IPSC-basiertes Kokulturmodell zur Erforschung der lymphatisch-epithelialen Interaktionen der sporadischen LAM (Lymphangioleiomyomatose)
Strukturbiologie
Zusammenfassung der Projektergebnisse
Lymphangioleiomyomatosis (LAM) is characterized by the formation of neoplastic lesions comprising of tumorous smooth muscle-like (LAM-SMC) and epithelioid-like cells that leads to invasion of bronchioles, blood, and lymphatic vessels, resulting in severe obstructive lung disease. To date there are limited therapeutic options for LAM patients, and it remains a severely debilitating and progressive disease. Investigating rare lung diseases is challenging with a lack of disease-relevant and patient-specific tissues available and animal model systems that poorly replicate the LAM-phenotype observed in humans. The cellular and molecular mechanisms that drive the extensive cystic tissue remodelling of the pulmonary parenchyma are largely unknown and therefore, there is a paucity of known molecular targets for therapeutic intervention. LAM nodules consist of aggregations of epithelioid cells expressing HMB-45 (human melanoma black-45), with smooth muscle-like cells (smooth muscle alpha actin expressing) located centrally, have been shown to be invaded by lymphatic endothelial cells (LEC). The impact of the lymphatic endothelium on the progression of the disease and the epithelial destruction is poorly understood and the recurrence of LAM lesions in transplanted lungs suggests that lymphatic involvement may influence metastasis in the lung. We therefore aimed to investigate the impact of the lymphatic endothelium on LAM progression by creating a 3-dimensional organoid model representing the LAM nodule. In lieu of vast supply of primary patient tissues and cells we proposed to generate TSC2-deficient induced pluripotent stem cells (iPSC) to mimic TSC2-LAM phenotypes. 3-D LAM organoids were generated integrating a core of LAM cells with LECs and these organoids were used to evaluate the impact of a multikinase inhibitor, Sorafenib, on LAM nodule invasion into the surrounding matrix. Sorafenib was found to significantly block invasion of LAM nodules in both the presence and absence of coculture of LECs. Importantly, this data suggests that multi-kinase inhibitors, such as Sorafenib, could be a potential therapeutic option to inhibit the progression of LAM. During the DFG funded fellowship several LAM-specific iPSC lines were also created. First, LAM patient-specific iPSCs were generated by reprogramming patient peripheral blood mononuclear cells in which we identified LAM-driving mutations in both TSC2 and FGFR2. Second, TSC2-knock out iPSCs were created using CRISPR-Cas9 gene editing targeting the first exon of TSC2. These iPSC lines provide a valuable resource for creating patient specific LAM nodules and cysts in vitro. To facilitate the development of efficient methods to generate iPSC-derived LECs a PROX-1-GFP reporter line was also created. A modified differentiation protocol in which NOTCH signalling was temporally improved LEC differentiation in the TSC2-deficient lines which will now be used to investigate the impact of TSC2 mutations on lymphangiogenesis in LAM by integrating these into our LAM nodules in the presence or absence of iPSC-derived alveolar and epithelial cells.
