Objective ·To establish a mouse model with dormant cancer and no obvious metastasis by combining the preimmune strategy with the mVenus-p27K^－ cell G₀ phase indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system. Methods ·The KPC1199 mouse pancreatic cancer cell line was transfected with the mVenus-p27K^－ cell G₀ phase indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system to construct a stable expression cell line, KPC1199-PDL. After being cultured in the serum-free condition, KPC1199-PDL cells were sorted into mVenus (+) cells and mVenus (-) cells by flow cytometry, and the expression of G₀ phase-related genes was verified by real-time fluorescence quantitative PCR (qPCR). Sensitivity of KPC1199-PDL cells to diphtheria toxin (DTX) and ganciclovir (GCV) was evaluated by CCK-8 assay. A transsplenic portal vein-hepatic metastasis model was constructed in wild-type C57BL/6 mice to validate the function of KPC1199-PDL cells in vivo by immunofluorescence technology. The KPC1199-PDL cells were injected subcutaneously into C57BL/6 mice, followed by in situ injection of DTX and GCV to ablate subcutaneous tumors 5 d later, to obtain preimmunized mice. The transsplenic portal vein-hepatic metastasis models were constructed in these mice. Bioluminescence imaging was used to evaluate subcutaneous tumor ablation and hepatic metastasis in the mice, and immunofluorescence assay was used to detect the distribution and dormant state of tumor cells in the livers of preimmunize mice. Results ·The three tool systems were stably expressed in KPC1199-PDL cells, and their proliferative ability was not affected. In the serum starving condition, some KPC1199-PDL cells expressed the mVenus protein, indicating entry into the G₀ phase; the mVenus (+) cells sorted out by flow cytometry exhibited significantly higher expression of G₀ phase-related genes (all P<0.05) and significantly lower expression of the proliferation-related gene compared with mVenus (-) cells (P<0.05). The CCK-8 assay demonstrated high sensitivity of KPC1199-PDL cells to DTX and GCV. In vivo experiments confirmed that KPC1199-PDL cells could be effectively traced through tdTomato protein expression, and could indicate entry into the G₀ phase through mVenus protein expression. Following subcutaneous tumor implantation and drug ablation, preimmunized mice were successfully obtained. In the subsequent transsplenic portal vein-hepatic metastasis model, no metastatic signals were observed in the liver by bioluminescence imaging, but single or small clusters of G₀ phase tumor cells expressing both mVenus and tdTomato, not expressing the proliferation marker Ki67, were detected in liver tissue sections by immunofluorescence analysis. Conclusions ·A recognizable and traceable dormant cancer model is constructed with the combination of the preimmune mouse model of pancreatic cancer, the mVeneus-p27K^－ indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system.