Role of genomic and microenvironment factors in conferring acquired resistance to ferroptosis to chemoradiation in esophageal adenocarcinoma

Project 3 Summary Esophageal cancer is the sixth most common cause of deaths worldwide with a 5-year survival rate of less than 20%. Similar to other intrathoracic malignancies, such as lung cancer, chemoradiation therapy (CRT) is an effective tool for treating esophageal adenocarcinoma (EAC). However, CRT leads to complete responses in EAC in only approximately 25% of patients. Thus, characterizing the cellular and molecular changes that occur in the tumor and tumor microenvironment during CRT can provide highly accurate predictive tools to identify which EAC patients will develop acquired resistance to therapy. CRT induces the cell death pathway ferroptosis in cancer cells, and the hypothesis that ferroptosis is a key regulator in acquired radiation therapy resistance is a central theme of the Acquired Resistance to Therapy and Iron (ARTI) Center. Project 3 will contribute to this central theme by bridging the basic science mechanisms in preclinical models discovered in Project 1 and Project 2 with clinical EAC tumor biopsies, collected at baseline (before treatment) and during the middle of the CRT regimen. These EAC tumor biopsies will undergo single cell transcriptome profiling to identify expression of ferroptosis-related genes and to provide an understanding of the complex communications between tumor cells and cells of the tumor microenvironment that may drive and/or regulate ferroptosis (Aim 1). Identification of transcriptomes that are directly or indirectly related to ferroptosis may help to prognose EAC patients who may or may not respond to CRT. Further spatial cellular mapping and deciphering the relationship of differentially expressed genes with ferroptosis will provide evidence for the tumor’s ability to evade ferroptosis upon irradiation. Aim 2 will focus on determining whether adaptive resistance to therapy occurs due to selection of rare, but pre-existing tumor cells, or due to de novo acquisition of alterations in genes directly or indirectly related to ferroptosis signaling pathways. Aim 3 will focus on elucidating the tumor microenvironment mechanisms that may confer ferroptosis resistance to CRT, such as fibroblasts, which have been shown to regulate the cystine transporter SCL7A11 that promotes ferroptosis resistance. In addition, tumor-infiltrated immune cells with hypoxic signatures will be analyzed for their association with ferroptosis resistance to CRT. In EAC tumors resistant to CRT, myeloid cell expansion was observed, and the phenotype and activation status of myeloid cells will be assessed using orthotopically transplanted patient derived EAC tumors. Further application of an innovative positron emission tomography tracer from the Molecular Imaging Core of the ARTI Center to assess oxidative potential of tumors as related to the myeloid cell expansion observed in resistant tumors will help discover molecular and cellular pathways related to ferroptosis. Project 3 will iteratively strengthen and support the basic science/mechanistic Projects 1 and 2, and Project 3 will significantly contribute to the ARTI Center goal of identifying patients who are at greatest risk to develop acquired radiation resistance.