Calcium Modulates Apoptosis And Differentiation In Human Normal Colon 3d Organoids Irrespective Of Donor Sex And Biopsy Site.

Abstract Background: Observational studies indicate that calcium, a micronutrient involved in numerous cell-signaling cascades, may protect against colon cancer. Stratified analyses suggest the protective effect of calcium may depend on sex and anatomic subsite, but these hypotheses have been difficult to test experimentally. Here, we employ human-derived normal colon 3D organoids as a model of the healthy colon crypt to interrogate the effect of calcium exposure on gene expression and the effect of donor sex and biopsy anatomic subsite on the response to calcium. Methods: A total of 39 3D colon organoid lines derived from colon biopsies (22 proximal, 17 distal) obtained from unrelated subjects (21 female, 18 male) during screening colonoscopy were grown under low (1.7 mM) calcium conditions, and a matching set of the same lines were grown under high (5.0 mM) calcium conditions. After 72 hours, total RNA was extracted from organoid lines, libraries were prepared with poly-A selection, and bulk RNA-sequencing was used to measure gene expression. DNA was extracted from the same lines and genotyped using the OncoArray 500K beadchip. Missing genotypes were imputed to the TOPMed reference panel using the TOPMed Imputation Server. Polygenic risk scores for colorectal cancer were calculated for each organoid line using Plink and 135 of 141 genome-wide significant SNPs associated with colorectal cancer in recent genome-wide association studies. Relative expression between low and high calcium conditions was tested using generalized linear models as implemented in DESeq2. Functional enrichment of gene ontologies among differentially expressed genes was performed with ToppFun. The effects of donor sex, biopsy subsite, and organoid polygenic risk score on response to calcium were tested using single and multiple linear regression as implemented in the base R lm function. Results: Exposure to calcium induced differential expression of 767 genes with a false discovery rate of 5%. Differentially expressed genes were enriched for biological processes including extracellular structure organization (Bonferroni p=1.6E-3) and positive regulation of apoptosis (Bonferroni p=2.2E-2). Although stratified analyses demonstrated more differentially expressed genes in 3D colon organoids derived from male subjects, no statistically significant effect of sex, subsite, or polygenic risk score on response to calcium was observed. Conclusion: We tested the effect of calcium exposure on gene expression in human-derived normal colon 3D organoids and found that calcium modulated processes related to differentiation and apoptosis, but the response to calcium was not affected by sex, anatomic subsite, or polygenic risk score. Citation Format: Christopher H. Dampier, Matthew Devall, Jennifer Bryant, Stephen D. Eaton, Jeroen R. Huyghe, Andre E. Kim, Virginia Diez-Obrero, Franzel J. van Duijnhoven, Duncan C. Thomas, Steven M. Powell, W James Gauderman, Ulrike Peters, Graham Casey. Calcium modulates apoptosis and differentiation in human normal colon 3D organoids irrespective of donor sex and biopsy site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2578.