High-risk neuroblastoma (NB) is rare, it often relapses and becomes refractory to treatment, making it a significant contributor to childhood cancer mortality. Addressing the challenges posed by NB's resistance to conventional apoptosis-inducing therapies has become a pressing concern in pediatric oncology research. In recent years, the growing comprehension of alternative cell death modalities distinct from apoptosis has revealed a promising avenue in the endeavor to combat treatment-resistant cancers. One such mechanism, ferroptosis, has attracted increasing attention for its potential role in combating therapy-resistant cancer cells. High-risk NB typically aligns with the immune exclusion phenotype, where substantial immune cell infiltration is notably absent. While the role of immune cell infiltration in NB is of significant interest, the underlying mechanisms remain elusive.

Cell viability and proliferation were assessed using MTT and colony formation assays. Lipid peroxidation levels were measured to confirm ferroptosis induction. Transcriptomic analysis was performed using RNA sequencing to identify differentially expressed genes upon Erastin treatment. ULBP1 expression was validated using qRT-PCR and flow cytometry. Co-culture experiments with NK-92 cells were conducted to assess NK cell-mediated cytotoxicity using a standard cytotoxicity assay. ATF4 involvement in ULBP1 regulation was investigated using siRNA-mediated knockdown and western blot analysis. Statistical analyses were performed using GraphPad Prism.

Here, we show that induction of ferroptosis using Erastin, a cysteine-glutamate antiporter inhibitor reduced NB cell proliferation and foci formation. Furthermore, our transcriptomics analysis showed upon treatment with Erastin of NB cells showed increased expression of ULBP1 (NKG2D ligand). Co-culture of NK cells and NB cells upon treatment with Erastin showed a significant increase NK cell cytotoxicity. Additionally, we found transcription factor ATF4 driver ULBP1 expression in NB cells upon ferroptosis induction.

Our results suggest that the NK cell’s cytotoxic function can be enhanced with Erastin mediated ferroptosis which might be beneficial for NB patients.