Generation of functionally mature pancreatic organoids as a replacement strategy for animal-models of pancreatitis and pancreatic cancer

The pancreas consists of two organs in one. The endocrine part houses insulin-secreting cells controlling blood glucose levels and the exocrine part produces enzymes controlling the digestion. Diseases affecting the exocrine pancreas, such as pancreatitis and pancreatic cancer, present dramatic mortality rates, as highlighted by the 95% mortality of patients diagnosed with pancreatic cancer. To alleviate the societal burden caused by these diseases, research teams all over the world are trying to better understand pancreatic diseases and establish early diagnosis and innovative therapeutic strategies. To develop and test the efficacy of these strategies, genetically engineered mouse models replicating most human pathophysiological features are routinely used in research labs. Although necessary for research purposes, the use of these models leads to the culling of thousands of animals worldwide every year.
Organoids are emerging 3D in vitro systems derived from stem cells, which represent a very promising alternative to animal models. Organoids are simplified versions of organs, of which they should recapitulate the physiology and microanatomy. Unfortunately, the pancreatic organoids generated so far do not faithfully recapitulate the pancreas complexity. This strongly limits the adoption of pancreas organoids as valuable replacement models in the labs working on pancreatic diseases. The goal of this project is thus to develop physiologically relevant organoid models for studying pancreatitis and pancreatic cancer in vitro.

The formation of the pancreas in the embryo is known to be tightly regulated by its local microenvironment, whose precise composition is still poorly known. The first goal of the project is to define the pancreas microenvironment observed in embryos and to reproduce it artificially in vitro for the maturation of organoids. To that end, I will use cutting-edge sequencing technologies on mouse embryonic pancreas and precisely define the biochemical cues that compose its microenvironment. I will then use the identified cues to supplement artificial microcavities in which organoids will be cultured. Cultured organoids will be monitored for the acquisition of mature morphological and physiologic pancreatic markers. This approach will allow me to identify the best microcavities composition to mature pancreatic organoids. To model pancreatitis, organoids will be treated with caerulein, a drug used to induce pancreatitis in animal models. To model pancreatic cancer, I will generate organoids using genetically modified stem-cells that over-activate a variant of a gene known to induce pancreatic cancer in mice and humans. I will assess whether the obtained organoids are valuable models to study pancreatitis and pancreatic cancer by comparing their pathophysiological features with those observed in animal models. The completion of this project will surely enable a significant reduction of animal procedures and provide research labs with new tools to screen the effect of next-generation drugs for pancreatic diseases treatment.

The goal of this project is to partially replace animal procedures used for modelling pancreatitis and pancreatic cancer by developing a 3D in vitro model system. Organoids are advanced systems used to model the physiological features of organs in vitro. The protocols published so far produce pancreatic organoids with limited exocrine differentiation and structural organization. Hence, they do not recapitulate the physiological complexity of the pancreas, preventing the adoption of organoids as replacement models for pancreatic diseases. In the embryo, pancreas organogenesis is tightly regulated by its local microenvironment. Therefore, the in vitro maturation of pancreatic organoids should take place in a synthetic microenvironment functionally mimicking the embryonic one. The first aim of this project will thus be to use in situ transcriptomics to define in the mouse embryo the biochemical cues composing the pancreas microenvironment. Using the cues identified in vivo, I will promote the maturation of mouse pancreatic organoids by culturing them in a 3D microarray platform. The structural and functional features of the organoids will be characterized by high-content imaging, immunostaining and in situ hybridization. Using this phenotyping pipeline, I will modulate the composition of the microenvironment to optimize the maturation of organoids. I will then assess the value of matured organoids as pancreatitis and pancreatic cancer models. To that end, I will analyze the phenotypic response of the organoids to caerulein treatment and Kras overactivation and compare it to the phenotypes of animal models. Lastly, the organoid models will allow me to use live-imaging to capture acinar-to-ductal metaplasia events, dynamic processes crucial in pancreatic diseases. As this has been so far impossible, it will highlight that more than just being valuable replacement models, organoids also bring technical advantages to the study of pancreatic diseases.