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Supplementary Figure from Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer

Supplementary Table from Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti–PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti–PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

Supplementary Figure 1 and 2. Generation and Characterisation of ECCre+;FAKY397F/Y397F and ECCre+;FAKY861F/Y861F mice.

Supplementary Figure 6. Knocking down Ang-2 in B16F0s cells ablates angiogenic response in ECCre+;FAKY861F/Y861F mice in vivo.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F–mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased β1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in β1 integrin activation. This corresponds with a decrease in Vegfa–stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell–stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo.Significance:Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

This work aims to develop safety shoes, with thermal regulation systems, namely innovative heating and cooling systems. Heating system was developed using printing techniques; and cooling system was developed using the integration of Peltier modules in the shoe structure. These materials are based on the Peltier effect, in which, when an electric current is applied, the heat moves from one face to the other, being subsequently removed using thermal dissipation methods. This effect allows an active cooling. Given the high technological challenge of integrating cooling systems into footwear, this paper will present only developments related to cooling system.

A correction to this paper has been published: https://doi.org/10.1007/s10456-021-09802-9

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrβCre+;FAKWT/WT, PdgfrβCre+;FAKY397F/Y397F and PdgfrβCre+;FAKY861F/Y861F mice, our data demonstrate that tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrβCre+;FAKY861F/Y861F but not PdgfrβCre+;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of a pericyte derived ‘pericrine’ signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

A adolescência, é conceptualizada como o período ocorrido entre a infância e a vida adulta, sendo uma etapa do ciclo de vida, marcada por profundas transformações físicas, psicológicas, sociais, afetivas e emocionais, podendo constituir-se, momento de vulnerabilidade, foco do cuidado da Enfermagem. Estudos recentes, desenvolvidos no decorrer da realidade pandémica, pela infeção COVID -19, causada pelo SARS-COV-2, veem espelhar o grande impacto que esta situação de saúde pública, trouxe à sociedade e em particular aos adolescentes, traduzindo-se em comportamentos desadaptativos, preditores de sofrimento mental. Sensível a estas necessidades emergentes, o Enfermeiro Especialista em Enfermagem de Saúde Mental e Psiquiátrica, tem um papel fulcral em promover/facilitar/capacitar uma transição mais equilibrada e harmoniosa, junto do adolescente em sofrimento mental. Este, detentor de uma compreensão singular do fenómeno de sofrimento e de quanto isto implica no projeto de vida do indivíduo, desenvolve conhecimentos e vivencias que lhe conferem competências, e que por sua vez lhe permitem mobilizar-se como instrumento terapêutico, junto do individuo em sofrimento. Posto isto, contrariando o paradigma vivencial de algumas realidades atuais, as abordagens terapêuticas não farmacológicas adquirem particular importância, onde o recurso a mediadores expressivos, nomeadamente a música, se apresenta como um precioso instrumento na intervenção com adolescentes, por esta assumir parte integrante da sua vida. O percurso construído e descrito neste relatório, tem como objetivo: desenvolver competências na intervenção clínica especializada, mobilizando a música como mediador expressivo com adolescentes em sofrimento mental. Dividido em dois grandes momentos, contexto clínico e comunitário, em Unidades de Psiquiatria da Infância e da Adolescência, conseguiu-se demonstrar, através da narrativa dos jovens, que a música é um impactante mediador expressivo mobilizado pelo Enfermeiro, junto dos adolescentes em sofrimento mental, em intervenções individuais ou grupais, facilitando a expressão e a transição.

Funding: The present study was funded by the Center for Research, Innovation, and Development in Nursing, in Portugal, by means of grants provided to some of the authors (CIDNUR, Safe2transition_2021). The increasing incidence of chronic and dependence leads to the need for hospitalization and adaptation in the process of returning home, as well as transition between care levels to ensure continuity of care. The World Health Organization has been warning about this problem since 2016, and consider reorganizing the care model as one of the solutions. The present study aimed to analyse the nurses’ perspective on transitional care for dependent people with rehabilitation care needs after hospital discharge. Methods: A focus group was developed with the participation of Rehabilitation Nurses from the hospital and community context, and content analysis was defined a posteriori. Results: From the content analysis emerged four related categories: promotion of continuity of care, nurse of advanced practice as a care manager, capacitation of the person and caregiver, and promotion of the care coordination. Conclusions: The present study allowed the strategies identification that minimize fragmentation risk of care and promote the person participation in transitional care. Ensuring transitional care is imperative to increase the quality of care, the satisfaction of professionals, clients, and the development of a system of sustainable health. info:eu-repo/semantics/publishedVersion

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John WileySons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Simple Summary Although immunotherapy has given the highest rate of improvement in cancer treatment in recent years, there is an urgent need to further improve its efficacy. Numerous strategies aim to transform non-responsive, immunosuppressive tumours into sensitive, immunopermissive tumours. The modulation of the tumour microenvironment, and especially the tumour vasculature offers opportunities for improved sensitivity to immunotherapy. Modulation of tumour blood vessels can enhance tumour oxygenation and T cell infiltration. Additionally, maturation of tumour blood vessels is thought to be involved in the efficient delivery of therapeutic agents. This review compiles the current strategies of vascular modulation to improve the efficacy of different immunotherapies: PD-1/PD-L1 and CTLA-4 antibodies, CAR T cells and cancer vaccines. Abstract Several strategies have been developed to modulate the tumour vasculature for cancer therapy including anti-angiogenesis and vascular normalisation. Vasculature modulation results in changes to the tumour microenvironment including oxygenation and immune cell infiltration, therefore lending itself to combination with cancer therapy. The development of immunotherapies has led to significant improvements in cancer treatment. Particularly promising are immune checkpoint blockade and CAR T cell therapies, which use antibodies against negative regulators of T cell activation and T cells reprogrammed to better target tumour antigens, respectively. However, while immunotherapy is successful in some patients, including those with advanced or metastatic cancers, only a subset of patients respond. Therefore, better predictors of patient response and methods to overcome resistance warrant investigation. Poor, or periphery-limited, T cell infiltration in the tumour is associated with poor responses to immunotherapy. Given that (1) lymphocyte recruitment requires leucocyte–endothelial cell adhesion and (2) the vasculature controls tumour oxygenation and plays a pivotal role in T cell infiltration and activation, vessel targeting strategies including anti-angiogenesis and vascular normalisation in combination with immunotherapy are providing possible new strategies to enhance therapy. Here, we review the progress of vessel modulation in enhancing immunotherapy efficacy.

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti–PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti–PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

Objective: To analyze the needs and facilitating and hindering elements related to transitional rehabilitation care. Methods: Integrative literature review oriented toward answering the question “What nursing interventions guarantee transitional rehabilitation care to dependent adult or elderly people when they return home after hospitalization?”. Results: The patients did not participate much in the planning of hospital discharge and decision-making when they had to return home. Informal caretakers reported that professionals showed detachment during hospitalization and delayed guiding instructions. Health professionals mentioned lack of time to offer this care modality as a difficulty. Final considerations: Ensuring training, follow-up, and coordination between care levels is essential. Care integration can reduce hospital stay and the impact of post-discharge complications. Transitional care contributes to a sustainable health system, higher care quality, and client satisfaction.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F–mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased β1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in β1 integrin activation. This corresponds with a decrease in Vegfa–stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell–stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. Significance: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

RESUMO Objetivo: compreender como os estudantes percepcionam o seu envolvimento em atividades de investigação e o contributo para o desenvolvimento de uma prática baseada na evidência. Método: estudo qualitativo, transversal, descritivo e exploratório, realizado em Portugal, em agosto de 2019, que recorreu ao grupo focal, com oito participantes, para responder à questão: “Quais as vantagens da participação dos estudantes de enfermagem em atividades de investigação para o desenvolvimento de conhecimentos, atitudes e competências de utilização da evidência?”. A análise de conteúdo foi realizada com o software NVivo. Resultados: da análise qualitativa dos achados, emergiram cinco categorias e subcategorias: autoaprendizagem, integração teórico-prática, trabalho interdisciplinar, tomada de decisão baseada na evidência e literácia científica. Conclusão: a criação de uma experiência eficaz de aprendizagem ajuda na construção do conhecimento e potencialmente contribui para a saúde da comunidade, os resultados de aprendizagem do estágio e o desenvolvimento de competências essenciais para uma Prática Baseada na Evidência. RESUMEN Objetivo: comprender como estudiantes perciben su envolvimiento con actividades investigativas y su contribución para desarrollar una práctica basada en evidencias. Método: estudio cualitativo, trasversal, descriptivo y exploratorio, hecho en Portugal en agosto de 2019, utilizando un grupo focal con ocho participantes para responder a la cuestión: “Cuales las ventajas de la participación de los estudiantes de enfermería en actividades de investigación para desarrollar conocimientos, actitudes y competencias de utilización de la evidencia?” Se hizo al análisis de contenido con el software NVivo. Resultados: el análisis cuantitativo de los datos generó cinco categorías y subcategorías: autoaprendizaje, integración teórico-práctica, interdisciplinariedad, tomada de decisiones basadas en evidencia, y alfabetización científica. Conclusión: la creación de una experiencia eficaz de aprendizaje ayuda en la construcción del conocimiento y potencialmente contribuye para la salud de la comunidad, para los resultados del aprendizaje en la pasantía y para el desarrollo de competencias esenciales para una Práctica Basada en Evidencias. ABSTRACT Objective: to understand how students perceive their involvement in investigation activities and how they contribute for the development of evidence-based practices. Method: qualitative, cross-sectional, descriptive, and exploratory study, carried out in Portugal, in August 2019. A focus group of eight participants was formed to answer the question: “What are the advantages of the participation of nursing students in investigations for the development of knowledge, attitudes, and performance in the use of evidence?” The software NVivo was used for a content analysis. Results: five categories and subcategories emerged from the qualitative analysis of findings: self-learning, integration of theory and practice, interdisciplinary work, evidence-based decision making, and scientific literacy. Conclusion: creating an effective learning experience helps building knowledge and can contribute for the health of the community, for improved educational results during the internship, and for the development of the abilities necessary for Evidence Based Practice.

Pathological angiogenesis contributes to tumour progression as well as to chronic inflammatory diseases. In this issue of EMBO Molecular Medicine, Esteban and co‐workers identify endothelial cell MT1‐MMP as a key regulator of intussusceptive angiogenesis (IA) in inflammatory colitis. Thrombospondin 1 (TSP1) cleavage by MT1‐MMP results in the binding of the c‐terminal fragment of TSP1 to αvβ3 integrin, which induces nitric oxide (NO) production, vasodilation and further initiation of IA. This novel control mechanism of inflammatory IA points towards promising new therapeutic targets for inflammatory bowel disease., Prof. Hodivala‐Dilke and colleagues discuss the findings from Esteban et al (in this issue of EMBO Molecular Medicine) that targeting intussusceptive angiogenesis via the TSP1‐nitric oxide axis exhibits therapeutic efficacy in the context of inflammatory bowel disease.

Angiogenesis is one important hallmark of cancer progression which explains the relevance of developing methods to efficiently analyze the neo-angiogenic process. In this report we make use of the transgenic adenocarcinoma of the murine prostate (TRAMP) model, considered a good model for studying clinical prostate cancer progression, to describe in detail the methods used to study angiogenesis in this type of solid tumor development. In this report we provide step-by-step procedures on the basis of previous work in our laboratory for: the mouse urogenital sinus (UGS) collection; microdissection of the prostate; preparation of the prostatic samples for immunofluorescence (to analyze vascular density, morphology, maturation, functionality, hypoxia, and others); preparation of prostatic samples to histopathological analysis and/or immunohistochemistry; and endothelial and vascular mural cell sorting and isolation by fluorescent associated cell sorting (FACS) to further analysis (mRNA, protein, or other) or to maintain in culture.

Angiogenesis is one important hallmark of cancer progression which explains the relevance of developing methods to efficiently analyze the neo-angiogenic process. In this report we make use of the transgenic adenocarcinoma of the murine prostate (TRAMP) model, considered a good model for studying clinical prostate cancer progression, to describe in detail the methods used to study angiogenesis in this type of solid tumor development. In this report we provide step-by-step procedures on the basis of previous work in our laboratory for: the mouse urogenital sinus (UGS) collection; microdissection of the prostate; preparation of the prostatic samples for immunofluorescence (to analyze vascular density, morphology, maturation, functionality, hypoxia, and others); preparation of prostatic samples to histopathological analysis and/or immunohistochemistry; and endothelial and vascular mural cell sorting and isolation by fluorescent associated cell sorting (FACS) to further analysis (mRNA, protein, or other) or to maintain in culture.

O conceito de saúde oral relacionada com a qualidade de vida refere-se ao impacto que a saúde oral tem na qualidade de vida da população. Distúrbios da cavidade oral, como a má oclusão, podem afetar o bem-estar dos indivíduos afetados. A má oclusão é considerada um problema de saúde pública, visto que tem alta prevalência e pode afetar de forma negativa a qualidade de vida dos indivíduos. Os adolescentes consideram os diastemas e a mordida cruzada anterior como as más oclusões mais urgentes no que toca a necessidade de tratamento ortodôntico. Objetivos: Avaliar a perceção da necessidade do tratamento ortodôntico e estética do sorriso por parte dos alunos de uma escola básica e secundária. Materiais e Métodos: Entrega de questionários constituídos por 4 perguntas relativas ao sorriso dos alunos, 6 imagens referentes a más oclusões distintas e 5 imagens de sorrisos, em que foi pedido aos alunos que avaliassem quanto a necessidade de tratamento ortodôntico nas primeiras seis imagens e escolhessem o sorriso mais estético nas outras cinco imagens. Resultados/Conclusão: 28% dos alunos são portadores de aparelho fixo e/ou removível, sendo que são as faixas etárias de 14 e 15 anos em que se encontram a maioria desses alunos. 68% dos alunos gosta do seu sorriso, sendo que a maioria são do género feminino. Os alunos consideram o diastema inter-incisivo como a má oclusão mais urgente no que toca a necessidade de tratamento ortodôntico (93%) e a mordida aberta como a menos urgente (57%). 85% dos alunos escolheram o sorriso nº4 (sorriso no qual se reduziu a visibilidade dos corredores bucais) como o mais estético. O sorriso gengival e sorriso baixo não foram considerados os mais estéticos por nenhum aluno.

Background The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1–4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis. Methods Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence. Results We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector. Conclusions Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease. Prostate © 2015 Wiley Periodicals, Inc.

// Ana-Rita Pedrosa 1 , Alexandre Trindade 1,2 , Catarina Carvalho 1 , Jose Graca 1 , Sandra Carvalho 1 , Maria C. Peleteiro 1 , Ralf H. Adams 3,4 and Antonio Duarte 1,2 1 Centro Interdisciplinar de Investigacao em Sanidade Animal (CIISA), University of Lisbon, Lisbon, Portugal 2 Instituto Gulbenkian de Ciencia, Oeiras, Portugal 3 Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Muenster, Germany 4 Faculty of Medicine, University of Muenster, Muenster, Germany Correspondence to: Antonio Duarte, email: // Alexandre Trindade, email: // Keywords : Jagged1, Notch, TRAMP, tumor angiogenesis, angiocrine Received : May 21, 2015 Accepted : May 29, 2015 Published : June 08, 2015 Abstract Angiogenesis is an essential process required for tumor growth and progression. The Notch signaling pathway has been identified as a key regulator of the neo-angiogenic process. Jagged-1 ( Jag1 ) is a Notch ligand required for embryonic and retinal vascular development, which direct contribution to the regulation of tumor angiogenesis remains to be fully characterized. The current study addresses the role of endothelial Jagged1-mediated Notch signaling in the context of tumoral angiogenesis in two different mouse tumor models: subcutaneous Lewis Lung Carcinoma (LLC) tumor transplants and the autochthonous Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). The role of endothelial Jagged1 in tumor growth and neo-angiogenesis was investigated with endothelial-specific Jag1 gain- and loss-of-function mouse mutants ( eJag1 OE and eJag1c KO). By modulating levels of endothelial Jag1 , we observed that this ligand regulates tumor vessel density, branching, and perivascular maturation, thus affecting tumor vascular perfusion. The pro-angiogenic function is exerted by its ability to positively regulate levels of Vegfr-2 while negatively regulating Vegfr-1 . Additionally, endothelial Jagged1 appears to exert an angiocrine function possibly by activating Notch3/Hey1 in tumor cells, promoting proliferation, survival and epithelial-to-mesenchymal transition (EMT), potentiating tumor development. These findings provide valuable mechanistic insights into the role of endothelial Jagged1 in promoting solid tumor development and support the notion that it may constitute a promising target for cancer therapy.

Objective— Notch signaling controls cardiovascular development and has been associated with several pathological conditions. Among its ligands, Jagged1 and Dll4 were shown to have opposing effects in developmental angiogenesis, but the underlying mechanism and the role of Jagged1/Notch signaling in adult angiogenesis remain incompletely understood. The current study addresses the importance of endothelial Jagged1-mediated Notch signaling in the context of adult physiological angiogenesis and the interactions of Jagged1 and Dll4 on angiogenic response and vascular maturation processes. Approach and Results— The role of endothelial Jagged1 in wound healing kinetics and angiogenesis was investigated with endothelial-specific Jag1 gain-of-function and loss-of-function mouse mutants ( eJag1 OE and eJag1c KO). To study the interactions between the 2 Notch ligands, genetic mouse models were combined with pharmacological inhibition of Dll4 or Jagged1, respectively. Jagged1 overexpression in endothelial cells increased vessel density, maturation, and perfusion, thus accelerating wound healing. The opposite effect was seen in e Jag1 cKO animals. Interestingly, Dll4 blockade in these animals led to an increase in vascular density but induced a greater decrease in perivascular cell coverage. However, Jagged1 inhibition in Dll4 gain-of-function (e Dll4 OE) mutants, with reduced angiogenesis, further diminished angiogenic growth and hampered perivascular cell coverage. Our findings suggest that as Dll4 blocks endothelial activation through Notch1 signaling, it also induces Jagged1 expression. Jagged1 then blocks Dll4 signaling through Notch1, allowing endothelial activation by vascular endothelial growth factor and endothelial layer growth. Jagged1 also initiates maturation of the newly formed vessels, possibly by binding and activating endothelial Notch4. Importantly, mice administered with a Notch4 agonistic antibody mimicked the mural cell phenotype of e Jag1 OE mutants without affecting angiogenic growth, which is thought to be Notch1 dependent. Conclusions— Endothelial Jagged1 is likely to operate downstream of Dll4/Notch1 signaling to activate Notch4 and regulate vascular maturation. Thus, Jagged1 not only counteracts Dll4/Notch in the endothelium but also generates a balance between angiogenic growth and maturation processes in vivo.

The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1-4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis.Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence.We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector.Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease.

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing.

Angiogenesis is an essential process required for tumor growth and progression. The Notch signaling pathway has been identified as a key regulator of the neo-angiogenic process. Jagged-1 (Jag1) is a Notch ligand required for embryonic and retinal vascular development. Although its role as a pro-angiogenic ligand has been thoroughly studied in developmental contexts, the real contribution of this Notch ligand to tumor angiogenesis remains unknown. The current study addresses the role of endothelial Jagged1-mediated Notch signaling in the context of tumoral angiogenesis in two different mouse tumor models: subcutaneous Lewis Lung Carcinoma (LLC) tumor transplants and the autochthonous Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). The role of endothelial Jagged1 in tumor growth and neo-angiogenesis was investigated with endothelial-specific Jag1 gain- and loss-of-function mouse mutants (eJag1OE and eJag1cKO). We show that the tumor vasculature of eJag1OE mutants presents increased vessel density, branching, and perivascular maturation, causing increased tumor perfusion. Ultimately, this leads to increased proliferation and survival of tumor cells, and increased epithelial-to-mesenchymal transition (EMT) resulting in increased LLC tumor growth as well as development and progression of prostate adenocarcinoma in mice. In contrast, in eJag1cKO mutants the vasculature is characterized by decreased vessel density, branching, and perivascular maturation, which restricts tumor vascular perfusion and increases vascular extravasation. This vascular phenotype leads to decreased proliferation and increased death of tumor cells, reduced EMT culminating in decreased LLC growth as well as inhibited development and progression of prostate adenocarcinoma in mice. Therefore, endothelial Jagged1 may constitute a novel promising target for solid cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Antonio Duarte, Ana-Rita Pedrosa, Alexandre Trindade, Catarina Carvalho, José Graça, Sandra Carvalho, Maria C. Peleteiro, Ralf H. Adams. Endothelial-specific Jagged1 blockade prevents solid tumor growth in pre-clinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2015-5225

Dissertação de mestrado em Psicologia Aplicada, As experiências adversas na infância (EAI) abarcam a exposição a um conjunto de circunstâncias desfavoráveis para o desenvolvimento normativo de um indivíduo e tanto homens como mulheres podem ter experienciado na infância algum tipo de adversidade. Estas experiências constituem um fator de risco para a perpetração de comportamentos agressivos ou violentos. Défices nas funções executivas (FE) estão associados a comportamentos agressivos e a EAI. Para este estudo estabelecemos como objetivo avaliar FE “frias”, flexibilidade cognitiva, atualização, inibição, e fluência verbal. O objetivo principal deste estudo é perceber se existem diferenças entre os homens e mulheres a cumprir pena efetiva de prisão em relação às EAI, crime e funções executivas. Para efeitos deste estudo foi utilizada uma amostra de 66 reclusos, 33 do sexo feminino e 33 do sexo masculino, emparelhados em termos de idade e escolaridade vertical. Não encontrámos diferenças significativas entre homens e mulheres em relação às EAI. Relativamente ao crime, encontramos diferenças significativas entre homens e mulheres em relação ao total do D-CRIM e em relação ao crime não violento, sendo que os homens relatam ter cometido mais crimes e mais crimes não violentos durante a vida do que as mulheres. Relativamente ao crime violento, não foram encontradas diferenças. Em relação às FE, houve diferenças entre homens e mulheres na inibição, sendo que as mulheres tiveram um pior desempenho do que os homens., Adverse childhood experiences (ACE) include exposure to a set of unfavorable circumstances for the normative development of an individual and both men and women may have experienced some type of childhood adversity. These experiences are a risk factor for the perpetration of aggressive or violent behaviors. Deficits in executive functions (EF) are associated with aggressive behavior and ACE. We evaluated e “cold” EF, namely shifting, updating and inhibition, and verbal fluency. The main objective of this study is to test if there are differences between men and women serving a prison sentence concerning ACE, crime and executive functions. For this study, we analyzed data from a sample of 66 prisoners matched for age and education (33 female). No significant differences were found between men and women concerning ACE. We found significant differences between men and women about the total D-CRIM and in relation to non-violent crime, with men reporting having committed more crimes and more non-violent crimes during their lifetime than women. Regarding violent crime, no differences were found. Regarding EF, women performed significantly worse than men for inhibition.