Functional and molecular characterization of Oncofetal Stem Cells in CRC Project Summary. Background. Colorectal cancer (CRC) is the second most deadly cancer worldwide, with nearly one million deaths annually1. CRC develops as a result of accumulating genetic and epigenetic aberrations in the colonic epithelium leading to formation of benign adenomas that evolve to invasive adenocarcinomas 2,3. Despite the advances in treatment options, resistance to therapy and recurrence remain inevitable in patients with advanced disease, accounting for the high mortality rates 4,5. The underlying malignant features have been often attributed to the LGR5+ CSCs. However, the unexpected findings that certain LGR5- cells sustain tumor growth upon selective ablation of this population 6,7 are consistent with the recent description of a potential second stem cell population, with fetal-like characteristics 8-10. While this is intriguing from a therapeutic perspective, existence of a second, plastic, stem cell population in a more primitive state (hereafter referred to as OnFSCs for OncoFetal Stem Cells) is solely based on transcriptional signatures. Gap. Despite recent studies describing its existence, the molecular underpinnings, functional relevance, and therapeutic implications of the OnF cellular state in CRC remain largely unexplored. Hypothesis. We hypothesize that OnFSCs work in tandem with the LGR5+ stem cells (SCs) to fuel tumor evolution in CRC. OnF cells emerge early during intestinal tumorigenesis (i.e. upon inactivation of the APC gatekeeper). Their resistance to therapy and high regenerative potential makes them a critical driver of tumor plasticity and cancer recurrence. Specific Aims. We here propose to functionally characterize the OnFSCs in CRC. In Aim1, we will first generate phenotypic transcriptional reporters to track the dynamics of the LGR5+ CSCs and OnFSCs. Then, by coupling these reporters to a suicide gene (i.e. DTR), we aim to dissect the functional interplay between these different stem cell pools in CRC. In Aim2, we will use a multi-omic approach to determine the molecular drivers of the OnF state and phenotypic plasticity in CRC. In Aim3 we will perform targeted phenotypic screening to identify compounds able to selectively deplete OnFSCs. The top hits will be tested in combination with the current standard of care or LGR5+SC-targeting therapies11,12. We will validate our top drug combinations in vivo using both mouse tumoroids and human PDOs and/or PDXs. The aim is to determine a selective combination therapy superior to the current standard of care. Impact. The findings of these studies will uncover a new layer of complexity in CRC biology and will establish a mechanistic foundation for designing effective combination therapies with enduring impact on CRC treatment.