Time-Series Multi-Omics Identifies ZG16 as a Prognostic Immunoregulatory Driver of Colitis-associated Colon Cancer Development and Checkpoint Blockade Response

Chronic inflammation is a known driver of colitis-associated cancer (CAC), yet the trajectory of immune suppression and its reversibility remain poorly defined. Here, we performed a time-series multi-omics analysis combining paired transcriptomic and proteomic profiling, flow cytometry, and cytokine measurements in an AOM/DSS-induced model, further supported by single-cell transcriptomic and epigenomic datasets. This roadmap delineates a stepwise progressive evolution of immune suppression, capturing coordinated changes in immune cell composition, cytokine dynamics, and inflammation-associated mutagenesis throughout CAC progression. We identified ZG16 as a previously undervalued immune modulator that is progressively downregulated during CAC and CRC development. Analysis of CRC cohorts in the TCGA database confirmed that ZG16 loss correlates with PD-L1 upregulation, suppression of T cell costimulatory/proliferative programs, and both poor prognosis and resistance to immunotherapy. Functionally, ZG16 suppresses PD-L1 expression and promotes CD8+ T/NK cell infiltration, thereby reshaping the tumor immune microenvironment. In both inflammation-driven and syngeneic tumor models, recombinant ZG16 reduces tumor burden and enhances the efficacy of PD-1 blockade. These findings position ZG16 as a rheostat-like immunoregulatory factor and translationally relevant biomarker in inflammation-driven cancers. Eight to twelve weeks-old male C57BL/6 mice were used for the establishment of AOM/DSS murine model with mild amendments. We randomly assigned the mice into a control group (before administration, W0) and 4 experimental groups (2/5/8/11 weeks after administration, W2/5/8/11). The mutagen AOM (8.5 mg/kg body weight) was injected intraperitoneally into mice on days 1 and 8 of the modeling process. A week later, in order to cause damage to the colonic epithelium, the mice were given drinking water that contained 1.5% (w/v) DSS for seven days in a row. The mice were then provided regular drinking water for a period of 14 days. The mice thereafter received drinking water containing DSS for two or more cycles. Throughout the entire trial, the W0 group continuously was offered distilled water, and were euthanized simultaneously with the AOM/DSS-treated mice. During the trial, we selected 3 mice in each group for further RNA and protein analysis.