Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

Prohibitin (PHB) is a highly conserved, ubiquitously expressed, multifunctional protein with a well‐characterized function as a chaperone‐stabilizing mitochondrial proteins. Recently it was reported that nuclear PHB participates in HIRA chaperone complexes and regulates downstream gene expression via cell cycle independent deposition of H3.3 into DNA. However, the role of PHB in cancer progression remains controversial with conflicting reports in the literature, perhaps due to its cell type‐dependent subcellular localization. Here, we report that the increased expression of nuclear PHB is positively correlated with metastasis of breast cancer cell lines. We showed PHB participates in the HIRA complex by interacting with HIRA through the linker region of the PHB domain and stabilizes all components of the HIRA complex in breast cancer. Overexpression of nuclear PHB resulted in a higher enrichment of histone H3.3 deposited by the HIRA complex at the promoters of mesenchymal markers. This coincided with an increased gene expression level of these markers, and induced EMT in breast cancer. Overall, these molecular and structural mechanisms suggest that nuclear PHB could hold promise as a potential target for cancer therapy.

Cell pyroptosis has a reciprocal relationship with various cancer treatment modalities such as chemotherapy. However, the tumor microenvironment, characterized by hypoxia, substantially restricts the development and application of tumor therapies that integrate cell pyroptosis. Therefore, the cascade amplification of oxidative stress by interfering with redox homeostasis in tumors may be a promising approach. In this study, black phosphorus (BP) nanosheets and a glutathione peroxidase 4 inhibitor (RSL3) were coloaded into a thermosensitive PDLLA-PEG-PDLLA (PLEL) hydrogel (RSL3/BP@PLEL). Owing to the photothermal property of BP nanosheets, the RSL3/BP@PLEL hydrogel may trigger the release of loaded drugs in a more controllable and on-demand manner. Investigation of the antitumor effect in a mouse liver tumor model demonstrated that local injection of the hydrogel formulation in combination with near infrared laser irradiation could efficiently suppress tumor growth by interfering with the redox balance in tumors. Mechanistic study indicated that the combined treatment of photothermal therapy and glutathione depletion based on this hydrogel efficiently induced cell pyroptosis through both caspase-1/GSDMD and caspase-3/GSDME pathways, thereby triggering the repolarization of tumor-associated macrophages from M2 to M1. Overall, we developed a biocompatible and biodegradable hydrogel formulation for application in combination cancer treatment, providing a new platform for enhancing the efficacy of cancer therapy by amplifying cell pyroptosis and apoptosis.