Epiploic Adipose Tissue (EPAT) in Obese Individuals Promotes Colonic Tumorigenesis: A Novel Model for EPAT-Dependent Colorectal Cancer Progression

Figures

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  Figure 1. (A) Cross-sectional model of Epiploic (EPAT) Colorectal Cancer Microphysiological System (CRC-MPS). (B) Representative images of CRC-MPS with HT29-GFP cells and EPAT obtained from obese (BMI 34.2) or lean (BMI 24.2) individuals vs. HT29-GFP grown with EPAT-derived stromal cells (ESC) only (48 h). Graphs represent the intensity of GFP pixels from CRC-MPS images, three independent wells for each EPAT, obtained from three obese or three lean individuals relative to ESC (Scale bar = 100 µm, BMI = Body Mass Index). (C) All individual values from CRC-MPS were normalized to matched ESC-only GFP signal, then normalized to lean average. Box and whisker plot represents the median, 25% and 75% quartiles, and highest and lowest values of each group. Thick dark grey bar within the box and whisker plot reports confidence interval (CI, 95%) with range in brackets above each box.

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  Figure 2. (A) Representative images and quantification of BrdU incorporation during DNA synthesis in HT29 cells treated with conditioned media (CM) from CRC-MPS (obese) vs. CM from ESC. Oleic acid (OA) treatment represents positive control and serum-free media (Con) represents negative control (24 h, n = 3, **** p < 0.0001, * p < 0.05 vs. Con; #### p < 0.0001, # p < 0.05 vs. OA, scale bar = 50 μm). (B) Representative images and quantification of EdU incorporation during DNA synthesis in HCT116 cells treated with CM from CRC-MPS (obese) vs. CM from ESC. Oleic acid (OA) treatment represents positive control and serum-free media (Con) represents negative control (24 h, n = 3 wells in 2 independent experiments, **** p < 0.0001, *** p < 0.001 vs. Con; #### p < 0.0001 vs. OA, scale bar = 50 μm). (C) Representative images and quantification of confluency disruption of HT29 monolayers via scratch assay (24 h, n = 3, **** p < 0.0001 vs. Con; #### p < 0.0001 vs. ESC, scale bar 200 = μm). (D) Transwell migration assay of HCT116 cells, with CM from CRC-MPS (obese), ESC, fetal bovine serum (FBS) as a positive control, and serum-free media as a negative control (Con) (24 h, n = 3, **** p < 0.0001 vs.…

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  Figure 3. (A) Top diseases representing differentially expressed genes (DEGs) in colon cancer HT29 cells mediated by CM from CRC-MPS from obese patients relative to control (p < 0.05, IPA). (B,C) Top canonical pathways representing these DEGs compared to DEGs from colon and colonic tumors (AOM/DSS) of HFD obese mice (n = 3 for each group, FC > |1.5|, FDR < 0.05, IPA). In (B), Column 1 contains pathways representing DEGs in HT29 cells treated with CM from CRC-MPS (vs. ESC), and Column 2 contains pathways in HFD obese mouse colon (vs. RD colon). In (C) Column 1 contains pathways representing DEGs in HT29 cells treated with CM from CRC-MPS (vs. ESC), and Column 2 contains pathways in HFD obese mouse tumors (AOM/DSS model vs. RD normal colon). (D) Shared pathways representing DEGs from HT29 cells treated with CM from CRC-MPS (Column 1) and human colon cancer relative to normal colon (Column 2, GSE4183, n = 23; Column 3, GSE141174, n = 198; fold change > |1.5|, FDR < 0.05, IPA). (E,F) Unsupervised hierarchical clustering and heatmap showing hOEC867 signature separating transcriptome of colon cancer samples from normal. Orange boxes highlight normal samples. Principal Component Analysis (PCA) of…

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  Figure 4. (A) Validation of DEGs in HT29 cells treated with CM from CRC-MPS (obese) and ESC (24 h) by qPCR. The following transcripts were assessed: SNAI1, TRIB2, OAS1, CENPE, BCCIP, and NNMT (qPCR, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). Similar significant alterations of these transcripts were found in colon cancer patient tissue (grey box plots) (TCGA: normal colon (n = 41) and tumors (n = 457)). (B) Transcriptomic data of samples from colon cancer patients that included both weight and height information (TCGA) were further classified by BMI (obese: BMI > 30, n = 84, and non-obese: BMI < 30, n = 198). Selected genes were analyzed for expression levels (log2 TPM) in various clinical cancer stage subsets associated with tumor dissemination (Box plots).