PROJECT SUMMARY/ABSTRACT Esophageal squamous cell carcinoma (ESCC) is a lethal cancer with a 10%-25% 5-year survival rate. Given the importance of early detection, the initiation process of ESCC must be better defined. To study this process, we focused on preneoplastic cells, which have the potential to progress to neoplastic cells. We comprehensively analyzed single-cell RNA-sequencing (scRNA-seq) datasets from human ESCC and healthy donors, as well as normal adjacent to cancer datasets, to reveal the spatial characteristics of preneoplastic cells. We also analyzed scRNA-seq data from a mouse model of carcinogen-induced ESCC to identify features of preneoplastic cells in a temporal context. Strikingly, molecular analysis revealed that preneoplastic cells, though histologically normal, exhibited elevated copy number variations and reduced TP53 and CDKN2A signaling compared to healthy normal samples in both spatial and temporal datasets. The CELF2 gene emerged as a robust marker of preneoplastic cells across both datasets, validated by an increased presence of CELF2+ cells in Trp53 and Cdkn2a double knockout (PC) organoids. These CELF2+ cells showed increased plasticity, acquiring cancer stem cell features and undergoing epithelial-mesenchymal plasticity (EMP). Aim 1 is to determine the cellular reprogramming in CELF2+ cells when TP53 and CDKN2A are ablated. The reprogrammed stemness will be validated by comparing the tumorigenicity of CELF2+ and CELF2- cells of PC organoids. Additionally, tumorigenicity of a genetically engineered mouse model with Trp53 and Cdkn2a deletion will be tested in the presence or absence of CELF2+ cells. Cellular reprogramming will also be evaluated by comparing the exact cell lineage of wild-type and PC organoids and PC-derived tumors. Employing cutting-edge genetic barcoding, we will trace the entire cellular trajectory. Cell lineage established in Aim 1 will be the most reliable reference for preneoplastic cells and normal esophageal cells. Furthermore, cell lineage and population analyses of PC-derived tumors will elucidate the mechanism of clonal selection during tumorigenesis. Aim 2 will address the role of EMP-undergone CELF2+ cells in immune evasion. Although EMP is conventionally known for its function in invasion and metastasis, its role in immune evasion is less studied. Our preliminary experiment identified mesenchymal-CELF2+ cells in the tumor niche release chemokines and cytokines. Aim 2 will disclose the efficiency of tumorigenicity when mesenchymal-CELF2+ cells are co-cultured with immune-rejected cells. In addition, the candidate chemokine and cytokine genes will be manipulated in preneoplastic cells to test our working model. Collectively, the proposed study will elucidate the cellular and genetic mechanisms of ESCC initiation. Under the guidance of Dr. Jae-Il Park, a mentor, Dr. Ko's research skillset, such as mouse genetics, organoids, and cancer signaling, as well as the skills of writing, teaching, and mentoring, will be expanded during the K99 phase. Completing the research and training will significantly facilitate Dr. Ko’s transition to an independent investigator.

PROJECT NARRATIVE Despite the importance of early diagnosis, genetic and molecular mechanisms of esophageal squamous cell cancer (ESCC) initiation remain elusive. The proposed research will elucidate the dual role, both autonomous and non-autonomous, of preneoplastic cells in driving ESCC development, which is expected to establish a novel foundation to develop new types of ESCC detection and treatment. Therefore, this study is related to public health and to the part of NIH’s mission that pertains to fostering fundamental creative discoveries and innovative research strategies as a basis for ultimately protecting health.