Chemical Specific Measurements of Cellular Heterogeneity in Organoids

Project Summary The central theme of this proposal is to develop technologies that will enable the extraction of chemical infor- mation from organoids. Organoids are cell cultures that are grown directly from human or animal tissues and can be generated from either healthy or diseased specimens. They are disease-agnostic and have been gen- erated from almost every organ in the human body, making them a valuable resource to study fundamental bi- ology, structure, and reactivity. Because they can be grown directly from human patients, there is considerable buzz surrounding them for their applications in disease prognosis and evaluating transplantation success. They also hold great promise in the arena of drug development, especially given new changes in legislation. In late 2022, the legislation governing drug testing in the United States was altered with the FDA Modernization Act 2.0. Previously, all new therapeutics had to be tested with an animal model prior to reaching the American pub- lic. However, with these laws in place, the use of organoids to evaluate new drugs is not just allowed but en- couraged. Despite these advances, the analytical strategies used to analyze organoids are lacking, especially protein analysis. In most organoid studies to date, protein analysis is completed by immunohistochemistry or cytokine arrays. While these methods offer information on a specific, pre-selected short list of proteins, they do not allow any novel protein discoveries. Especially in the arena of drug testing, where evaluation of off-target effects is critical, a discovery-based method that allows broad sampling of the proteome is needed. In this pro- posal, we outline the development of a suite of mass spectrometry approaches that will enable exploration of the organoid proteome, in a cell specific manner. Using magnetic sorting to separate the different cellular spe- cies of interest, followed by global mass spectrometry profiling, we will parse the relative contributions to the proteome for the different constituent cell types that encompass the multicellular organoid. The proteomic changes will then be validated by means of imaging mass spectrometry analysis on the organoid sections. The research to be conducted will be with lung organoids, based on a current successful collaboration between our laboratory and a group of pulmonologists, however the methods we develop will be applicable to organoids grown from any organ in the human body, thus impacting a wide range of human diseases. As we develop our methods, we will explore proteins involved in senescence, a fundamental cellular process characterized by a gradual deterioration of the functional characteristics in a mammalian cell. Senescence occurs throughout the human body yet the protein markers commonly used to characterize it lack specificity. With the combination of relevant lung organoid models and global discovery proteomics, we will elucidate alternative and better mark- ers for senescence in the human lung. Again, while our methods will be developed with lung cells, these ap- proaches will be applicable to organoids grown from any organ. With these advances, organoids will be poised to expand on their clinical promise in the areas of disease prognosis, transplantation, and drug screening.