Ageing is associated with molecular signatures of inflammation and type 2 diabetes in rat pancreatic islets.

AIMS/HYPOTHESIS: Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas. METHODS: Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods. RESULTS: Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1). CONCLUSIONS/INTERPRETATION: Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.Biotechnology and Biological Sciences Research Council (Grant ID: BB/H003312/1), British Heart Foundation, FP6 Epigenome Network of Excellence programme, GlaxoSmithKline, Nuffield Foundation, Royal Society, Medical Research Council (Grant ID: MRC_MC_UU_12012/4)This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00125-015-3837-

Establishment of pancreatic cancer zebrafish xenografts for personalized medicine in oncology practice

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2021O cancro do pâncreas é das malignidades mais agressivas e mortais. Esta doença raramente é diagnosticada num estadio em que a resseção cirúrgica é viável. A maioria dos doentes, a quando do diagnóstico, encontram-se num estadio avançado onde as opções terapêuticas são limitadas. Para além disso, as características peculiares do microambiente tumoral do cancro do pâncreas, com um estroma fibrótico extremamente denso, compromete a distribuição eficaz dos fármacos anticancerígenos. A quimioterapia sistémica é a única opção terapêutica para doentes com cancro do pâncreas avançado – FOLFIRINOX ou gemcitabina+nab-paclitaxel. No entanto, ainda não existe na clínica marcadores eficazes com valor preditivo que permitem identificar qual a melhor terapêutica para cada doente. Consequentemente, os doentes são submetidos a múltiplas rondas de tratamento e toxicidades desnecessárias, até encontrar a terapia que seja mais eficaz. A imunoterapia também tem sido explorada como terapia complementar para o tratamento do cancro do pâncreas, incluindo inibidores de checkpoint imunológicos. Contudo, o microambiente tumoral rico em fibroblastos e células imunes com atividade imunossupressora, constitui um obstáculo significativo. Além disso, muitos dos doentes não são elegíveis para este tipo de terapia e portanto estratégias mais personalizadas estão a ser a investigadas em ensaios clínicos. Desta forma, um teste capaz de prever as respostas de cada doente antes do tratamento, seria de grande valor para o tratamento personalizado do cancro do pâncreas. O principal objetivo deste projeto de investigação foi testar as principais opções terapêuticas para o cancro do pâncreas em estadio avançado - FOLFIRINOX e gemcitabina+nab-paclitaxel - utilizando o modelo xenógrafo de peixe-zebra. Com este objetivo, xénografos de peixe-zebra foram gerados utilizando linhas celulares humanas de cancro do pâncreas (Panc-1 e MIA PaCa-2), e várias características tumorais foram analisadas por microscopia confocal, incluindo dinâmica tumoral – proliferação e morte celular – e composição do microambiente tumoral. Os efeitos citotóxicos do nivolumab em monoterapia e em combinação com gemcitabina+nabpaclitaxel (ensaio clínico a decorrer) também foram avaliados. Os resultados demonstraram que as linhas celulares de cancro do pâncreas apresentam diferentes capacidades de implantação no modelo de xénografos de peixe-zebra. Relativamente às terapias anticancerígenas, os nossos resultados demonstraram que os xénografos de peixe-zebra são capazes de revelar respostas tumorais ao FOLFIRINOX e gemcitabina+nab-paclitaxel, incluindo comprometimento da proliferação celular e indução da apoptose. Neste projeto, testámos também a imunoterapia com o anticorpo anti-PD-1- nivolumab. Surpreendentemente os xénografos de peixe-zebra submetidos ao nivolumab em monoterapia e em combinação com gemcitabina+nab-paclitaxel também revelaram sensibilidade celular, com indução significativa da apoptose e redução do tamanho tumoral. De seguida, decidimos caracterizar o microambiente tumoral em particular o infiltrado de neutrófilos e macrófagos. Aos 4 dias pós-injeção, a percentagem de neutrófilos aumentou em relação ao primeiro dia, e os macrófagos do tipo M2 (atividade pró-tumoral) passaram a dominar o microambiente tumoral. Para estudar o papel destes infiltrados na tumorigénese, gerámos xenógrafos em mutantes hipomórficos. A redução de neutrófilos, levou a um aumento do tamanho tumoral, enquanto que a redução de macrófagos, levou a um efeito contrário – diminuição do tamanho tumoral. Estes dados sugerem que os neutrófilos e macrófagos têm um papel antagónico, os neutrófilos com um papel anti-tumoral e os macrófagos pró-tumoral. Sumariamente, os nossos resultados realçam a viabilidade de usar xénografos de peixe-zebra como um modelo in vivo para o screening de respostas tumorais às opções terapêuticas do cancro do pâncreas, e para o estudo da complexidade do microambiente tumoral.In the modern era of cancer research, pancreatic cancer has proven to be one of the most aggressive and lethal malignancies. Pancreatic cancer is rarely diagnosed at a time when surgical resection is feasible. Therefore, most of the patients present with an advanced disease, at the time of diagnosis, in which treatment options are limited. In addition, the pancreatic cancer microenvironment has peculiar characteristics with a thick layer of stroma, which builds up around the tumor and compromises an efficient drug delivery. Systemic chemotherapy remains the only treatment option for patients with advanced pancreatic cancer – FOLFIRINOX or gemcitabine+nab-paclitaxel. However, effective biomarkers to help predict treatment responses for each patient are still lacking. Consequently, patients go through several trial-and-error approaches and subjected to unnecessary side effects, until the best therapy is found. Immunotherapy has also been explored as a complementary therapy for the treatment of pancreatic cancer, including immune checkpoint inhibitors. But, the tumor microenvironment enriched in fibroblasts and immune cells with immunosuppressive activity poses a major obstacle. Besides, many patients are not eligible for this type of treatment, and therefore more personalized regimens are being investigated in clinical trials. In this way, a test able to predict individual responses before treatment would be of great value for personalized pancreatic cancer treatment. The ultimate goal of this research project was to screen the major therapeutic options for PC treatment, FOLFINIROX and gemcitabine+nab-paclitaxel, using the zebrafish xenograft model. Additionally, the cytotoxic effects of nivolumab as a monotherapy, and in combination with gemcitabine+nab-paclitaxel (clinical trial ongoing) were also evaluated. To address this, zebrafish xenografts were generated with established human pancreatic cancer cell lines (Panc-1 and MIA PaCa-2), and several cancer hallmarks were analyzed through confocal microscopy, including tumoral dynamics – proliferation and cell death – and composition of the tumor microenvironment. Data revealed that pancreatic cancer cell lines have different capacities to engraft in the zebrafish xenograft model. Regarding anticancer therapies, results showed that zebrafish xenografts are able to reveal anti-tumor responses to both FOLFIRINOX and gemcitabine+nab-paclitaxel regimens, leading to impaired cell proliferation and induction of apoptosis. In this project, we also tested anti-PD-1-nivolumab immunotherapy. Surprisingly, zebrafish xenografts subjected to nivolumab and nivolumab in combination with gemcitabine-nab-paclitaxel also revealed cellular sensitivity, with significant induction of apoptosis and tumor size shrinkage. Next, we decided to characterize the tumor microenvironment, in particular neutrophil and macrophage populations. At 4 days post-injection, the percentage of neutrophils increased in comparison with the first day, and M2-like macrophages (protumoral activity) started to dominate the tumor microenvironment. To study the role of both populations in tumorigenesis, zebrafish xenografts were generated using hippomorphic mutants as hosts. Reduction of neutrophils induced an increase in the tumor size, while reduction of macrophages induced an opposite effect – decrease of the tumor size. These results suggest that neutrophils and macrophages are playing opposing roles, neutrophils as anti-tumoral and macrophages as pro-tumoral. Altogether, and most importantly, our results highlight the feasibility of using the zebrafish xenograft model as an in vivo screening platform for pancreatic cancer therapy, and to study the complexity of the tumor microenvironment

The TWEAK/Fn14/CD163 axis - Implications for metabolic disease

TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF superfamily that controls a multitude of cellular events including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. TWEAK control of these events is via an expanding list of intracellular signalling pathways which include NF-κB, ERK/MAPK, Notch, EGFR and AP-1. Two receptors have been identified for TWEAK – Fn14, which targets the membrane bound form of TWEAK, and CD163, which scavenges the soluble form of TWEAK. TWEAK appears to elicit specific events based on the receptor to which it binds, tissue type in which it is expressed, specific extrinsic conditions, and the presence of other cytokines. TWEAK signalling is protective in healthy tissues, but in chronic inflammatory states become detrimental to the tissue. Consistent data show a role for the TWEAK/FN14/CD163 axis in metabolic disease, chronic autoimmune diseases, and acute ischaemic stroke. Low circulating concentrations of soluble TWEAK are predictive of poor cardiovascular outcomes in those with and without diabetes. This review details the current understanding of the TWEAK/Fn14/CD163 axis as one of the chief regulators of immune signalling and its cell-specific role in metabolic disease development and progression

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra)

Attribution of ghrelin to cancer; Attempts to unravel an apparent controversy

Ghrelin is an endogenous peptide hormone mainly produced in the stomach. It has been known to regulate energy homeostasis, stimulate secretion of growth hormone, and mediate many other physiologic effects. Various effects attributed to ghrelin contribute to many aspects of cancer development and progression. Accordingly, a large body of evidence has emerged about the association of ghrelin with several types of cancer in scales of cell-line, animal, and human studies. However, existing data are controversial. This controversy occurs in two main domains: one is the controversial results in local effects of ghrelin on different types of human cancer cell-lines; the second is the apparent disagreement in the results o

Evaluation of the Toxicological Consequences of Dietary Exposure to Individual Classes of Polycyclic Aromatic Hydrocarbons or a Mixture in the Fischer-344 Rat.

There is special concern about the potential health effects of by-products released into the environment after combustion or conversion of fossil fuels. Polycyclic aromatic hydrocarbons (PAH), while initially released largely into the atmosphere, are subsequently deposited in soil and water and on foods. Although the significance of PAH contamination with regard to environmentally caused diseases and cancer in humans is difficult to estimate, it is suggested that the potential hazards may be underestimated. In an effort to understand the potential adverse health effects of PAHs, a comprehensive study was undertaken to determine the metabolic fate of selected PAHs as individual agents and as components of a mixture following oral exposure in the Fischer-344 rat. Studies included radioisotope binding studies to evaluate the absorption, distribution, and elimination of selected compounds, acute toxicity trials to define maximum tolerated chemical concentration levels, and chronic studies to determine temporal effects of individual compounds and the mixture. Selected compounds included 2-aminoanthracene (2-AA), benz (a) anthracene (BA), and the 1,3-; 1,6-, and 1,-8-dinitropyrenes (DNP). Results of radioisotope binding studies and ingestion studies clearly indicated that toxicity of BA or DNP were minimal in exposed rats. Administration of 50$\mu$Ci per kg (\sp3H) -BA (10mg/kg) or (\sp3H) -DNP (10mg/kg) via oral gavage resulted in a rapid excretion of greater than 70% of the recovered label at 48 h. Similarly, when rats were fed BA or dinitropyrenes (DNP) at concentrations up to 0.1% of the diet toxic effects were not observed following 14-, 30-, or 80-days of exposure. In contrast administration of 2-AA at concentration levels as low as 0.0075% of the diet resulted in a temporal and dose response progression in toxic effects of the liver and pancreas. Hepatocellular hyperplastic nodules were detected in rat liver following 80 days of ingestion of 2-AA at 0.01% and 0.0075% of diet. 2-AA pancreatic toxicity resulted in large cytoplasmic vacuoles and almost complete depletion of insulin stores in the endocrine pancreas and necrotic changes in the exocrine pancreas. Attenuation of the toxic effects of 2-AA was demonstrated in rats fed a mixture of the same compounds at the same dietary level of incorporation

Two novel therapies for the treatment of type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is caused by an autoimmune destruction of islet β cells. Current treatments are based on replacement therapy using insulin analogs but, due to the impossibility to simulate physiological glucose control, it leads to diabetes complications. Thus, novel treatments are required. As a difference of what happens with the immunosuppressor therapy, which have limited clinical efficacy, the induction of antigen specific tolerance (AST) can specifically block the activation of autoreactive T cells, preserving the survival and function of the pancreatic β cells and preventing the development of T1DM. Here, two different strategies are followed, in order to induce a tolerogenic environment. The expression of the self-Ag insulin under non-inflammatory conditions, using the SV40 background allows the restoration of the AST in the RIPB7.1 mouse model. Also, the protective role in pancreatic islets of the liver receptor homologue 1 (LRH1) against apoptosis was considered, as well as the prevention of hyperglycemia in T1DM mouse models by the induction of an anti-inflammatory environment promoted by the LRH1 agonist BL001. However, the limitations of BL001 as potential medication prompted us to develop an in vitro drug-screening platform that allowed the identification of two novel LRH1 agonists, BL002 and BL003. These non-toxic agonists protect mouse islets from cytokines-induced apoptosis, improving their survival. Thus, the AST induced by SV40 vector as well as the new generation of LRH1 agonists BL002 and BL003 must be considered as two promising therapies for the treatment of T1DM