Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.

The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.

SENP1 inhibition suppresses the growth of lung cancer cells through activation of A20-mediated ferroptosis.

Background: Ferroptosis is a type of cell death driven by iron accumulation and lipid peroxidation, which is involved in the pathogenesis of various tumors. Small ubiquitin-like modifier (SUMO)-specific protease 1 (SENP1) is a critical SUMO-specific protease, which controls multiple cellular signaling processes. However, the roles and mechanisms of SENP1-mediated protein SUMOylation in the regulation of cell death and ferroptosis remain unexplored. Methods: The gene expression of SENP1 and ferroptosis-related genes in samples of lung cancer patient and cells were determined by immunohistochemical staining, real-time polymerase chain reaction (RT-qPCR) and Western blot. The association of gene expression with the survival rate of lung cancer patients was analyzed from public database. The erastin and cisplatin was used to induce ferroptosis, and cell ferroptosis were determined by evaluated lipid-reactive oxygen species (ROS), cell viability and electron microscopy. The protein interaction was determined by immunoprecipitation (IP) and shotgun proteomics analysis. An in vivo tumor transplantation model of immunodeficient mice was used to evaluate the effect of SENP1 on tumor growth in vivo. Results: SENP1 is aberrantly overexpressed in lung cancer cells and is associated with the low survival rate of patients. SENP1 inhibition by short hairpin RNA transduction or a specific inhibitor suppressed the proliferation and growth of lung cancer cells both in vitro and in vivo. SENP1 overexpression protected lung cancer cells from ferroptosis induced by erastin or cisplatin. Transcriptome and proteomics profiles revealed the involvement of SUMOylation regulation of the inflammation signal A20 in SENP1 inhibition-induced ferroptosis. Functional studies proved that A20 functions as a positive inducer and enhances the ferroptosis of A549 cells. A20 was shown to interact with ACSL4 and SLC7A11 to regulate the ferroptosis of lung cancer cells. Conclusions: SENP1 was identified as a suppressor of ferroptosis through a novel network of A20 SUMOylation links ACSL4 and SLC7A11 in lung cancer cells. SENP1 inhibition promotes ferroptosis and apoptosis and represents a novel therapeutic target for lung cancer therapy.

NUDT21 suppresses the growth of small cell lung cancer by modulating GLS1 splicing.

The mRNA precursor 3'-end modification factor NUDT21 is a major regulator of 3'UTR shortening and an important component of pre-mRNA cleavage and polyadenylation. However, its role in pathologic progress of small cell lung cancer (SCLC) remains unclear. In this study, we observed that NUDT21 expression is downregulated in SCLC tissues. Hypoxia-induced down-regulation of NUDT21 through HIF-1α. NUDT21 shRNA transduction promotes proliferation and inhibits apoptosis of A549 cells. NUDT21 inhibition also promotes tumor growth in a mouse xenograft model. Furthermore, we clarified that HIF-1α mediated NUDT21 downregulation which altered the expression patterns of two isoforms of GLS1, GAC and KGA. These results link the hypoxic tumor environments to aberrant glutamine metabolism which is important for cellular energy in SCLC cells. Therefore, NUDT21 could be considered as a potential target for the treatment of SCLC.