Year: 2024
PI Name: Nils Rouven Hansmeier
Mentor Name: René Haegerling
Project Title: Spatial Distribution of PIK3CA-mutated Cells in Lymphatic Malformations
Abstract: Lymphatic malformations are clinically very heterogeneous ranging from small local ‘birthmarks’ to life-threatening entities affecting entire limbs, organs or larger parts of the human body. Often, these conditions require surgical and/or pharmacological intervention to prevent further disease progression and to increase patients’ quality of life. Therefore, an in-depth understanding of the underlying vascular pathology is crucial for appropriate treatment and management. However, our current knowledge on lymphatic malformations and in particular spatial cellular distribution of individual mutated and non-mutated cells within the lesion is very limited. This is due to insufficient tools and technologies to visualize and investigate individual cells at a spatial multicellular level. To overcome these limitations and gaps of knowledge, the proposed project aims to generate novel state-of-the-art nanobody-based reagents suitable for immunofluorescence staining of entire tissue biopsies and combine this with cutting-edge imaging and visualization technologies using lightsheet microscopy. Nanobodies raised against wildtype and mutated PIK3CA, i.e the most frequent cause for somatic lymphatic malformations, will be used to visualize the spatial distribution of individual cells and its expression of wildtype and mutated protein in diseased tissue samples using optical sectioning. This will allow a spatial clonal analysis in lymphatic malformations positive for PIK3CA mutation. In summary, the proposed project represents an innovative approach that has the great potential to provide for the very first time novel insights into spatial cellular arrangement of somatic PIK3CA mosaicism and to allow spatial phenotyping as well as clonal analysis in patients with PIK3CA-associated lymphatic malformations.
Funding from the LGD Alliance Europe.
Year: 2024
PI Name: Yarelis Gonzalez-Vargas
Mentor Name: Brandon Dixon
Project Title: Towards a Precision Medicine Approach for the Study and Management of Pediatric Lymphatic Malformations
Abstract: This project aims to develop a high-throughput testing platform for lymphatic malformations (LMs), a rare type of vascular anomaly caused by genetic mutations. Our goal is to create a patient-specific Micro-Physiological System (pMPS) using advanced biomaterials and artificial intelligence (AI) to enhance pre-clinical drug testing. Current treatments for LMs, often repurposed from cancer therapies, are not well-tolerated by children. By using patient-derived organoids (PDOs) embedded in a specially designed hydrogel, we can replicate LM conditions in vitro. These PDOs will be used to test drug responses, with data collected to train a deep learning model that automates image analysis and reduces human error. This innovative approach leverages the combined expertise of bioengineering and medical genetics, aiming to streamline the development of effective and safer treatments for pediatric patients with LMs. The project not only seeks to improve drug efficacy predictions but also to advance understanding of LM biology, potentially leading to better therapeutic strategies.
Funding from the Lymphatic Malformation Institute
Year: 2024
PI Name: C. Griffin McDaniel
Mentor Name: Tim Le Cras
Project Title: Mutant to wild-type endothelial cell interactions in the pathogenesis of kaposiform lymphangiomatosis
Abstract: Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly (CLA) that can cause fluid leakage around the heart and lungs and severe bleeding problems. A mutation in NRAS, an important gene for cell growth and survival, has been found in affected tissue from patients with KLA. This mutation has also been identified in many cancers. However, only a small percentage of the cells within the diseased tissues of KLA patients have the NRAS mutation. Furthermore, bleeding problems in KLA patients are unexpected as the lymphatic system does not normally transport blood. We hypothesize that the NRAS mutant cells in KLA communicate with surrounding normal cells to drive abnormal vessel growth and contribute to disease pathogenesis. To address this hypothesis, we will use human cells cultured in petri dishes and mouse models. First, we will mix mutant and normal human cells and determine the proportion of mutant cells that is required to cause abnormal vessel structures in a three-dimensional cell culture model of vessel formation. Then, mutant and normal cells will be mixed and implanted into mice to determine the proportion of mutant cells needed to cause abnormal vessel growth in vivo. Finally, we will culture normal and mutant cells on nearby beads and determine whether the mutant cells secrete factors that cause the normal cells to contribute to abnormal vessel growth. The goal of these studies is to improve our understanding of the disease process in KLA patients, gain insights into other CLA, and identify new therapeutic targets.
Funding from LGDA.
Year: 2023
PI Name: Nour C. Bacha
Mentor Name: Carrie Shawber
Project Title: Assessment of Lysosomal Dysfunction in Lymphatic Anomaly Pathogenesis
Abstract: Lymphatic anomalies are congenital slow-flow diseases, including Generalized Lymphatic Anomalies (GLA). Genetic studies have shown that somatic mutations leading to PI3K/AKT/mTOR and/or RAS/MAPK pathway hyperactivation are causal to GLA pathogenesis. Due to crosstalk between the RAS and PI3K pathways, most mutations in GLA lead to AKT and mTOR hyperactivation. Our team’s preliminary data revealed that hyperactivation of PI3K signaling led to the abnormal cytoplasmic accumulation of CD31 and VE-CADHERIN in lymphatic anomaly patient-derived lymphatic endothelial cells (LA-LECs) with PIK3CA variants or variants leading to RAS hyperactivation compared to control cells. This correlated with reduced expression of two essential proteins at the cell surface, CD31 and VE-CADHERIN, and an increase of CD31 in endosomes and lysosomes of LA-LECs. As PI3K/AKT/mTOR signaling inhibits lysosomal degradation, these data suggest that LA-LECs have lysosomal insufficiency downstream of PI3K/mTOR hyperactivation that contributes to GLA pathogenesis. We hypothesize that hyperactivation of PI3K signaling leads to lysosomal dysfunction in LA-LECs that contributes to their pathogenesis and can be reversed by improving lysosomal protein degradation. Therefore, we propose to assess CD31 and VE-CADHERIN degradation and expression related to lysosomal dysfunction and to inhibit mTOR signaling pathway to evaluate this dysfunction downstream PIK3CA hyperactivation. We will also use drugs to improve lysosomal degradation and determine if they normalize CD31 and VE-CADHERIN expression at the cell surface which is crucial for lymphatic vasculature structure integrity.
Funding from LGDA.