Targeted delivery of anti-miRNA21 sensitizes PD-L1high tumor to immunotherapy by promoting immunogenic cell death.

Rationale: Growing evidence has demonstrated that miRNA-21 (miR-21) upregulation is closely associated with tumor pathogenesis. However, the mechanisms by which miR-21 inhibition modulates the immunosuppressive tumor microenvironment (TME) and improves tumor sensitivity to immune checkpoint blockade therapies remain largely unexplored. In this study, we demonstrate the precise delivery of anti-miR-21 using a PD-L1-targeting peptide conjugate (P21) to the PD-L1high TME. Methods: Investigating miR-21 inhibition mechanisms involved conducting quantitative real-time PCR, western blot, flow cytometry, and confocal microscopy analyses. The antitumor efficacy and immune profile of P21 monotherapy, or combined with anti-PD-L1 immune checkpoint inhibitors, were assessed in mouse models bearing CT26.CL25 tumors and 4T1 breast cancer. Results Inhibition of oncogenic miR-21 in cancer cells by P21 efficiently activates tumor suppressor genes, inducing autophagy and endoplasmic reticulum stress. Subsequent cell-death-associated immune activation (immunogenic cell death) is initiated via the release of damage-associated molecular patterns. The in vivo results also illustrated that the immunogenic cell death triggered by P21 could effectively sensitize the immunosuppressive TME. That is, P21 enhances CD8+ T cell infiltration in tumor tissues by conferring immunogenicity to dying cancer cells and promoting dendritic cell maturation. Meanwhile, combining P21 with an anti-PD-L1 immune checkpoint inhibitor elicits a highly potent antitumor effect in a CT26.CL25 tumor-bearing mouse model and 4T1 metastatic tumor model. Conclusions: Collectively, we have clarified a miR-21-related immunogenic cell death mechanism through the precise delivery of anti-miR-21 to the PD-L1high TME. These findings highlight the potential of miR-21 as a target for immunotherapeutic interventions.

Assessing the cytotoxicity of aerosolized carbon black and benzo[a]pyrene with controlled physical and chemical properties on human lung epithelial cells.

Atmospheric particulate matter (PM) is a complex mixture of hazardous particles containing hundreds of inorganic and organic species. Organic components, such as carbon black (CB) and benzo[a]pyrene (BaP), are known to exhibit diverse genotoxic and carcinogenic effects. The toxicity of CB and polycyclic aromatic hydrocarbons has been well studied, however the combined toxicity is much less understood. A spray-drying system was used to control the size and chemical composition of PMs. PMs were prepared by loading BaP on three different sized CBs (0.1 µm, 2.5 µm, and 10 µm) to obtain BaP-unloaded CB (CB0.1, CB2.5, and CB10) and BaP-loaded CB (CB0.1-BaP, CB2.5-BaP, and CB10-BaP). We analyzed cell viability, levels of oxidative stress, and pro-inflammatory cytokines using human lung cells (A549 epithelial cells). Cell viability decreased when exposed to all PMs (PM0.1, PM2.5, and PM10), regardless of the presence of BaP. The increase in PM size due to BaP-adsorption to CB resulted in insufficient toxic effects on human lung cells compared to CB alone. Smaller CBs reduced cell viability, leading to reactive oxygen species formation, which can cause damage to cellular structures deliver more harmful substances. Additionally, small CBs were predominant in inducing the expression of pro-inflammatory cytokines in A549 epithelial cells. These results indicate that the size of CB is a key factor that immediately affects the inflammation of lung cells, compared to the presence of BaP.

The effect of PhIP precursors on the generation of particulate matter in cooking oil fumes at high cooking temperatures and the inflammation response in human lung cells.

Cooking Oil Fumes (COFs) contain carcinogenic organic substances such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs), of which 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is known as mainly meat-borne carcinogens. In this work, to identify the mechanisms to induce the inflammation response in human lung cells (A549) exposed to COFs, we investigated the physicochemical and biological characteristics of COFs generated with PhIP precursors (L-phenylalanine, creatinine, and glucose) at high cooking temperatures (300 °C and 600 °C). Interestingly, we found that PhIP was not formed both at 300 °C and 600 °C, while a large number of carbon nanoparticles were generated from soybean oil containing the PhIP precursors at 600 °C. From the biological analysis, COFs generated with the PhIP precursors at 600 °C induced the most significant pro-inflammatory cytokine (IL-6). This result indicates that the particulate matter in COFs generated with the PhIP precursors above the smoke temperature is the primary factor directly affecting the lung inflammatory response rather than PhIP. This study demonstrates for the first time a novel principle of the inflammatory response that the PhIP precursors can aggravate lung injury by affecting the physical properties of COFs depending on cooking temperature. Therefore, our finding is a significant result of overcoming the bias in previous studies focusing only on the chemical toxicity of PhIP in the inflammatory response of COFs.