Stage-associated SERPINF1, NFATC2, and LGALS9 and immune features in colorectal adenoma-carcinoma progression: Transcriptomic analysis and pilot in vitro perturbation

Background The adenoma-carcinoma sequence involves coordinated epithelial and microenvironmental changes, but a SERPINF1/PEDF-NFATC2-LGALS9 relationship has not been established in colorectal epithelium. We integrated a stage-resolved public transcriptomic series with pilot perturbation experiments in HT29 colorectal adenocarcinoma cells, fibroblasts, and CD8 + T cells. Methods GSE41657 (normal mucosa, low-grade adenoma, high-grade adenoma, and adenocarcinoma; n = 12/21/30/25) was analyzed for four-stage heterogeneity, predefined composite scores, correlations, and high-grade versus low-grade contrasts. HT29 cells received si-PEDF with or without INCA-6, a calcineurin-NFAT interaction inhibitor. Conditioned-medium assays compared si-NC-CM with si-PEDF-CM. Numerical values from the pilot experiments are reported approximately from the plotted group summaries. Results SERPINF1, LGALS9, and NFATC2 showed four-stage heterogeneity (Kruskal-Wallis P = 1.90 × 10 −7 , 3.48 × 10 −4 , and 3.49 × 10 −3 ), but none reached FDR significance in the direct high-grade versus low-grade adenoma comparison. The source-defined CAF and ECM scores also differed across stages; high-grade versus low-grade adenoma showed a source-reported AKT difference of +0.427 (FDR = 0.030) and cytotoxic T-cell difference of −0.401 (FDR = 0.030). SERPINF1 and LGALS9 were uncorrelated in lesion tissue (rho = −0.004, P = 0.969). In HT29 cells, si-PEDF was accompanied by lower SERPINF1 mRNA and higher LGALS9 mRNA and NFATc2 protein signals; the INCA-6 condition attenuated the LGALS9 and NFATc2 changes. si-PEDF-CM increased α-SMA and FAP signals in fibroblasts and reduced IFN-γ+Granzyme B + CD8 + cells from approximately 42.7% to 19.4%. PD-1+TIM-3+ cells also decreased, from approximately 23.0% to 11.0%. Conclusions The data identify stage-associated bulk-tissue patterns and an NFAT-associated epithelial response to PEDF perturbation in HT29 cells. They do not establish a transition-specific signaling switch, NFATC2-specific transcriptional control, or LGALS9-mediated stromal or immune effects. The simultaneous reduction in IFN-γ+Granzyme B+ and PD-1+TIM-3+ CD8 + populations is more consistent with altered acute T-cell state than with a canonical checkpoint-defined exhaustion phenotype.