Ferroptosis induction enhances anti-PD-1 efficacy in NSCLC via HIF-1α/PD-L1 modulation.

[BACKGROUND] The monotherapeutic efficacy of immune checkpoint inhibitors (ICIs) remains unsatisfactory in patients suffering from non-small-cell lung cancer (NSCLC). Ferroptosis, an iron-dependent cell death process, has been identified as a promising immunotherapy adjuvant; however, ferroptosis inducers (such as erastin, RSL3) may paradoxically up-regulate hypoxia-inducible factor 1α (HIF-1α) and programmed death ligand 1 (PD-L1) to propel tumor immune evasion. It is critical to explore the molecular mechanism of ferroptosis in NSCLC immunotherapy and verify the efficacy of combined regimens for overcoming ICI limitations clinically.

[METHODS] This work analyzed 162 NSCLC patients receiving immunotherapy retrospectively to evaluate correlation between PD-L1 expression and progression-free survival (PFS). In vitro, CCK-8 assay, flow cytometry, qPCR, and Western blotting were utilized to measure impacts of ferroptosis inducers (Erastin, RSL3) upon cell viability, reactive oxygen species (ROS) levels, and PD-L1/HIF-1α expression in A549/H1299 NSCLC cells; ferroptosis-specific effects were validated by means of iron chelators (DFO, ferrostatin-1). In vivo, subcutaneous tumor models were built in C57BL/6 mice with LLC cells; therapeutic effects of ferroptosis inducer (IKE) alone or combined with anti-PD-1 antibody were evaluated through tumor weight measurement and immunohistochemistry (IHC). HIF-1α binding to PD-L1 promoter was confirmed via chromatin immunoprecipitation (ChIP); related signaling pathways were explored using transcriptome sequencing and KEGG enrichment analysis.

[RESULTS] For NSCLC patients who received immunotherapy, high PD-L1 expression correlated with longer PFS, and 4-HNE was associated positively with PD-L1 and CD8⁺T infiltration. In vitro, Erastin/RSL3 induced dose-dependent cell death, ROS accumulation, and PD-L1 up-regulation, reversed by iron chelators. In vivo, IKE+anti-PD-1 inhibited tumor growth significantly, whereas it increased CD8⁺T infiltration. Mechanistically, Erastin up-regulated HIF-1α via PI3K-AKT, which bound PD-L1 promoter (ChIP-verified), reversed by iron chelators.

[CONCLUSIONS] Ferroptosis inducers have dual effects in NSCLC, namely, promoting tumor cell death and triggering PD-L1-dependent immune evasion via the PI3K-AKT-HIF-1α pathway. However, combining ferroptosis inducers with anti-PD-1 antibodies retains the anti-tumor effect of ferroptosis and overcomes immune evasion by obstructing the PD-L1 pathway, offering a novel strategy for enhancing NSCLC immunotherapy efficacy.