. This study has not demonstrated the presumed critical role of MS therapy in the course of COVID-19 but evidenced that people with MS with advanced age and disease, in progressive course, and those who are more disabled have a higher probability of severe and even fatal diseas

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

p38α limits the contribution of MAP17 to cancer progression in breast tumors

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER (SAF2009-08605), Consejeria de Ciencia e Innovacion and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142). AC's laboratory is also funded by a fellowship from the Fundacion Oncologica FERO, supported by Fundació Josep Botet.Peer reviewe

Structural analysis of rLCC111.

<p>Gel filtration chromatogram profile, as measured by UV absorbance at 280 nm (blue trace), and molar mass (red trace), as measured by MALLS (only the data at the central part of the peak is shown, and can be read at the right hand axis, in red color) The mass measured at the centre of the peak is 335 kDa (A). The inset shows a coomassie stained SDS-PAGE under reduction conditions of the input and the indicated separately collected fractions. Circular dichroism spectrum recorded at 25°C and thermal denaturation measured by the change in the ellipticity at 222 nm (B).</p

ELISA to assay binding of rabbit anti-laminin antibody (A) and the anti-laminin scFv antibody L36 (B) to plastic coated with LM111 or with rLCC111.

<p>As control, we used rabbit anti-fd-bacteriophage antibody (A) and the anti-p32 scFv antibody 2.15 (B). ELISA to demonstrate the presence in the same rLCC111 molecule of two truncated laminin chains, tα1 and tβ1 or tγ1 and tb1 (C).</p

rLCC111 substrates do not support cell attachment.

<p>Adhesion of HT0180 cells to plastic-coated LM111 or rLCC111 (ranging from 0.08 to 20 μg/ml), or BSA was measured by bioluminescence. (A). Data are plotted as the log of fold change in adhesion relative to BSA. The coating efficiency of LM111 and rLCC111 was monitored by ELISA using a polyclonal anti-laminin antibody (B). Soluble rLCC111 (5 to 20 mg/ml) inhibits adhesion of HT1080 cells (C) to plastic immobilized intact LM111 (10 mg/ml). Data shown are from a representative experiment out of three independent ones. (*, p<0,05; **, p<0,005).</p

Representative gelatin zymogram showing MMP9 and MMP2 activities in HT1080 cells were cultured under serum free conditions for 72 hours under substrate-independent conditions on uncoated wells (1) or on LM111 (2) or rLCC111 (3) coated wells (A).

<p>Densitometric evaluation of MMP-9 (B) and latent MMP-2 (C).</p