ObjectiveTo construct a novel KPC mouse model of type 2 diabetes mellitus （T2DM） comorbid with spontaneous pancreatic cancer based on the gene editing-metabolic intervention dual-driven strategy， and to compare it with traditional models. MethodsA total of 14 male KPC mice were randomly divided into novel model group （T2DM-KPC group with 7 mice） and control group （KPC group with 7 mice）， and 14 male BALB/c-nu nude mice were randomly divided into traditional model group （T2DM-pancreatic cancer group with 7 mice） and control group （pancreatic cancer group with 7 mice）. The mice in the KPC group and the pancreatic cancer group were fed with normal diet， and those in the T2DM-KPC group and the T2DM-pancreatic cancer group were fed with a high-fat diet. After 4 weeks， the mice in the T2DM-KPC group and the T2DM-pancreatic cancer group were given intraperitoneal injection of streptozotocin. Subsequently， the mice in the KPC group and the T2DM-KPC group developed primary pancreatic tumor spontaneously over time， while those in the T2DM-pancreatic cancer group and the pancreatic cancer group were inoculated with tumor cells to form subcutaneous tumor xenograft at 2 weeks after stabilization of blood glucose. The 4 groups were observed in terms of tumor formation rate， tumor formation time， body weight， and the change in blood glucose； RNA sequencing was performed for tumors from the KPC group and the T2DM-KPC group， and then molecular subtyping was performed； HE staining， Masson staining， and immunohistochemical staining were used to assess the histopathological features and tumor microenvironment of pancreatic tumor from the T2DM-KPC group， which were compared with those of the T2DM-pancreatic cancer group. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups； the Fisher’s exact test was used for comparison of categorical data between multiple groups. ResultsThe T2DM-KPC group had a tumor formation rate of 85.71% and a tumor formation time of 104.40±2.87 days， while the T2DM-pancreatic cancer group had a tumor formation rate of 71.43% and a tumor formation time of 95.20±9.47 days， and there were no significant differences between the two groups in tumor formation rate， tumor formation time， body weight， and blood glucose （all P>0.05）. Molecular subtyping showed that the model in the KPC group highly resembled the pancreatic progenitor subtype of human pancreatic ductal adenocarcinoma （PDAC）， and the model in the T2DM-KPC group highly resembled the immunogenic subtype of PDAC. HE staining showed that tumor cells in the T2DM-KPC group were arranged into glandular tubular structures of varying shapes， exhibiting significant cellular atypia， and this model faithfully recapitulated the pathological features of primary pancreatic cancer and showed greater invasiveness than the KPC group. Immunohistochemical staining and Masson staining showed that compared with the T2DM-pancreatic cancer group， the T2DM-KPC group had significantly higher degrees of tumor proliferation （assessed by Ki-67 expression） and fibrosis （assessed by α-SMA and Masson） （all P<0.05）， suggesting that the mouse model in the T2DM-KPC group could better recapitulate the features of hyperproliferation and pronounced desmoplasia in human pancreatic cancer. ConclusionA novel KPC mouse model of T2DM comorbid with spontaneous pancreatic cancer is successfully constructed in this study. This model can accurately mimic the histopathological architecture and stromal microenvironment of T2DM comorbid with pancreatic cancer， realize the longitudinal simulation of the progression of pancreatic tissue from intraepithelial neoplasia to invasive carcinoma and metastasis in the presence of T2DM， and support the translational research on immunotherapy， thereby providing a novel experimental carrier for in vivo studies on spontaneous pancreatic cancer in T2DM.

Objective To establish a nude mouse model of type 2 diabetes mellitus(T2DM)and pancreatic cancer that allows dynamic observation of tumor formation process and facilitates in vivo research.Methods At first,human pancreatic cancer PANC-1 cells were transfected with lentiviral vector GV260 to construct the pancreatic cancer cell line PANC-1-Luc with stable expression of firefly luciferase.Then,36 specific pathogen-free nude mice were randomly divided into control group with 12 mice and model group with 24 mice(nude mice with T2DM and pancreatic cancer).The mice in the control group were fed with breeding diet and were then given ectopic subcutaneous implantation of PANC-1-Luc cells,and those in the model group were first given high-fat diet and intraperitoneal injection of 1%STZ,followed by ectopic subcutaneous implantation of PANC-1-Luc cells.The fluorescence in vivo imaging system and the manual measurement method were used for simultaneous and dynamic monitoring of the growth of pancreatic cancer in nude mice in the two groups,and the tumor growth curve was plotted to investigate the correlation between fluorescence value and tumor volume.Subcutaneous tumors and pancreatic islets were observed under a microscope to verify whether the model was successfully established,and immunohistochemistry was used to measure the expression of Ki-67 in tumor tissue to investigate the influence of hyperglycemia on the growth of pancreatic cancer in nude mice.The independent-samples t test was used for comparison of normally distributed continuous data between groups,and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups.Results The optimal virus titer was determined as 5×107 TU/mL for the stable transfection of lentiviral vector in PANC-1 cells,and the optimal concentration selected with puromycin was 20 μg/mL,with an optimal selection time of 9 days.The fluorescence value of PANC-1-Luc cells was linearly and positively correlated with the number of cells,with the linear equation of y=42.56x-42 504(r=0.977,P=0.004).The blood glucose value of T2DM nude mice was 23.05(19.25-26.40)mmol/L,with a blood glucose level of＞11.1 mmol/L in each nude mouse,and there was a significant difference in blood glucose value between the T2DM nude mice and the control nude[6.15(5.20-7.30)mmol/L](Z=-8.45,P＜0.001).Compared with the control group,the model group had reductions in the number and volume of pancreatic islets,with irregular shapes and unclear boundaries,and pathological examination confirmed that the xenograft tumor was pancreatic cancer tissue,which showed that the model was established successfully.In the model group,there was a linear positive correlation between subcutaneous tumor size and fluorescence values,with the linear equation of y=232 348 691x-8 258 608(r=0.911,P=0.031).The model group had a significantly higher positive rate of Ki-67 than the control group(50.333%±7.808%vs 15.917%±4.055%,t=13.55,P＜0.001),suggesting rapid tumor proliferation in the model group.Conclusion The T2DM nude mouse model of pancreatic cancer established in this study can simulate the pathological process of the development and progression of pancreatic cancer in the context of T2DM and dynamically observe the influence of hyperglycemia on the growth of pancreatic cancer cells in vivo,thereby providing a new experimental vector for the in vivo study of the development and progression of pancreatic cancer in the context of T2DM.