Materials Today Bio | Synergistic Regulation of Tumor Immune Microenvironment through PD-L1 Targeting and Epigenetic Inhibitors

This study designed a ROS-responsive prodrug polyplex that combines CRISPR interference (CRISPRi) system targeting PD-L1 with epigenetic inhibitor AZA. The system effectively converts 'cold tumors' to 'hot tumors', significantly enhancing antitumor immunotherapy outcomes and providing a novel combination strategy for triple-negative breast cancer treatment.

 

Literature Overview
This study, titled 'Reshaping tumor immune microenvironment through ROS-responsive prodrug polyplexes via synergistic effect of CRISPRi system and epigenetic inhibitor for breast cancer therapy', published in Materials Today Bio, reviews PD-L1 targeting regulation and epigenetic drug synergy in triple-negative breast cancer immunotherapy. The research proposes a ROS-responsive poly(beta-amino ester) (PBAE) co-delivery system that synchronizes PD-L1 gene silencing with AZA release, establishing a new platform for tumor immunotherapy.

Background Knowledge
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options. Clinical application of immune checkpoint inhibitors like PD-L1 antibodies is restricted by individual variability and immune-related side effects. The CRISPRi system employs dCas9-KRAB to achieve transcriptional repression without DNA cleavage, improving safety. Epigenetic inhibitors such as AZA enhance antigen presentation and MHC I expression through demethylation, promoting T-cell recognition. However, single-agent AZA demonstrates low response rates, making combination therapies a research focus. Additionally, ROS-responsive carriers enable drug release in high-ROS tumor environments, improving delivery efficiency and reducing off-target toxicity. This study constructs a PD-L1-targeting CRISPRi system combined with AZA co-delivery platform to reshape tumor immune microenvironment and improve therapeutic outcomes.

 

 

Research Methods and Experiments
The research team synthesized a ROS-responsive PBAE-S-AZA copolymer and complexed it with CRISPRi plasmids targeting PD-L1 to form polyplexes. The complexes efficiently bind to tumor cells through electrostatic interactions and release AZA and plasmids in high-ROS environments. The plasmid-encoded dCas9-KRAB and sgRNA target PD-L1 promoter and 5′UTR regions to downregulate expression. PD-L1 suppression efficiency was validated in 4T1 cells, followed by penetration and endocytosis mechanism evaluation in 3D cell spheroid models. Flow cytometry, confocal microscopy, and Western blot analyses assessed PD-L1 expression, MHC I levels, and dendritic cell (DC) maturation. RNA-seq analyzed gene expression changes and immune pathway activation. In animal experiments, subcutaneous 4T1 tumor models evaluated treatment efficacy across groups, with histopathological analysis and immune cell infiltration assessment performed.

Key Conclusions and Perspectives

• PBAE-S-AZA/pMax-GFP polyplexes at 50:1 mass ratio exhibited 102.76 ± 0.08 nm particle size and 10.87 ± 0 mV zeta potential
• PD-L1 expression was significantly reduced by 49.6% after 72-hour sgRNA3 transfection, with AZA showing no significant impact on PD-L1 expression
• ROS-responsive polyplexes demonstrated efficient endocytosis in 4T1 cells, entering cells within 0.5 hours and escaping endosomes/lysosomes after 1 hour to release plasmids
• Synergistic AZA release and PD-L1 silencing substantially enhanced MHC I expression, antigen presentation, and DC maturation, thereby activating T-cell responses
• In animal models, PAC polyplex treatment significantly inhibited tumor growth, achieving notably smaller tumor volumes and higher survival rates compared to PBS, AZA, and antibody groups
• RNA-seq revealed upregulation of IFN-α/β signaling, antigen presentation, and MHC I-related genes, with simultaneous PD-L1 downregulation, indicating successful immune microenvironment remodeling

Research Significance and Prospects
This study demonstrates a ROS-responsive nanocarrier platform enabling synergistic CRISPRi-AZA delivery, offering a novel gene-editing-epigenetic combination strategy for TNBC treatment. Future optimization should focus on enhancing in vivo delivery efficiency and targeting specificity, exploring application potential in other solid tumors, and advancing personalized tumor immunotherapy platforms.

 

 

Conclusion
This study successfully developed a ROS-responsive nanocomplex that combines PD-L1 targeting via CRISPRi with AZA release, achieving PD-L1 expression silencing and immune microenvironment remodeling in tumor cells. The system demonstrated superior gene transfection efficiency and antitumor effects in both cellular and animal models, providing an innovative immunotherapy strategy for triple-negative breast cancer. This platform holds promise for treating other immune-cold tumors in future applications, enhancing precision and safety in cancer immunotherapy.

 

Huan Deng, Qianru Li, Bingxu Wang, Lihong Fan, and Songwei Tan. Reshaping tumor immune microenvironment through ROS-responsive prodrug polyplexes via synergistic effect of CRISPRi system and epigenetic inhibitor for breast cancer therapy. Materials Today Bio.