Journal: bioRxiv

Article Title: Inflammatory Stromal Aging in Ulcerative Colitis and Colitis-Associated Cancer

doi: 10.64898/2026.05.18.725891

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbents assay (ELISA) analysis of secreted CXCL8 protein present in the conditioned media of human primary colon fibroblasts isolated from the stroma of normal (n = 4), colitis (n = 16), and colon cancer samples (n = 8). Colon cancer samples include colitis-associated cancer (CAC; indicated by the red dots) and sporadic colon cancer samples (indicated by the black dots). ( B-E ) Quantitative real-time PCR (qRT-PCR) relative expression analysis of CXCL8, CDKN2A, TP53, CDKN1A mRNAs comparing the same primary fibroblasts as in (A) . Each dot represents an individual patient sample. ( F ) Principal component analysis (PCA) of bulk mRNA sequencing comparing normal colon fibroblasts, UCAFs, and CAFs. Ellipsoids show 95% confidence intervals. ( G ) Volcano plot of differentially expressed genes comparing normal vs. UCAFs with significance threshold of Log2FC ≥ 2.5 and P.adj < 0.05. ( H ) Gene Set enrichment analysis (GSEA) of the top 25 Hallmark pathways significantly upregulated or downregulated in normal colon fibroblasts compared to UCAFs ( H ). (I-L) Enrichment plots for four of the gene sets (indicated in red in H ). (M-R) Heatmaps of the top differentially expressed genes in six of the Hallmark gene sets comparing normal colon fibroblasts (normal), UCAFs, and CAFs. Statistical analysis was performed using Student’s t -test. Data are shown as mean ± standard deviation. ns, not significant; P >0.05; * P ≤ 0.05 and ** P ≤ 0.01.

Article Snippet: The human colon fibroblast cell line (CRL1541) and colon cancer epithelial cell line (HCT 116) were obtained from ATCC, validated via STR analyses and tested for mycoplasma contamination prior to use.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Inflammatory Stromal Aging in Ulcerative Colitis and Colitis-Associated Cancer

doi: 10.64898/2026.05.18.725891

Figure Lengend Snippet: (A-F) CXCL8 mRNA and secreted CXCL8 protein expression levels in colitis-associated colon fibroblasts (CT5-S/T), normal colon fibroblasts (CRL1541-S/T) and colon cancer epithelial cells (HCT116) following stimulation with Tumor Necrosis Factor α (TNFα, 20 ng/ml), Interleukin-1 β (IL-1β, 960 pg/ml), Lipopolysaccharide (LPS, 10 μg/ml), Flagellin (FliC,100 ng/ml), and Interferon-γ (IFNγ, 33 ng/ml) either individually or in combination. (G-H) Dose-response curves for serial dilutions of TNFα in CT5-S/T and CRL1541-S/T. (I) DESeq2-normalized mRNA-seq reads for TNF receptors ( TNFRSF1A & TNFRSF1B ) in CT5-S/T, CRL1541-S/T fibroblasts and HCT116 colon cancer epithelial cells. (J-K) Dose-response curves for serial dilutions of IL-1β on CT5-S/T and CRL1541-S/T fibroblasts. (L) DESeq2-normalized mRNA-seq read counts for the interleukin-1 receptor family members ( IL1R1 & IL1R2 & ILRN ) comparing CT5-S/T, CRL1541-S/T fibroblasts and HCT116 colon cancer epithelial cells. Statistical analyses were performed as indicated in each panel. Error bars represent standard deviation of at least three independent experiments. Dose-response curves were obtained by nonlinear regression analysis with R 2 and EC50 values indicated.

Article Snippet: The human colon fibroblast cell line (CRL1541) and colon cancer epithelial cell line (HCT 116) were obtained from ATCC, validated via STR analyses and tested for mycoplasma contamination prior to use.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Inflammatory Stromal Aging in Ulcerative Colitis and Colitis-Associated Cancer

doi: 10.64898/2026.05.18.725891

Figure Lengend Snippet: (A) Immunofluorescence of colon tissue from a patient with ulcerative colitis showing the presence of mycoplasma (red) with the interstitial colon fibroblasts visualized by PDGFRA (green). In the merged panel, nuclei were counterstained with DAPI (blue). ( B) Mycoplasma colonization status in normal colon fibroblasts (CT303-Prox) and colitis-associated colon fibroblasts (CT306-Dist and CT307-Dist). (C) ELISA of secreted CXCL8 protein in matched fibroblasts with or without mycoplasma. (D-G) qRT-PCR analysis of CXCL8 mRNA, miR-20a, miR-93 and miR-20b . Statistical analysis was completed using the student T-test; Data are shown as mean ± standard deviation. ns, not significant, P > 0.05; * P ≤ 0.01; ** P ≤ 0.001; ** P ≤ 0.0001

Article Snippet: The human colon fibroblast cell line (CRL1541) and colon cancer epithelial cell line (HCT 116) were obtained from ATCC, validated via STR analyses and tested for mycoplasma contamination prior to use.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation

Journal: bioRxiv

Article Title: Inflammatory Stromal Aging in Ulcerative Colitis and Colitis-Associated Cancer

doi: 10.64898/2026.05.18.725891

Figure Lengend Snippet: (A-C) qRT-PCR of CXCL8 in a normal colon fibroblast line (CT303-Prox) and two colitis-associated colon fibroblast lines (CT306-Dist & CT307-Dist) in the presence or absence of mycoplasma treated with NF-κB inhibitor, IKK-16 ( D-F ) CXCL8 mRNA levels following stimulation with TNFα or LPS in the same fibroblast lines. (G ) Volcano plot comparing gene expression changes as a consequence of mycoplasma with a significance threshold of 2.5 for Log2FC and 0.05 for P.adj. (H-L) GSEA depicting the top upregulated and downregulated Reactome gene sets in the mycoplasma-positive and -negative colon fibroblasts ( H , NES, Normalize Enrichment Score). Enrichment plots and corresponding heatmaps for two gene sets highlighted in red in (H), REACTOME_TNF_RECEPTOR_SUPERFAMILY_TNFSF_MEMBERS_MEDIATING_NON_CANONICAL_NF_KB_PATHWAY (I-J) and REACTOME_CHEMOKINE_RECEPTORS_BIND_CHEMOKINES (K,L) . All cell culture experiments were performed with at least three biological replicates; data are shown as mean ± standard deviation. Statistical analysis was completed using the Student’s t-test. ns, not significant, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: The human colon fibroblast cell line (CRL1541) and colon cancer epithelial cell line (HCT 116) were obtained from ATCC, validated via STR analyses and tested for mycoplasma contamination prior to use.

Techniques: Quantitative RT-PCR, Gene Expression, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Inflammatory Stromal Aging in Ulcerative Colitis and Colitis-Associated Cancer

doi: 10.64898/2026.05.18.725891

Figure Lengend Snippet: (A) Schematic of the generation of Mesenchymal-Derived Fibroblasts (MDFs) from induced pluripotent stem cells (iPSCs) reprogrammed from normal colon fibroblasts and UCAFs. UC, ulcerative colitis; FB, fibroblasts; DE, definitive endoderm; SPH, spheres; iHNO, induced human normal colon organoids; iHUCO, induced human ulcerative colitis organoids; MDF, mesenchymal-derived fibroblasts. (B,C) Dose response curves for TNFα stimulation for MDFs derived from CT5 (UCAF) and CRL1541 (normal colon fibroblast) compared to their immortalized parental counterparts. ( D ) Comparative TNFα dose-response of MDFs from iPSC-derived normal colon fibroblasts (CRL1541-MDF, DPNL-MDF) vs. UCAFs (CT5-MDF, KHCT-MDF) shows a significantly stronger CXCL8 induction in MDFs derived from UCAFs than normal colon fibroblasts. Data represent the mean of at least three biological replicates; error bars indicate standard deviation. Dose-response curves were obtained by nonlinear regression analysis, with E max values indicated. Differences in E max values were assessed extra sum-of-squares F test; all E max values were significant from each other with P <0.0001.

Article Snippet: The human colon fibroblast cell line (CRL1541) and colon cancer epithelial cell line (HCT 116) were obtained from ATCC, validated via STR analyses and tested for mycoplasma contamination prior to use.

Techniques: Derivative Assay, Standard Deviation