ABSTRACT Cancer has been characterized as a chronic, poorly healing wound where inflammatory and proliferative processes continue unchecked without normal healing and resolution of inflammation. In one sense, cancer represents an imbalance of pro-inflammatory over pro-resolving processes. In colorectal cancer (CRC), we propose this pro-inflammatory imbalance may be driven by arachidonic acid (AA)-rich Western diets and associated downstream lipid metabolism. The role of AA in cancer development and progression, however, is still controversial when in vitro, in vivo, and human data are considered. Our preliminary LC-MS/MS data suggest a disordered lipoxygenase pathway CRC lipid metabolism, where AA, its derivatives (e.g, LTB4, 5- HETE), and principal genes (e.g., ALOX5, ALOX5AP) are largely over-expressed in tumor vs. normal samples. Recent in vivo murine data suggest dead cancer cell membranes (apoptotic debris), commonly generated from rapidly growing or treated tumors, may be the main source of AA that promotes tumor growth. Surprisingly, single cell RNASEQ data suggest the majority of AA lipid modulating genes are expressed in the immune cells of the tumor microenvironment (TME) rather than in the tumor cells themselves, suggesting a role for AA in regulating the immune TME. Moreover, our preliminary analysis of > 2000 CRCs suggest the immune activity of the TME is a strong predictor of long-term CRC survival. The recent proliferation of checkpoint inhibitors has fully demonstrated the potential to modulate the TME to enable cancer cures. Understanding precisely how disordered AA lipid metabolism impacts the immunity of the CRC TME is thus a critical unmet need that could lead to novel therapeutic approaches for CRC based on lipid metabolism and signaling. Here we propose to: 1) add a novel feature of quantified AA pathway lipid mediators to a large “reference” set of human highly clinically- and molecularly curated CRCs. We will determine their ability to distinguish, and possibly refine, subclasses defined by: a) consensus molecular subtyping (CMS) with enriched vs. diminished immune TMEs; b) TME immune activation scores; c) CRC genotypes; 2) assess the effect of AA-based pro-inflammatory vs. pro-resolution mediators on paired, isogeneic patient derived organoids (PDOs), with and without intact immune TMEs, to clarify mechanisms of action; 3) define the role of AA and ALOX5 in tumor growth and metastatic progression in a murine, syngeneic CRC orthotopic model. We will anchor the experimental plan on AA supplementation resembling the Western diet in order to demonstrate its inflammatory effects on CRC. These aims will decipher the potential of regulating AA and its lipid derivatives as a novel therapeutic approach to enhance the immune activity of the TME.

PROJECT NARRATIVE In one sense, cancer represents an imbalance between pro-inflammatory and pro-resolving processes that promote tumor growth and suppress the immunity of the tumor microenvironment (TME). Here, we plan to understand how AA-rich Western diets are metabolized by ALOX5 and related genes that regulate the TME. These studies will lead to new therapeutic approaches to CRC through modulation of lipid-signaling in the TME.