Transcriptomic Response to Calcium in Normal Colon Organoids is Impacted by Colon Location and Sex Open Access

Colorectal cancer is among the leading causes of cancer-related death in the United States. Although some studies have found no association between calcium and colorectal cancer (1, 2), a recent meta-analysis concluded that there was strong evidence that calcium intake reduces risk of colorectal cancer (3). This inverse association with risk has been observed for total, dietary, and supplemental calcium in men and women and in proximal and distal colon. Some studies have shown that the chemopreventive effects of calcium may be specific to different regions of the colon; however, findings of colon location-specific associations have not been consistent (4).

A limited number of randomized controlled trials have tested the effect of supplemental calcium on recurrence of colon adenoma, a precursor to colon adenocarcinoma. Two early trials that each enrolled several hundred men and women found modest, protective effects of calcium supplementation, although the association in the smaller trial was not statistically significant (5, 6). The largest randomized controlled trial testing the effect of supplemental calcium on colorectal cancer incidence found no effect in over 36,000 postmenopausal women (7). Reconciliation of these inconsistent trial results with the conclusions of observational studies is complicated by an incomplete knowledge of the molecular mechanisms underlying the putative protective effects of calcium.

There is incomplete knowledge of the pathways through which calcium may modulate colorectal cancer risk, and the relationship between environmental and inherited genetic risk. Approximately 140 independent genetic loci have been associated with colorectal cancer risk in genome-wide association studies (GWAS) of Europeans and East Asians (8–10), but identification of the genes and biologic pathways affected by inherited risk variants has been slow.

To improve understanding of the underlying molecular responses to extracellular calcium in epithelial cells of the colon crypt, we interrogated gene expression differences associated with different calcium levels in patient-derived normal colon organoids from 36 subjects. To the best of our knowledge, our study represents the largest to date to directly assess molecular events induced by extracellular calcium in an organoid model of the colon stem cell niche. We identified differences in gene expression, and found a significant association between increased calcium and differentiation of colon organoids. This response was both colon location and sex dependent, with the strongest impact in colon organoids derived from males and from the right colon. Finally, we observed an overlap between differentially expressed genes (DEG) following calcium supplementation and genes mapping to colorectal cancer GWAS loci, suggesting a potential relationship between calcium supplementation and inherited genetic risk biology. Our results provide potentially important insight into the role of calcium in reducing colorectal cancer risk.

Healthy subjects undergoing screening or surveillance colonoscopy at the Digestive Health Center of the University of Virginia Health System (UVA) during the period September 2017 to December 2019 were enrolled under an approved Institutional Review Board for Health Sciences Research protocol following informed consent (IRB-HSR #19439). Subjects were considered healthy when they were without acute gastrointestinal symptoms, personal history of colorectal cancer, inflammatory bowel disease, or other known colon pathology, and when they had no more than three tubular adenomas, each fewer than 10 mm in diameter. Male and female subjects of any race were considered for enrollment. Ultimately, 36 healthy subjects, 18 male and 18 female, were enrolled into the study. The effect of genetic ancestry on drug response (11) was an important consideration for this study. Only White subjects are included due to insufficient enrollment of non-White subjects and the need to reduce sample heterogeneity. Race-specific differences in calcium-related genes have previously been reported, which would have been challenging to adjust given the framework and size of our study (12). Recruitment was performed in accordance with relevant guidelines and regulations and was consistent with those required by both the NIH and University of Virginia. Written, informed consent was obtained from each patient and the study was conducted in accordance with U.S. Common Rule.

Biopsy collection from grossly normal colonic mucosa was conducted via standard endoscopic forceps. Biopsies were collected within 5 cm of the hepatic flexure (right colon) or within 5 cm of the splenic flexure (left colon) during colonoscopy. Upon retrieval, biopsies were placed in DMEM/F12 media with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin on ice and transported to the laboratory. Sample collection was prioritized to improve sample integrity. As such, the process, from biopsy to plate takes approximately 1 to 2 hours. For each subject included in this study, organoids were established from biopsies of either the right or left colon and stored at low passage number under liquid nitrogen prior to experimentation. In the cases of biopsy collections from individuals with polyps, the biopsy was collected 10 cm away from the polyp.

Organoids were established from biopsies using a modification of the method described by Sato and colleagues (13). The modified method is described in detail in Devall and colleagues (14–17). Samples were washed three times with DPBS (Gibco, #14190–136) at room temperature. Following this, 10 mL of 9 mmol/L Ultra-Pure EDTA (Invitrogen, #155750–038) in room temperature DPBS was added to each colon biopsy. Biopsies were then incubated in EDTA and DPBS for 20 minutes at room temperature. Manual inversions of the samples were carried out every 2 to 3 minutes. During incubation, all tubes, tips, and pipets were conditioned with wash media [DMEM/F-12 (Gibco, #12634–010), 10% FBS (Gibco, #16000–044), 10 mmol/L HEPES (Gibco, #15630–080), 2 mmol/L L-glutamine (Cellgro, #25–005-CI), 1X Pen-Strep (Gibco, #15140–122: (100 U/mL penicillin, 100 μg/mL streptomycin)), 1X Glutamax (Gibco, #35050–061)] to avoid crypt adhesion. DPBS/EDTA was subsequently removed following incubation. 10 mL of DPBS without EDTA was added. This solution was then manually pipetted up and down performed 8 to 10×. Tissue was allowed to settle and supernatant was collected in a 15 mL tube. This process was repeated a total of 3×. Collected supernatant was spun at 1,200 rpm at 4°C for 5 minutes. All but 2 mL of supernatants from each 15 mL tube was then removed and combined in a clean 50 mL tube, without disturbing the pellet. Using a conditioned 5 mL pipet, the remaining 2 mL from each tube was then combined into one 15 mL tube and spun at 1,200 rpm at 4°C for 5 minutes. The resulting supernatant was carefully removed. 10 μL of complex media [45% Wash Media, 50% L-WRN conditioned media, 10 nmol/L Gastrin (Sigma, #G9020), 10 μmol/L Y27632 dihydrochloride (R&D, #1254), 1× B27 Supplement (Gibco, #17504–44), 1X N2 Supplement (Gibco, #17502–048), 1 mmol/L n-acetylcysteine (Sigma, #A9165), 50 ng/μL EGF (Life Technologies, #PHG0311L), 10 mmol/L nicotinamide (Sigma, #N6636), 500 nmol/L A83 (R&D, #2939), 10 μmol/L SB202190 (R&D, #1264)] was added. To reduce the clumping of the colon crypts, this mixture was then manually pipetted several times. Two hundred microliters of Matrigel (Corning, #356237) was then added and mixed. 35 to 50 μL of each sample was then plated at the center of wells of a 48-well plate and incubated at 37°C for 15 minutes. 300 to 500 μL of complex media was then added and samples were returned to the incubator. Organoids were checked and fed after 24 hours. Following this, they were passaged as needed (every 3–5 days) then frozen.

Organoids were thawed and grown in 48-well culture plates in standard growth media. They were passaged as needed (approximately once every 72 hours) with a needle and TrypLE, as described in Devall and colleagues (14–17). One day prior to initial exposure, all organoids were passaged and grown for 24 hours in standard growth media with an ambient calcium concentration of 1.66 mmol/L. After 24 hours, one set of organoids per subject was selected for higher calcium treatment. Standard growth media was removed from wells of the treatment group and replaced with growth media fortified with 3.34 mmol/L additional calcium chloride (Sigma, CAS #10035–04–8) to bring the total calcium concentration in the media to 5 mmol/L. This concentration was based on prior studies of human colonic epithelial cells in culture in which calcium was shown to have antiproliferative effects at a concentration of 2.2 mmol/L in the absence of other additives and 10 mmol/L in the presence of butyrate and deoxycholic acid for up to 4 hours (18–20). The other set of colon organoids were maintained at an ambient calcium concentration of 1.66 mmol/L. Both sets of organoids (i.e., lower and higher calcium concentrations) were grown for 72 hours under experimental conditions. This period of time was chosen so as to avoid the effects of passaging on colon organoids following treatment, which may add stochastic noise into the system. After 72 hours, residual media was removed, Matrigel was mechanically disrupted, and 200 μL of RNA Lysis Solution RA1 (with 4 μL TCEP; Clontech/Machery-Nagel RNA XS Kit) was added to each well. The contents of each well were then collected in a sterile Eppendorf tube, vigorously vortexed (five pulses), and stored in a −80°C freezer prior to RNA extraction.

Imaging was taken at the beginning and end of calcium treatment in a subset of samples using Lumenera Infinity2–2C 2.0 Megapixel CCD Color Camera (catalog no. #95107) and Infinity Analyze software at 100× magnification.

Total RNA was extracted using the NucleoSpin RNA Mini Kit (Macherey-Nagel). Initial RNA integrity numbers (RIN) were measured with an Agilent 4200 Tapestation instrument. RIN values ranged from 8.4 to 10.0 with a mean of 9.9. Libraries were prepared using the TruSeq Stranded Total RNA Gold Kit, and sequencing was carried out according to Illumina protocols at the Northwest Genomics Center of the University of Washington. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on a NovaSeq 6000 instrument to provide 100bp paired-end fragments. Resulting FASTQ files were aligned to GENCODE v29 (21) using STAR (v2.6.1d; ref. 22) and RSEM (v1.3.1; ref. 23). A median mapping rate of 94% was observed, yielding 22.5 uniquely mapped reads per sample.

Cell-type composition was inferred for samples sequenced in bulk by deconvolution using CIBERSORTx and a curated single-cell RNA-sequencing (scRNA-seq) dataset (24), as reported previously (14, 16, 17, 25). Pre-processing and cell selection of scRNA-seq data used for single-cell deconvolution has been described previously (14). Initial analysis failed to accurately capture cell scores for the original six epithelial cell populations. As such, we made use of a reduced matrix consisting of four cell populations: cycling transit-amplifying (TA), mature enterocyte (colonocytes of the colon), mature goblet cells, and stem cells. scRNA-seq (24) and bulk transcript per million (TPM) data were uploaded to CIBERSORTx (26). A signature matrix was defined using largely default parameters with two exceptions: minimum gene expression was set to 0.8 and sampling was performed across the whole dataset. For estimation of absolute cell scores, S-mode batch correction was used, and 500 permutations were required for significance analysis. The effect of different calcium concentrations on cell composition was estimated by fitting mixed-effect linear models for absolute cell-type proportions using the lmerTest package in R (27).

Gene expression analysis was carried out in R (version 4.1.1; ref. 28). Gene abundance estimates from RSEM were converted to gene counts using tximport (29) and a paired regression within DESeq2 (30). Sample pairing was used as a blocking factor, which allowed for the comparison of gene expression differences across treatment conditions while accounting for differences observed within sample pairs. Genes were considered significant if they survived a 5% adjusted FDR in DESeq2 (30). To adjust for the effect of cell composition on gene expression, absolute cell scores were centered, scaled, and added as additional covariates to each regression model. Pathway analysis was performed by uploading nominally significant (P < 0.05) DEGs to Toppfun (31) and performing our analysis under default settings. Our provided gene lists were compared with backgrounds consisting of genes curated by Toppfun for each category of enrichment analysis in humans. For more information, please see the original manuscript.

For validation of RNA-seq, RNA was isolated from a subset of samples (n = 4) treated with different concentrations of calcium using TRizol reagent (Thermo Fisher Scientific), and cDNA was synthesized from 2 μg of total RNA using the High-Capacity Reverse Transcriptase cDNA Kit (Thermo Fisher Scientific). qPCR was performed using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) with TaqMan assays for the following genes that showed differential expression in bulk RNA-seq data from right and left colon organoids: heme oxygenase 1 (HMXO1; assay ID: Hs01110250_m1), 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2; Hs00985427_m1), Serine protease 1 (PRSS1; Hs00605631_g1), CEA cell adhesion molecule 7 (CEACAM7; Hs03988977_m1), Keratin 80 (KRT80; Hs01372365_m1), S100 calcium binding protein A4 (S100A4; Hs00243202_m1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1; internal control; Hs02800695_m1). The PCR reactions were prepared for each RNA sample on QuantStudio 5 (Thermo Fisher Scientific). Reactions were normalized using the control gene HPRT1, and calculations were performed according to the 2^–ddCT method. Each assay was performed in duplicate. For a subset of genes: PLEK2, HSD17B2, EGLN3, CA9, ACAA2, and DNHD1, qPCR was also performed on four matched left, right colon organoid pairs derived from the same individual. Data were analyzed for statistical differences using an empirical bayes regression in limma, while accounting for sample pairing, as described previously (14, 16, 17).

Colorectal cancer GWAS index SNPs were downloaded from the GWAS catalog (32) and from Huyghe and colleagues (8). Genes were considered if they had at least a single nucleotide of one exon overlapping a 1 Mb interval, which was centered on the index SNP. The genomic location of SNPs was based on their hg38 coordinates. BiomaRt (33, 34) was used to determine GrCH38 gene coordinates of nearby genes.

Raw data for this manuscript has been uploaded to Gene Expression Omnibus and is available for download using accession no. GSE196168.

To test whether calcium treatment induces cell composition changes in colon organoids, we inferred cell-type proportions in each organoid sample (n = 36 pairs; demographics are listed in Supplementary Table S1) by single-cell deconvolution and modeled the effect of calcium supplementation on cell proportions. Our analysis of all 36 sample pairs (herein full analysis) revealed a significant increase in goblet cells (FDR = 7.98E^–03) and a concomitant decrease in the stem cell population (FDR = 0.012) in those samples exposed to higher calcium concentrations. By stratifying our analysis, we found that these significant differences were primarily driven by right colon organoids (Supplementary Table S2). We also identified a significant increase in colonocytes in males treated with higher calcium concentrations (FDR = 0.022), which appeared to be consistent across right and left colon. However, larger studies will be necessary to validate these additional findings which are based on smaller sample sizes. However, no noticeable differences in macroscopic appearance of colon organoids were observed (Supplementary Fig. S1).

Previously, we have shown that variation in cell composition can impact findings from RNA-seq analysis of bulk datasets (14). To address this, we incorporated cell scores into our regression model, as performed previously (14, 16, 17, 25). Expression of 485 genes were significantly associated with the different calcium concentrations in our full analysis, including: CEACAM7 (BH = 4.75E^–03), KRT80 (BH = 1.43E^–07), and pleckstrin homology like domain family A member 1 (PHLDA1; BH = 1.48E^–03; Fig. 1; Supplementary Table S3), all of which have been associated with colonic epithelial health and colorectal cancer (35–39). We validated significant differences in gene expression in four of six selected DEGs using qPCR in a small subset of samples (n = 4), whereas consistency for direction of effect was confirmed in the remaining two genes (Supplementary Table S4). We also cross-referenced DEGs with results generated in a previous, small study of calcium treatment in human colon organoids, which assessed the effect of calcium on the human proteome (40). Of the 40 proteins found to be differentially regulated in at least one of three calcium conditions, five [carbonic anhydrase 1 (CA1), solute carrier family 26 member 2 (SLC26A2), prostate stem cell antigen (PSCA), CEACAM7 and HMGCS2] were significantly overexpressed in our larger study, whereas a trend towards significant overexpression was also observed for trefoil factor 2 (TFF2). These findings were concordant with the observed protein upregulation (40), whereas only one DEG, caveolae-associated protein 1 (CAVIN1), was discordant for direction of effect between the two studies. The reasons for this discordance are unknown.

To begin to elucidate the relationship between common genetic variation, calcium exposure, and colorectal cancer risk, we intersected calcium response genes with genes mapping to colorectal cancer GWAS loci. Here, 40 of the 485 DEGs impacted by calcium treatment in our full analysis mapped to colorectal cancer GWAS loci (Table 1), indicating a potentially important interaction between colorectal cancer genetic variation and nutritional factors that may modulate colorectal cancer risk. Fisher test for enrichment showed that this overlap was significantly greater than expected by chance (P = 0.016).

Epidemiologic studies suggest subject sex and colon location may influence the effect of calcium intake on colorectal cancer risk (41, 42). To qualitatively assess whether sex and colon location influence the response of organoids to calcium, we performed stratified analyses of each subset of interest. The total number of DEGs in the male cohort (n = 124) was higher than in the female cohort (n = 61; Supplementary Table S5). A total of 21 DEGs were common to both male and females, and all shared the same direction of effect.

Differences across strata were also observed for the colon location from which the organoids were derived, with 249 calcium-responsive DEGs identified in right colon-derived organoids compared with 55 identified in the left colon-derived organoids. Only 18 DEGs were significant in both (Table 2; Supplementary Table S6). More calcium dose-responsive DEGs were identified in the right colon organoids, and only right-sided DEGs were enriched for calcium-related Gene Ontology processes such as response to calcium ion (FDR = 0.023) and mitochondrial calcium ion homeostasis (FDR = 0.038; ref. 31; Supplementary Tables S7A and S7B). Beyond calcium-related genes, several interesting DEGs such as pyruvate kinase M1/2 (PKM) were found to be significant in right (FDR = 0.011) but not left colon-derived organoids (FDR = 0.999). The M2 isoform of pyruvate kinase (PKM2) is known to play a role in glycolysis, and it has been suggested that its loss may function in intestinal stem cells to promote colitis-associated colorectal cancer (43). To define whether these differences were driven by colon location, we performed a secondary qPCR analysis of six genes that were only significant in right colon organoids (Supplementary Table S8) using matched right and left colon organoids derived from the same individuals (n = 4). Here, we found that four of the six genes assayed were concordant for direction of effect with RNA-seq data in both colon locations, but only HSD17B2 was significant in right colon organoids (P = 0.02). No gene assayed was significant in matched, left colon organoids.

We present the largest study to date of calcium supplementation in normal, human colon organoids. Previously, we have shown that outlier responses to chemical treatments driven by inter-individual variability are greatly diminished following increased sample size in the colon organoid model (14). By performing our analysis in a similarly large in vitro setting and exposing normal colon organoids to a strict dose under controlled conditions, we believe that our robust results provide improved insight into the short-term effects of increased calcium concentrations in colon epithelial cells. By relating these findings to colorectal cancer GWAS loci, we provide a unique insight into common genes that may be affected by both colorectal cancer risk variants and dietary habits.

We show that calcium supplementation impacts cell composition by increasing goblet cell and reducing stem cell populations, implying increased cellular differentiation. This result is somewhat consistent with a previously published study of normal colon organoids exposed to calcium, where higher concentrations of calcium correlated with increased differentiation (40). Despite a difference in calcium concentrations used between the two studies, our results support and expand upon previous findings. Note that the increased size of our study (36 vs. 5 independent lines) offers significant power advantages for statistical analysis. In addition, the previous study was limited to colon organoids derived from left colon biopsies, whereas our study included organoids derived from both right and left colon. We were able to show that the increase in goblet cell and reduction of stem cell populations were more pronounced in right than left colon-derived organoids. This effect was more pronounced in males, where we observed not only a decrease in the stem cell and increase in goblet cell populations, but also an increase in the colonocyte (enterocytes of the colon) population. It has been reported that calcium may indirectly reduce cell proliferation as a function of reduced damage to the gastrointestinal tract, owing to the binding of calcium to toxic agents, which leads to their eventual excretion (44). However, our findings add weight to the hypothesis that calcium supplementation may act to reduce colorectal cancer risk through promoting differentiation and reducing stem cell populations (with a greater impact in male, right colon).

There is independent epidemiologic evidence that colon location impacts response to different environmental risk factors. These side differences in response to environmental factors may have broader implications, and there is a growing recognition of differences in the molecular phenotypes of tumors arising in the right and left colon (25, 45, 46). It is important to note that our finding of more DEGs occurring in organoids derived from the right than left colon following increased calcium does not necessarily imply an improved chemopreventive outcome in the right colon. Indeed, several studies have reported a stronger chemopreventive role for calcium in left colon (7, 41, 46). Although some of the differential expression observed here may be driven by differences in sample size (21 right vs. 15 left), it is unlikely that the resulting improvement in power would explain all of our findings. For example, increased PKM expression was only found in right colon organoids and an increased sample size in left colon organoids seems unlikely to affect this result based on our analysis. However, whether this alteration in differential expression is chemopreventive is up for debate. Increased PKM expression is observed in colon tumors versus normal-adjacent tissue (47). However, a recent study has shown that the deletion of the PKM2 isoform in both Lgr5⁺ and Villin⁺ cells actually served to promote tumor progression in a murine model by enhancing the Wnt/β-catenin pathway (43). The tumor-preventive role of genes such as insulin-like growth factor binding protein 2 (IGFBP2), which was overexpressed only in right-colon organoids, is perhaps more complex. Some experimental evidence suggests IGFBP2 promotes proliferation and migration of colorectal cancer cells (48). Further studies will be needed to clarify the role of these genes in calcium response and their role in colorectal cancer oncogenesis/prevention.

We also observed an effect of biologic sex of organoids on response to different concentrations of calcium, which was maintained even following adjustment for cell composition. Reports of the effect of sex on associations between calcium intake and colorectal cancer are inconsistent. An inverse association has been observed for total, dietary, and supplemental calcium in men and women and in both the right and left colon (7). More studies are warranted to address this issue in light of our findings.

Calcium treatment also revealed a number of results that appeared largely consistent across subgroups such as HMGCS2. HMGCS2 expression has previously been found to be significantly reduced in colorectal cancer tumor versus normal adjacent tissue and is reportedly regulated by Wnt/β-catenin/PPARγ signaling in both normal intestinal organoids and colorectal cancer cell lines (49). Higher expression of HMGCS2 was found to be a consequence of Wnt/β-catenin inhibition. This finding is consistent with the observed reduction in the stem cell population within our calcium-treated colon organoid model. HMGCS2 has also been implicated in intestinal cell differentiation (50), whereby knockdown of HMGCS2 led to reduced differentiation of Caco-2 cells, although most notably for the enterocyte lineage. Hmgcs2 loss has previously been shown to drive Lgr5⁺ stem cells towards a secretory lineage (51), thus the somewhat paradoxical findings of increased HMGCS2 expression identified in our study and the increased number of goblet cells require further examination.

We examined the potential relationship between calcium response and inherited genetic risk of colorectal cancer. Colorectal cancer GWAS have led to the identification of over 140 genomic loci, however there is limited data on the likely causal genes mapping within these GWAS loci. We identified 40 calcium dose-responsive DEGs in our full analysis that mapped to colorectal cancer GWAS loci, suggesting a potential mechanistic overlap between genes implicated in inherited and environmental risk of CRC. We observed similar overlaps between DEGs and genes mapping to GWAS loci in our aspirin, ethanol, and carcinogens exposure studies in organoids (14, 16, 17). Of the 40 DEGs observed in our calcium study that mapped to GWAS loci, 11 DEGs were also seen in at least one of our previous studies, further supporting a causal role for those genes in inherited risk (CEACAM7) (aspirin); (FYVE), rhoGEF and ph domain containing 2 (FGD2), flotillin 1 (FLOT1), ferratin heavy chain 1 (FTH1), HLA-F anti-sense RNA 1 (HLA-F-AS1), neuraminidase 1 (NEU1), ENSG00000285761, and rhophilin rho GTPase binding protein 2 (RHPN2) (carcinogens); 24-dehydrocholesterol reductase (DHCR24), HLA-G and parathymosin (PTMS) (ethanol). That CEACAM7 was also overexpressed in aspirin exposed colon organoids, a drug whose chemopreventive role is still a cause for debate (52), may be of importance. Similarly, the opposing directions of effect for DHCR24 and HLA-G (ethanol) as well as FLOT1, HLA-F-AS1, RHPN2, and ENSG00000285761 (carcinogens) to that observed for calcium may indicate how differing exposures interact with genes in the vicinity of colorectal cancer GWAS loci to confer prevention/risk. However, we are limited by a lack of functional characterization of these genes, and additional studies will be needed to confirm that this overlap between genes responsive to environmental factors and those mapping to GWAS loci represents meaningful colorectal cancer risk biology.

Both colorectal cancer incidence and the overall risk of colorectal cancer risk factors are affected by colon location (53). An important limitation within the study was therefore our decision not to treat matched colon organoids derived from both left and right colon of the same individual when performing stratified analysis. A potential colon location-specific response to various treatment regimens has been an unexpected outcome of our first series of large colon organoid experiments (16, 17). Although this does not always appear to be the case for all drug treatments (14), the differences in response for aspirin, ethanol, and now calcium treatment are intriguing. Unfortunately, their interpretation is somewhat mired by the use of nonmatched colon organoid pairs. Subject-to-subject variability may play a role in driving this signal. Future studies should consider sample pairing across locations and look to further increase sample sizes to limit the potential of outliers on DEG reporting. To somewhat address this limitation, we performed a secondary qPCR analysis of matched left and right colon organoids treated with and without supplementary calcium. This analysis was limited to a subset of individuals (n = 4) and was not performed using RNA-seq technology. However, it did reveal that HSD17B2 was only significant in right, not left colon organoids. Although a trend to significance for this gene occurred in left colon organoids, the fold change was almost doubled in right colon. This finding adds weight to our hypothesis that colon location impacts response to environmental exposure in the colon organoid model. If further validated, this finding may have important clinical ramifications and should serve as a point of caution for future organoid research and study designs. Despite this, further research into how the effect of subject-specific variability on calcium response is warranted. A number of additional limitations are also present within this study. We exposed organoids to a single dose of calcium for 72 hours. This dose was selected as an approximately two-fold supplement to the dose present in our standard media. However, pharmacodynamic studies of the colon would need to be undertaken to determine the relevance of this dose to that which colon epithelial cells are exposed to following calcium supplementations in randomized control trials (54, 55). Further, a number of randomized control trials have used co-supplementation of Vitamin D₃ (54, 55), which was not performed here, where increased study power was favored over introducing further complexity into the study design (timeline, dose, co-supplementation). This should be considered when interpreting our results. For example, here we find that calcium favors colon epithelial cell differentiation, a shift that is implicated in colorectal cancer reduction (40). We also see similar results in aspirin (chemopreventive; ref. 17) and opposing findings in ethanol (risk; ref. 16), both of which were studied over 72 hours. However, a 24-hour treatment of carcinogens to colon organoids led to an unexpected reduction in proliferative cell populations and an increase in differentiated populations (14). Cell compositional shifts are a dynamic process and it remains possible that our well-powered snapshot of the colon organoid response to exposures does not recapitulate entirely what is occurring within colon epithelial cells exposed to each condition over a prolonged or immediate period.

Finally, heterogeneity exists in the literature about the role of calcium supplementation in colorectal cancer. Some previous studies have provided strong evidence to indicate an important role of calcium in colorectal cancer prevention (3), whereas others have considered that calcium treatment alone is not sufficient (56, 57). Further still, some evidence for increased polyps have been found in randomized control trials of calcium treatment with and without Vitamin D₃ (58). Our study has attempted to address these discrepancies through the use of a controlled dose of calcium to cell populations of the colon crypt. Here, we identify cell population shifts indicative of a chemopreventive effect, as well as gene expression differences that oppose those seen in colorectal cancer tumors (e.g., HMGCS2); however, our study design limits further evaluation of the chemopreventive effects of these genes/shifts. Future studies may need to consider coupling genetic editing techniques for colorectal cancer driver mutations with calcium treatment (59), either alone, or in combination with other minerals, to more fully address this relationship.

In summary, we present data supporting the hypothesis that the potential protective effects of calcium may relate to induction of differentiation and a reduction in proliferative cells of the colon crypt including stem cells. Calcium response appears to be affected by colon-location and biological sex, with the strongest effect being seen in males and right colon organoids. We also observed an overlap between genes involved in calcium response and inherited risk, implying a biological relationship between environmental and inherited risk. Further exploration of these genes may provide greater mechanistic insight into the role that calcium has in risk modulation of colorectal cancer and potentially could provide insight into novel therapeutic targets for colorectal cancer prevention.

M.A.M. Devall reports grants from NIH during the conduct of the study. S.J. Plummer reports grants from NCI during the conduct of the study. G. Casey reports grants from NIH during the conduct of the study; as well as grants from NIH outside the submitted work. No disclosures were reported by the other authors.

M.A.M. Devall: Conceptualization, data curation, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. C.H. Dampier: Conceptualization, data curation, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. S. Eaton: Data curation, investigation, methodology, writing–review and editing. M.W. Ali: Investigation, methodology, writing–review and editing. S.J. Plummer: Data curation, investigation, methodology, writing–review and editing. J. Bryant: data curation, investigation, methodology, writing–review and editing. W.J. Gauderman: Funding acquisition, investigation, writing–review and editing. U. Peters: Conceptualization, funding acquisition, investigation, writing–review and editing. S.M. Powell: Data curation, investigation, writing–review and editing. G. Casey: Conceptualization, data curation, funding acquisition, investigation, methodology, project administration, writing–review and editing.

The authors would like to thank all of the individual's who's graciously made donated biopsies for use in this study. This work was supported by funding through NIH grants: NIH/NCI R01 CA201407 to U. Peters, W.J. Gauderman, and G. Casey as well as NIH/NCI R01 CA143237 and NIH/NCI R01 CA204279 to G. Casey. Study sponsors had no role in the study design, collection, analysis, or interpretation of data.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.