Epithelial cells are essential orchestrators of organ physiology in the gastrointestinal tract, but impaired epithelial function or malignant transformation is associated with severe diseases with high mortality. Among the different GI disorders inflammatory and malignant diseases of the pancreas emerge due to their therapeutic difficulties, potentially lethal outcome and thus their social burden. Therefore our lab seeks to understand how epithelial cells sense physiological stimuli and stress factors and how they integrate and transduce these signals to physiological or pathological responses. We believe that our work will provide significant understanding of multiplex epithelial functions in physiology and pathophysiology, which shall have significant multidisciplinary and translational consequences and will help to develop novel therapeutic approaches to treat inflammatory and malignant diseases.
Epithelial Ion Secretion
Physiological pancreatic exocrine fluid and ion secretion and it’s damage is the trigger (such as cystic fibrosis) or booster (acute and chronic pancreatitis) of pancreatic diseases. Thus, restoration of pancreatic ductal secretion in inflammatory pancreatic diseases would be of primary importance. To achieve this we need detailed molecular characteristics of human pancreatic ductal secretion, which is currently not available only indirectly suggested relying on animal models. A recently developed model system – 3 dimensional organoid cultures based on Lgr5+ adult stem cells – will give us a unique possibility to analyse the epithelial cell physiology in an organ-like structure. Second – even more importantly – it will give us the possibility to expand our investigations to study the human relevance of our findings and overcome the limited translational potency of 2D cell lines and animal models.
Calcium and cAMP signalling are the hallmarks of epithelial signal transduction, however only limited information is available how they organize to microdomains (MDs) and how this localisation affect their activity and interactions with each other. It has been described that Orai1 and the PM bound Ca2+ activated adenylyl-cyclase 8 (AC8) physically interact, which maximizes the efficiency of the response, whereas the activation of Stim1 recruits AC3 resulting in enhanced cAMP accumulation and PKA activation. These suggest tight interrelated communication between the two signalling pathways. Although likely, the role of ER/PM junction MDs in this communication has not been investigated yet. Similarly, we have very limited information about the role of MDs in the regulation of secretory processes. Key components of the epithelial ion secretion are two Cl- channels, ANO1 and cystic fibrosis transmembrane regulator (CFTR). These channels are expressed mostly on the apical PM of different epithelial cells. Although we know that signalling complexes form MDs to maximize the efficiency of response to stimulation, there is absolutely no information about the MD localization of these channels, or about the role of MD localisation in channel function and regulation. On the other hand, the ion channels can also shape intracellular signalling. Our group showed that CFTR regulates the activity of PMCA pump, and thus determines Calcium ion extrusion. The nature and role of this interaction is not known. To address these important physiological questions we combine cutting edge imaging techniques (such as super-resolution microscopy, or correlative light and electron microscopy) with state-of-the-art cell physiology methods and aim to investigate the composition and functions of ER/PM junction MDs.
Epithelial cells in pancreatic diseases
Impaired epithelial function or malignant transformation is associated with severe diseases with high mortality. Among the different GI disorders inflammatory and malignant diseases of the pancreas emerge due to their therapeutic difficulties, potentially lethal outcome and thus their social burden. Chronic pancreatitis (CP) is a progressive inflammatory disease accompanied by the functional and morphological of the pancreas. CP patients suffer from malabsorption, post-pancreatitis diabetes mellitus, debilitating pain and a significant decrease of the quality of life. Natural history of inflammatory pancreatic diseases suggest that ~ 20%-30% of patients with acute pancreatitis have a recurrence and ~10% develop CP. Medical treatment of CP is limited to supportive therapy, and there is no definitive therapy yet, however the incidence of CP is increasing. Pancreatic ductal adenocarcinoma (PDAC) exhibits one of the poorest prognosis of all solid tumors with an overall 5 year survival of ~5-6% and represents a major challenge in cancer medicine. Mortality due to PDAC is projected to surpass that of breast and colorectal cancer by 2030 in the United States due to the aging population and to the therapy improvements of other malignancies. Combination chemotherapies moderately improved patient survival, but this benefit is often abolished by severe side effects of the therapy. Although basic research of molecular mechanisms improved our understanding of pancreatic diseases, translation of this improvement into patients benefit is still to be achieved.
There are three major bottlenecks hindering translational pancreatic research. The limited access to human tissue for research use, the deficient knowledge of human pancreatic epithelial physiology combined with the lack of human-relevant disease models eventuate in poor translation of preclinical findings and limited efficiency of drug screening. Animal models of inflammatory pancreatic diseases have significant shortcomings, whereas in vitro models utilize mainly isolated rodent pancreatic acinar and ductal cells and are not applicable for CP. Whereas, current models for PDAC consist of 2D immortalized cell lines, patient-derived xenografts, and genetically engineered mouse models. These models yielded significant advances in PDAC research, but they do not recapitulate relevant features of human disease and fail to predict therapeutic responses. In the recent years organoid cultures (OCs) derived from tissue specific Leucine-rich repeat-containing G-protein coupled receptor 5 positive (Lgr5+) adult stem cells emerged as novel models of organ development and disease. By maintaining the activity of Wnt/β-Catenin signal transduction cascade – a key drivers of most types of tissue stem cells – OCs can be grown in vitro for long-term in 3D extracellular matrix based hydrogels; whereas, epithelial cells in the culture maintain the original cellular diversity and organization of the organ of origin 16. Originally small intestinal adult stem cells were used to generated crypt–villus like structures in vitro. Since then OCs have been established from a wide range of organs in the GIT, including colon, esophagus, stomach, liver and pancreas. Patient-derived PDAC organoid cultures had a major impact on the field and enabled the study and correlation of in vitro responses of PDAC OCs to chemotherapeutics. PDAC OCs therapeutic profiles paralleled patient outcomes suggesting the applicability of OCs to prospective therapeutic selection. However this system still have limitations. OCs consist of epithelial cells, which could be beneficial for studying epithelial functions, but the reductionist nature might limit the use of OCs for more complicated research questions (such as the effect of stroma on drug sensitivity of tumor cells).
Therefore in this complex research project our mission is to capitalize the advantages of patient-derived pancreatic OCs and improve the culture system by establishing disease-relevant cocultures of stromal and epithelial cells. These cocultures will be used to study disease mechanisms, intercellular communication and therapeutic possibilities in inflammatory and malignant pancreatic diseases.