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

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.

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.

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.

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.