Room 1.1: Cancer
Presenter: Pieter Schol
Title: AR ENHANCER HETEROGENEITY IN PROSTATE CANCER: IS IT FUNCTIONAL?
Lab: Division of Oncogenomics, The Netherlands Cancer Institute
Androgen receptor (AR) is a main driver of prostate cancer (PCa), a major cause of death in men worldwide. PCa therapies inhibit AR signaling, but tumors often develop into castration resistant PCa (CRPC), which is untreatable and ultimately lethal. Understanding CRPC development is therefore of major research interest.
When bound to its ligand DHT, AR regulates gene transcription by relocating to the nucleus and binding to AR binding sites (ARBS). Only a small minority of ARBS are found at gene promoters, and most are located distally from a gene’s transcription start site. This suggests that AR mediates transcription predominantly through enhancers. ChIP-seq analysis of patient samples shows an interpatient variability in ARBS. Many are patient-unique, but some are shared by the majority of patients. It is currently unknown whether these different classes of ARBS also have different functional consequences on transcription.
In this project, we interrogate such functional differences by deleting individual enhancers with CRISPR/Cas9, and assess individual ARBS’ contribution to gene transcription. Understanding ARBS heterogeneity in PCa could open avenues to assessing the clinical effect of ARBS heterogeneity, and ultimately enable more personalized treatment.
Presenter: Marlies Oosthoek
Title: PANCREATIC CANCER-ASSOCIATED FIBROBLASTS INFLUENCE CHEMOKINE RECEPTOR EXPRESSION IN T CELLS
Lab: Laboratory of Medicine, Karolinska Institutet
Pancreatic cancer is known for its dense and immunosuppressive tumour microenvironment. Amongst other cell types, cancer-associated fibroblasts (CAFs) make up a great deal of the stroma of pancreatic cancer. CAFs have been known to influence the tumour microenvironment by releasing cytokines, chemokines and extracellular matrix proteins. The aim of this study was to investigate how CAFs influence chemokine receptor expression on T cells. We investigated this by comparing chemokine receptor expression on peripheral blood mononuclear cells (PBMCs) co-cultured with CAFs or tumour spheroids or both. We also examined the functionality of T cells in the presence of CAFs and PANC-1 spheroids. Finally, the expression of chemokine receptors was investigated on tumour-infiltrating lymphocytes isolated from tissue samples. CAFs upregulated CXCR4 expression on CD8+ T cells and CCR4 expression on CD4+ T cells in the presence of tumour spheroids, while there was a decrease in CXCR3, CCR5, and CCR6 expression on CD8+ T cells in the presence of CAFs and tumour spheroids. We also show there was lower expression of granzyme B, interferon-γ and perforin in the presence of CAFs and tumour spheroids. Functionality was further impaired in the population that highly expressed CXCR4, as there was lower granzyme B and interferon-γ production. Furthermore, there was a distinct chemokine receptor expression pattern on tumour infiltrating lymphocytes in pancreatic cancer compared to other cancer types. Combined, these findings show that CAFs impair functionality partly via increasing CXCR4 and that CAFs potentially influence immune cell recruitment via affecting their chemokine receptor expression
Room 1.2: Neuro(muscular)
Presenter: Marlies Wagener
Title: QUALITY ABOVE QUANTITY: DISSECTING INSOMNIA SYMPTOMS FROM SHORT SLEEP DURATION IN RELATION TO CORONARY ARTERY DISEASE: NEW EVIDENCE FROM MULTIVARIABLE-ADJUSTED AND FACTORIAL MENDELIAN RANDOMIZATION ANALYSES
Lab: Department of Internal Medicine, LUMC
Background: Multivariable-adjusted and Mendelian Randomization (MR) studies identified both insomnia symptoms and short sleep duration as risk factors for coronary artery disease (CAD). Since sleep is a modifiable risk factor, it is important for targeted treatment to understand how insomnia and sleep duration independently contribute to CAD risk. This study aimed to dissect insomnia symptoms from sleep duration in relation to CAD and serum lipid levels.
Methods: We stratified participants from the UK Biobank (consisting of over 500.000 participants aged 40-70) into subgroups based on the presence of insomnia symptoms and short (<7h), average (7-9h) and long (≥9h) sleep duration. Data on insomnia symptoms and total sleep duration was collected through self-reported questionnaires. We used multivariable-adjusted Cox proportional hazard models, linear regression models and factorial MR. MR analyses used genetic risk scores for self-reported total sleep duration and insomnia symptoms. In all analyses we used CAD and serum lipid levels as outcomes.
Results: In the multivariable-adjusted Cox proportional hazard model, a combination of short or long sleep duration was associated with the largest increase in CAD risk. In factorial MR analyses, effects of genetically-influenced insomnia symptoms and CAD were the same in groups of genetically-influenced short (OR: 1.15; 95% confidence interval: 1.11-1.19), average (1.09; 1.07-1.12) and long (1.08; 1.00-1.16) sleep duration. Short and long sleep duration were not associated with CAD in individuals with either a low or high genetically-influenced risk for insomnia symptoms. Furthermore, we found that insomnia symptoms were only associated with elevated blood triglycerides and not LDL cholesterol.
Conclusion: Our research shows that CAD risk and increased triglyceride levels are driven by insomnia symptoms and not sleep duration. Interventions targeting insomnia might therefore be more effective in CAD prevention than those targeting short sleep duration.
Presenter: Marloes Holierhoek
Title: ASSESSING BENEFICIAL EFFECTS OF DIETARY SUPPLEMENTATION OF BRANCHED AMINO ACIDS IN A SEVERE MOUSE MODEL FOR DUCHENNE MUSCULAR DYSTROPHY
Lab: Department of Human Genetics, LUMC
Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder resulting in progressive muscle weakness and atrophy. The absence of the dystrophin protein in DMD patients leads to contraction-induced muscle fiber degeneration. Regeneration of these muscle fibers is impaired, and eventually the fibers get replaced with fibrotic and adipose tissue. As a result of the continuous muscle fiber degeneration and regeneration cycles, protein demands in DMD patients are increased. It is believed that patients might have insufficient protein intake to meet these increased demands, resulting in amino acid deficits. Since there is no cure for DMD at the moment, patients often resort to nutritional supplements to improve their disease progression. However, effects of nutritional supplements on disease pathology are still unclear. Branched-chain amino acid (BCAA) supplementation is a promising candidate to meet the need for the increased protein demand. The BCAAs are among the essential amino acids and are demonstrated to be involved in the activation of protein synthesis pathways and to promote muscle repair in mdx mice. In this study, we investigated whether BCAA supplementation can improve muscle pathology in D2-mdx mice, a relatively new DMD mouse model. In addition, we assessed if BCAA supplementation is affected of affects treatment with the corticosteroid prednisolone, as this drug is frequently used by DMD patients to slow down the progression of the disease. D2-mdx males were fed either a normal diet or a diet supplemented with BCAAs, for nine weeks. In addition, the mice received either prednisolone injections or saline injections. It was found that the modified diet could not improve in vivo muscle functionality, nor did it influence bodyweight or weight of individual muscles. Furthermore, preliminary findings suggest that the BCAA diet also did not affect the severity of muscle fibrosis, calcification, and regenerating muscle fibers in the D2-mdx mice.
Room 1.3: Immunology
Presenter: Tim van Tienhoven
Title: THE ROLE OF REGULATORY T CELLS IN IMMUNOTHERAPY RESISTANCE IN AN IN VIVO MESOTHELIOMA MOUSE MODEL.
Lab: Department of Pulmonary Medicine, Erasmus MC
Malignant pleural mesothelioma (MPM) is an aggressive cancer that originates from the mesothelial cells lining the pleura. Incidence rates are increasing worldwide, and the disease is generally characterized by a poor prognosis. Besides conventional therapies, it has been shown that the newly-emerged strategy of immunotherapy with checkpoint inhibitors (ICI) is also ineffective in the majority of MPM patients. To increase the number of patients with durable response following immunotherapy, we sought to determine mechanisms of ICI therapy resistance. As regulatory T cells (Treg cells) are an highly immunosuppressive subset of T cells that are able to express immune-checkpoints proteins, they could potentially be involved. It is hypothesized that ICI therapy unleashes Treg cells from checkpoint inhibition, allowing them to impede anti-tumor immunity and induce ICI therapy resistance.
To determine whether Treg cells play a role in the resistance against ICI therapy, we used an ICI-resistant mesothelioma murine model by which mice were inoculated in the peritoneal cavity with the mouse mesothelioma cell line AE17-OVA and were treated with αPD-L1 ICI therapy. Following therapy, Treg cells were isolated from tumors and secondary lymphoid organs and were examined for transcriptional alterations, expression of immunoregulatory proteins, and their functional suppressive capacity.
We demonstrate that αPD-L1 treatment increases the expression of immunosuppressive genes, including Il10, Havcr2, and Fgl2, especially in PD-1+ Treg cells. Besides, we reveal that Treg cells become highly proliferative upon αPD-L1 treatment, as indicated by an increased Ki67 protein expression, and become functionally more potent in suppressing naïve CD8 T cells. The enhanced suppressive capacity of Treg cells following αPD-L1 ICI therapy was not restricted to tumor tissue but was observed in secondary lymphoid organs, indicating a systemic phenomenon. Collectively, our data indicates that Treg cells may play an important role in the resistance against ICI therapy, and provides an insight on how these Treg cells could induce this resistance.
Presenter: Mette Dekkers
Title: DEVELOPMENT OF HYPOIMMUNOGENIC HUMAN INDUCED PLURIPOTENT STEM CELLS FOR TRANSPLANTATION PURPOSES
Lab: Department of Nephrology, LUMC
Introduction: Despite being the solely curative treatment for end-stage organ disease, the success of organ transplantation is hampered by shortage of donors and the need for immunosuppressive medication. The recent progress in cellular reprogramming has led to the idea of using induced pluripotent stem cell (iPSC)-derived tissue as an alternative to conventional organ transplantation. In this study, we aim to develop universal hypoimmunogenic human iPSCs, which are resistant to allogenic immune rejection.
Methods: To prevent attack by the complement system, complement regulatory protein CD55 was knocked-in in human iPSCs using CRISPR/Cas9 technology. To inhibit T cell mediated rejection, B2M, which encodes the light chain of HLA class I, was knocked-out. Following verification of the genetic modification, protection against complement activation, T cell and NK cell responses was evaluated in vitro in serum incubation and coculture assays.
Results: CD55 knock-in iPSCs show reduced C3 and C5-b9 deposition, reflecting inhibition of complement activation, compared to controls. Upon differentiation to endothelial cells (ECs), ECs display only modest complement deposition and CD55 knock-in has a minor additional protective effect. In B2M knock-out iPSCs, HLA class I expression is absent. HLA-A2 specific CD8+ T cells induce cytotoxicity and produce IFNγ in response to HLA class I-positive controls, but do not respond to B2M knock-out iPSCs. Although it was hypothesized that absence of HLA class I would trigger the “missing-self” response, no response of NK cells to iPSCs, irrespective of HLA class I expression, has been observed, but this needs to be verified.
Conclusion: Together, these results illustrate that CD55 knock-in and B2M knock-out protects iPSCs from the complement system and allogenic CD8+ T cells, respectively. Further research is required to evaluate the immunogenicity in iPSC-derived cells, which are more relevant for clinical application, such as kidney organoids or pancreatic beta cells.
Room 1.4: COVID-19
Presenter: Daphne Bouwens
Title: ANTIBODY-DEPENDENT CELLULAR PHAGOCYTOSIS OR CYTOTOXICITY IN SARS-COV-2: WHAT THE VACC?
Lab: Bioassay Development and Automation, Janssen Vaccines
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the pandemic of coronavirus disease 2019 (COVID-19) and needs little introduction: it has led to millions of infections, and hundreds of thousands of fatalities worldwide. Infection takes place when the SARS-CoV-2 Spike (S) glycoprotein attaches to host cells via the ACE2 receptor. Upon entry via endosomes, fusion of the viral membrane transfers viral RNA into host cytoplasm, where replication takes place.
The primary aim of vaccines against SARS-CoV-2 is to develop an effective and safe humoral response to control the worldwide pandemic. Antibodies that recognize the S protein can work in a (1) neutralizing manner, by directly engaging the receptor-binding domain of the S glycoprotein and preventing cell entry, or (2) non-neutralizing, by activating effector immune cells upon binding of the Fc region of the antibody to the immune cell (including macrophages and NK cells). Comprehensive understanding of these Fc-mediated effector function of antibodies against SARS-CoV-2 is critical in combatting disease, and these mechanisms are elucidated by developing an Antibody-Dependent Cellular Cytotoxicity or Phagocytosis assay (ADCC or ADCP, respectively, or ADCx in short).
The extent to which infection- or vaccine-elicited serum antibodies are able to induce ADCx activity is typically measured using a cell-based assay. In this research, the set-up of the ADCx assay is established, and each component of the assay is optimized. This optimization consists of choosing representative target cells, the optimal incubation time, and determining the titration range of convalescent sera and composition of the buffers. ADCx activity will be quantified using a luminescent read-out. In the end, this reporter-based ADCC and ADCP bioassay is used to characterize the Fc-mediated effector functions of (pre-) clinical serum samples of vaccinated donors against the SARS-CoV-2 virus.
Presenter: Nina de Beijer
Title: PURIFICATION OF CORONAVIRUS REPLICATION ORGANELLES
Lab: Department of Cell and Chemical Biology, LUMC
Coronaviruses are positive stranded RNA viruses that belong to the order of Nidovirales. Following the entry into the host cell, coronaviruses rearrange the host cellular membranes to establish replication organelles (RO), which mainly consist of double membrane vesicles (DMVs). Recently, it was proven that the double membrane vesicles (DMVs) that coronavirus induce are the sites of viral genome replication and that a pore connects the DMV interior to the cytoplasm. This pore, spanning the double membrane, could provide an exit for newly-made viral RNAs to be exported from the DMVs interior into the cytoplasm for translation and encapsidation. The structure of the pore was discovered using cellular cryo-tomography, allowing imaging of structures in their native state and their natural cellular context. However, the process of preparing samples from infected cells that are thin enough for cryo-EM analysis is incredibly laborious. To increase the resolution of the structure, the throughput of samples needs to be significantly higher and thus the sample preparation faster. To tackle this, we aim to purify coronaviral DMVs/ROs with intact pores, providing us with an in vitro system to study DMV and pore function. First, recombinant viruses, murine hepatitis virus (MHV) with an affinity tagged nsp3 were engineered and characterized. These tags could be used to pull down DMVs, since nsp3 is part of the pore complex spanning the DMV membranes. If the DMVs and pore remain intact, the created system can be used to study coronavirus replication in greater detail.
Presenter: Jonathan Benhaiem
Title: LAB SURVIVAL TACTICS
The lab is a treacherous place. At first sight, the biomedical lab is fun, exciting, and challenging. But underneath a layer of pipettes, 96 well plates and R code, lies a much more important and often overlooked aspect of the life at work: Lab Politics. Lab politics can be described as all diplomatic relationships between the stakeholders of a laboratory. As a student in the lab, you will encounter tricky situations, and everyone will have a different approach to solve these issues. Often, a solution can be found by following guidelines and recommendations from colleagues and your supervisors. But how do you deal when situations get out of hand, or when you have a problem with your supervisor? Understanding and reacting to motives and intentions is an invaluable tool to be successful in any environment. Managing your way through conflicts is learned mainly through experience. During the workshop, you may be assigned a role as a stakeholder in a scenario. In the end, the goal is to get acquainted with the power of Lab Politics and to be more confident in representing your ideals and motives in the lab!
Presenter: Soraya Schachtschabel
Title: HOW TO SPREAD THE AWESOMENESS OF (YOUR) SCIENCE
Imagine, you have worked in the lab for months and you finally get your most anticipated results. They are great and you can’t wait to share them with the world. But how? Research articles are great to convey your findings to fellow researchers, yet they are certainly not your average family member’s cup of tea. Letting the world (and your curious friends) know what you have done all that time behind a lab bench or a computer screen, and why your discoveries are worthwhile, is a key aspect of being a scientist. However, contrary to what you might think, it is not the easiest aspect. As all things in life, it takes practice to communicate science in the best way possible. But, once done well, science communication is a great tool to reach a broad audience and spark their enthusiasm in research. During this workshop we will discuss the further significance of science communication, look at tips on how to translate science to friends, family (and perhaps complete strangers) and put these tips into practice. At the end you’ll be ready to spread some science awesomeness!
Presenter: Esther Domenie
Title: LEARNING BY PRACTICE: CREATING INTERACTIVE SCIENCE LESSONS FOR CHILDREN
How to make science fun for children? I think we all remember science classes that were so boring that you rather would have done something else. Or, maybe, you do not remember them at all! However, as we all know, science is not boring but very interesting and exciting. At Technolab Leiden, where I am currently doing my internship, we want to engage children in science and teach them about science in an interactive way. At Technolab it is all about learning together, by DOING. In this workshop, we will create an interactive lesson for children about a scientific topic, following Technolab’s basic principles. What will be your role as a teacher, what is the goal of your lesson, how to engage children and what kind of methods are suitable for your topic? We will discover this and more during this workshop.