Abstract C030: Effect of palmitic acid exposure in normal colon organoids derived from young versus older individuals

Background: Diet and obesity play a role in the intestinal microenvironment. High-fat diet and metabolic changes associated with obesity has been shown to increased cellular remodeling and local inflammation. Given the rise in early onset colorectal cancer, here we explore the effect of a high-fat environment in colonic organoids derived from young versus older individuals. We propose that organoids derived from younger individuals have more cellular plasticity, allow them to proliferate in high-fat environment at the cost of increased mutational burden and an altered intestinal microenvironment that increases the risk of tumor development and progression. Method: We generated organoids obtained from normal colon tissue from patients undergoing colorectal resection for colon cancer, one £45 years old and the other >60 years old. After sufficient growth, organoids were chemically passaged, counted and seeded at 50,000 cells per 50µl of Matrigel in a 24-well plate. After 2 days of growth in standard organoid media, organoids were treated with 30µM of palmitic acid. Equivalent amounts of ethanol in 1% BSA served as a control. PDOs were examined under Celigo Image Cytometer (Nexcelom) at desired time points to quantify number of organoids per well. Following treatment, RNA was obtained using RNAqueous-Micro Kit and then converted to cDNA using Bio-Rad kit. qRT-PCR Bio-Rad was conducted using iCycler RT–PCR detection system. Results: A high-fat environment led to significantly increased organoid formation in normal colon organoids in £45YO group but not in the >60YO group (mean number of organoids 245 versus 314 in £45YO-control and £45YO-exposed respectively; p-value<0.0001 and 167 versus 197 in >60YO-control and >60YO-exposed respectively; p-value 0.0892). After 96hr of growth, this significant difference was lost. Following passage and despite adjustment to normal organoid media, the £45YO-exposed group had significantly increased organoid formation capacity compared to control (mean number of organoids 94 versus 182 in £45YO-control and £45YO-exposed; p-value=0.006). TLR4 gene expressions levels were quantified and found to be significantly increased by exposure to PA in the <45YO group but not the >60YO group (p-value 0.0299). Conclusion: Using palmitic acid, a known component of a high-fat diet, we demonstrate that exposure leads to increased proliferation in colonic organoids derived from young individuals but not older individuals. Despite normalization of the environment, the second generation of organoids derived from those exposed demonstrated continued proliferation compared to control. TLR4 gene expression was also significantly increased in this group. This receptor likely may modulate inflammation in the tumor microenvironment. Citation Format: Casidhe-Nicole R. Bethancourt, Yosuke Mitani, Michelle Zhang, Tianxia Li, Yoanna Pumpalova, Beatrice Dionigi, Joel Gabre. Effect of palmitic acid exposure in normal colon organoids derived from young versus older individuals [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: The Rise in Early-Onset Cancers—Knowledge Gaps and Research Opportunities; 2025 Dec 10-13; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(23_Suppl):Abstract nr C030.