In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-ras^G12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-ras^G12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Apoptosis is an essential process for organisms and animal development, and its dysregulation is related to the progression of several diseases. Therefore, methods to detect apoptosis are necessary for mechanistic research and drug development. To overcome the disadvantages of traditional methods for detecting apoptosis, a variety of imaging strategies based on enzyme-mediated fluorescence activation have been developed. This study aims to design a novel strategy for apoptosis fluorescent reporters which are inactivated by caspase-3. Four candidate EGFP mutations containing DEVD-similar sequences were selected by their structural positions and hydrophilicities, and an EGFP mutant was chosen by investigating fluorescence expression. This EGFP mutant was stably expressed in various cells using the safe harbor locus. To verify our apoptosis reporter system, protein levels, colocalization, and intensity were investigated in apoptosis-induced EGFP mutant-expressing cells. The fluorescence intensity of the mutant EGFP was decreased in a time- and concentration-dependent manner by staurosporine and H₂O₂, which induce apoptosis. Furthermore, compared to a dark-to-bright reporter of apoptosis (caspase-activatable GFP), our bright-to-dark system showed greater sensitivity for apoptosis. Our system is useful in various models, including other species. Our method does not require additional peptides, which makes it easily adaptable to other systems. And, our apoptosis reporter may be useful in a variety of research fields as well as drug screening.

Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer’s disease and Parkinson’s disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.