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

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

Transient display of chimeric proteins on biological surfaces as an effective strategy for modulations of innate and adaptive immune responses.

The major premise of this dissertation was to transiently display novel immunological ligands on biological membranes as a localized means of modulating innate and adaptive immune responses with applications to bone marrow and pancreatic islet transplantation. In Chapter two, we engineered donor allogeneic bone marrow cells to transiently display a novel form of FasL, SA-FasL, to efficiently purge out alloreactive donor T cells to prevent acute GVHD. In Chapter three, we engineered pancreatic islets with a novel form of CD47, SA-CD47, to modulate instant blood mediated inflammatory reaction (IBMIR) to prevent immediate islet graft loss following intraportal transplantation. GVHD is initiated and perpetuated by mature T cells in the bone marrow inoculum following transplantation into conditioned recipients. Upon activation, T cells upregulate Fas receptor and become sensitive to FasL-mediated apoptosis. Thus, we hypothesized that the display of SA-FasL on T cells in bone marrow will result in their apoptosis potentially in autocrine fashion following activation in response to recipient alloantigens and engagement of Fas with SA-FasL on the T cells, thereby resulting in the prevention of vi acute GVHD. We demonstrated that SA-FasL engineered T cells underwent apoptosis following response to alloantigens both in vitro and in vivo. Most importantly, in an haploidentical rodent setting where C57BL/6 bone marrow cells containing T cells transplanted into lethally irradiated F1 recipients, engineering cells with SA-FasL resulted in the prevention of lethal acute GVHD in 80% of recipients long term (\u3e100 days). We extended this observation to xenogeneic acute GVHD setting, where mice receiving SA-FasL-engineered human PBMCs were significantly protected. Significant islet mass loss following intraportal transplantation is a major barrier for clinical islet transplantation. IBMIR is initiated and perpetuated by innate immune cells. CD47-SIRPα axis known as innate immune checkpoint delivers “don’t eat me signal” to prevent phagocytosis and activation of myeloid cells. Thus, we hypothesized that engineering islets to transiently display SA-CD47 as an innate immune checkpoint will mitigate IBMIR and enhance engraftment following intraportal transplantation. In a syngeneic marginal mass model of intraportal transplantation, SA-CD47-islets showed better engraftment and function as compared with the control group (87.5 vs 14.3%). Engraftment was associated with low levels of intrahepatic inflammatory cells and mediators of islet destruction, including HMBG-1, tissue factor, and IL-1β. Overall, we show that transient display of immunological ligands on biological membranes is effective in modulating innate and adaptive immune responses with significant translational implication for multiple immune-based disorders

Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis.

Chronic pancreatitis (CP) is a progressive and irreversible inflammatory and fibrotic disease with no cure. Unlike acute pancreatitis (AP), we find that alternatively activated macrophages (AAMs) are dominant in mouse and human CP. AAMs are dependent on interleukin (IL)-4 and IL-13 signalling, and we show that mice lacking IL-4Rα, myeloid-specific IL-4Rα and IL-4/IL-13 were less susceptible to pancreatic fibrosis. Furthermore, we demonstrate that mouse and human pancreatic stellate cells (PSCs) are a source of IL-4/IL-13. Notably, we show that pharmacologic inhibition of IL-4/IL-13 in human ex vivo studies as well as in established mouse CP decreases pancreatic AAMs and fibrosis. We identify a critical role for macrophages in pancreatic fibrosis and in turn PSCs as important inducers of macrophage-alternative activation. Our study challenges and identifies pathways involved in crosstalk between macrophages and PSCs that can be targeted to reverse or halt pancreatic fibrosis progression

Molecular regulators of smoltification and viral infection management tools for salmon aquaculture

Accurate smoltification and disease management in Atlantic salmon (Salmo salar) are key issues for the aquaculture industry. Due to their anadromous lifecycle the transfer of salmon from fresh water (FW) to seawater (SW) is crucial to their survival; too early can cause mortality, too late can cause desmoltification and long-term health problems. Both scenarios can increase susceptibility to four viral diseases: Salmon alphavirus (SAV), Infectious salmon anemia virus (ISAV), orthoreovirus (PRV), and Piscine myocarditis virus (PMCV). They all show similar clinical and histopathological symptoms and can easily spread throughout farms. Understanding the initial innate immune response to these viruses may provide biomarkers that could help identify and monitor infections. An in house and onsite Na+/K+ ATPase (NKA) qRT-PCR assay was developed for the salmon biomarker ATPase to test smoltification readiness in salmon smolts. Tested against NKA enzymatic assays it showed a similar success rate over 3 years: NKA qRT-PCR (57%), NKA activity assay (60%). Onsite tests confirmed that the ATPase mRNA transcript is a useful biomarker for smoltification detection. An in-lab and mobile multiplex qRT-PCR assay was developed for detection of SAV, PRV and PMCV. The analytical sensitivity of the SAV (86.5% SE 0.11), PRV (90.94%, SE 0.09) and PMCV (100.46%, SE 0.19) assays was 102 copies for PMCV and 103 for SAV and PRV. Initial results suggest individual assays could be run on site at farms. Addition of an internal control, probit analysis and viral positive tests are still required for multiplex assay integration. Salmon erythrocytes were infected with ISAV, SAV and Poly I:C to investigate whether they induce and up-regulate innate immune response genes. All genes were expressed at low levels in all parameters investigated including non-infected control erythrocytes. These findings suggest erythrocytes act as an initial buffer to viral infections and may help stimulate the innate immune response

Cystic Fibrosis Impact on Cellular Function

The following literature review provides an account in support of the premise that the cystic fibrosis (CF) disease affects widespread areas of the body primarily due to the defective CFTR protein. Mutations in the CFTR gene lead to defects in CFTR protein that causes the disease. Lack of protein function or lack of functional protein cause variability in severity of the phenotype. The defective CFTR protein changes ion influx and efflux across the body’s cell membranes, which ultimately changes the internal environment of these cells. This change contributes to each cell’s production of proteins through transcription and translation. The simple changes in ion movement in and out of these cells have detrimental effects on the overall function of the cells. The cells create a system for maintenance of the body’s health, and if the cells do not function appropriately, the body fails to manage viral and bacterial infections. In this review, I will discuss how the defective CFTR channel works in the cells of the bodies of CF patients, and explain why the normal CFTR channel is essential for proper health and maintenance of the human body. In addition to the CFTR channel, other factors affect the severity of the disease. Genome-wide association studies explore other factors attributed to CF, including environmental factors, modifier genes, transcriptional regulation factors, and post-translational modifications. Potentiators and correctors target the CFTR channel in order to increase CFTR function, working alongside therapies in order to combat the effects of defective channels in CF patients

Thyroid Autoimmunity as a Window to Autoimmunity: An Explanation for Sex Differences in the Prevalence of Thyroid Autoimmunity

Autoimmune thyroid diseases (AITDs), predominately Graves׳ disease and Hashimoto׳s thyroiditis, comprise the most common autoimmune diseases in humans. Both have the production of anti-thyroid antibody as an important aspect and both are much more prevalent in females, being at least 10 times more common than in males. Using these two clues, a hypothesis for the initiation of thyroid autoimmunity is proposed that helps to make the case that the thyroid is one of the most sensitive sites for autoimmunity and helps account for the prevalence and the observed sex differences in AITDs and associated diseases, such as type 1 diabetes and Latent Autoimmune Diabetes in Adults (LADA). The primary mechanisms proposed involve the underlying state of inflammation as a result of the adipokines, especially leptin, TNF-α, and IL-6, and the receptors able to recognize pathogen-associated molecular patterns (PAMP׳s) and damage-associated molecular patterns (DAMP׳s) through Toll-like receptors (TLR) and others receptors present on thyrocytes. The adipokines are produced by adipose tissue, but have hormone-like and immune modulating properties. As the levels of leptin are significantly higher in females, an explanation for the sex difference in thyroid autoimmunity emerges. The ability of the thyrocytes to participate in innate immunity through the TLR provides an adjuvant-like signal and allows for the action of other agents, such as environmental factors, viruses, bacteria, and even stress to provide the initiation step to break tolerance to thyroid self-antigens. Seeing the thyroid as one of the most sensitive sites for autoimmunity, means that for many autoimmune disorders, if autoimmunity is present, it is likely to also be present in the thyroid – and that that condition in the thyroid was probably earlier. The evidence is seen in multiple autoimmune syndrome

Investigating the physiological and pharmacological effects of the gut hormone peptide YY (PYY)

The obesity epidemic is a critical and global public health burden. Drugs that safely promote weight loss are urgently needed to halt the rising prevalence of obesity and its associated complications, such as type 2 diabetes (T2D). Gut hormones are important regulators in metabolism and have therapeutic potential as treatments for obesity and T2D. The gut hormone peptide YY (PYY) is released from the intestine after a meal. Exogenous PYY3–36 suppresses food intake in both rodents and humans, including in the obese state. PYY3–36 suppresses appetite by acting on its receptor, the Y2R. Y2R is expressed in brain appetite centres but also in the afferent vagus nerve, the main neuroanatomical link carrying information from the gut to the brain. However, the relevant contribution of the afferent vagus to the overall effects of PYY3–36 is unknown. Chemogenetic activation of vagal afferent neurones results in reduced food intake (surpassing the effects of PYY) and might have altered the immune landscape of the gastrointestinal tract. To dissect the role of the Y2R expressed in the afferent vagus, we have developed a novel microsurgical technique in the mouse. Our work suggests that vagal Y2R mediates the anorectic effect of low dose and endogenous PYY3–36 and that this vagal signalling pathway regulates short-term feeding. This anorectic effect was not caused by an aversive response. In vitro calcium imaging confirmed that PYY3–36 directly activates vagal afferents. Chronic treatment of diet-induced obese (DIO) mice with a long-acting PYY3–36 analogue, Y242, did not cause a significant body weight loss. Longitudinal tracking of individual islet function using a novel imaging platform allowed to study the effect of diet and Y242 treatment. Chronic Y242 did not improve or worsen islet function in obese mice. Therefore, PYY-based treatments might not be suitable as a single agent but have potential in combination with other gut-hormones. Vagus nerve neuromodulation has shown potential as an anti-obesity therapy and the work in this thesis adds to a better understanding of vagal afferent function which will help optimise therapeutic interventions.Open Acces

Viruses Implicated in the Initiation of Type 1 Diabetes Affect β Cell Function and Antiviral Innate Immune Responses: A Dissertation

The increasing healthcare burden of type 1 diabetes (T1D) makes finding preventive or therapeutic strategies a global priority. This chronic disease is characterized by the autoimmune destruction of the insulin-producing β cells. This destruction leads to poorly controlled blood glucose and accompanying life threatening acute and chronic complications. The role of viral infections as initiating factors for T1D is probable, but contentious. Therefore, my goal is to better characterize the effects of viral infection on human β cells in their function of producing insulin and to define innate immune gene responses in β cells upon viral infection. These aspects were evaluated in various platforms including mice engrafted with primary human islets, cultured primary human islets, β cells derived from human stem cells, and a human β cell line. Furthermore, the contributions of cell-type specific innate immune responses are evaluated in flow cytometry-sorted primary human islet cells. Taken together, the results from these studies provide insights into the mechanisms of the loss of insulin production in β cells during virus infection, and characterize the antiviral innate immune responses that may contribute to the autoimmune destruction of these cells in T1D