Periodic Reporting for period 1 - IronKiller (Joining the forces of Natural Killer Cells and Ferroptosis to treat Refractory Neuroblastoma)
Reporting period: 2023-09-01 to 2025-08-31
Neuroblastoma is the most common extracranial paediatric solid tumour, and it accounts for over 15% of cancer childhood deaths. High-risk patients undergo extensive multimodal treatment, but relapse/progression rates remain over 70%. In the last decade, the use of cell immunotherapy has resulted in a significant increase in survival for haematological cancers, but solid tumours remain a challenge. This problem is enhanced in paediatric solid tumours due to the low mutational burden, the high heterogeneity, and the strongly immunosuppressive tumour microenvironment. This project aims to combine two novel therapeutic approaches to overcome these challenges.
Ferroptosis is a recently described type of programmed cell death that depends on iron accumulation and lipid peroxidation. Ferroptosis not only is immunogenic, being able to reverse the immunosuppressive tumour microenvironment, but can also be triggered by multiple drugs with different mechanism of action, opening the door to personalized therapy. Our main goal is to combine ferroptosis-inducing agents with natural killer (NK) cell immunotherapy. We hypothesize ferroptosis induction will promote NK cell infiltration in the tumours. Meanwhile, once the immunosuppressive environment has been overcome, the NK cells will be able to target neuroblastoma cells better than other immunotherapies, as they don´t depend on specific targets/mutations.
Furthermore, the high heterogeneity of neuroblastoma, along with the variety of ferroptosis induction mechanism, might complicate the personalized matching of patient and treatment. In the later years, mathematical oncology modelling has risen as a powerful tool to predict tumour response and improve personalized medicine.
The main objectives we expect to achieve with this project are:
1) To characterise the immune response triggered by ferroptosis in neuroblastoma
2) To identify and validate at least one combination of ferroptosis-inducing agents and NK cells
3) To generate a mathematical model that can assist combination selection based on tumour characteristics and mechanism of action of the drugs
On top of these scientific objectives, we also aim to achieve training and career development achievements and communication goals, both to the scientific community and the general society.
Ferroptosis is a recently described type of programmed cell death that depends on iron accumulation and lipid peroxidation. Ferroptosis not only is immunogenic, being able to reverse the immunosuppressive tumour microenvironment, but can also be triggered by multiple drugs with different mechanism of action, opening the door to personalized therapy. Our main goal is to combine ferroptosis-inducing agents with natural killer (NK) cell immunotherapy. We hypothesize ferroptosis induction will promote NK cell infiltration in the tumours. Meanwhile, once the immunosuppressive environment has been overcome, the NK cells will be able to target neuroblastoma cells better than other immunotherapies, as they don´t depend on specific targets/mutations.
Furthermore, the high heterogeneity of neuroblastoma, along with the variety of ferroptosis induction mechanism, might complicate the personalized matching of patient and treatment. In the later years, mathematical oncology modelling has risen as a powerful tool to predict tumour response and improve personalized medicine.
The main objectives we expect to achieve with this project are:
1) To characterise the immune response triggered by ferroptosis in neuroblastoma
2) To identify and validate at least one combination of ferroptosis-inducing agents and NK cells
3) To generate a mathematical model that can assist combination selection based on tumour characteristics and mechanism of action of the drugs
On top of these scientific objectives, we also aim to achieve training and career development achievements and communication goals, both to the scientific community and the general society.
The IronKiller project implemented a multi‐stage preclinical pipeline combining in vitro assays, in vivo studies and in silico analyses to evaluate how ferroptosis‐inducing agents can enhance natural killer (NK) cell therapy against high‐risk neuroblastoma (HR-NB). Technical activities included detailed characterization of ferroptotic compounds and iron nanoparticles in cell and organoid models, validation in patient‐derived xenografts (PDX), and preliminary mathematical and bioinformatic analysis to predict treatment responses. Key achievements include the identification of optimal drug candidates and mechanisms that boost NK cell infiltration, setting the stage for future clinical translation. The main scientific and technical achievements are:
Enhanced NK infiltration via ferroptosis
Demonstrated that pre‐treatment with Erastin and RSL3 significantly increases NK cell infiltration into neuroblastoma organoids; identified HMGB1 release as a key recruiter. Showed iron‐oxide nanoparticles reduce organoid viability and partially infiltrate tumor models without harming NK cells.
Identification of optimal drug candidates
Selected Erastin as the most consistent ferroptosis inducer in vitro and prioritized Sorafenib for its clinical applicability as it share the same mechanism of action. Confirmed Sorafenib’s potent HMGB1 induction despite needing sequential dosing to spare NK viability. Validated Sorafenib’s superior efficacy over Sulfasalazine across organoid models.
In vivo proof of concept
Established that NK92MI monotherapy delays tumor progression in PDX models, with greater effect in chemo‐resistant tumor models. Mapped expression of activating (e.g. DR4) and inhibitory (HLA-Class I) ligands post‐NK treatment, informing resistance mechanisms.
Correlative and predictive insights
Revealed that low expression of key NK‐activating genes associates with poorer survival in neuroblastoma patients. Identified neuroblastoma among the top three most sensitive cancers to Erastin and Sorafenib in large‐scale cell‐line screens.
These findings establish a robust preclinical foundation for combining ferroptosis inducers with NK cell therapy in refractory neuroblastoma and inform patient stratification strategies for upcoming clinical trials.
Enhanced NK infiltration via ferroptosis
Demonstrated that pre‐treatment with Erastin and RSL3 significantly increases NK cell infiltration into neuroblastoma organoids; identified HMGB1 release as a key recruiter. Showed iron‐oxide nanoparticles reduce organoid viability and partially infiltrate tumor models without harming NK cells.
Identification of optimal drug candidates
Selected Erastin as the most consistent ferroptosis inducer in vitro and prioritized Sorafenib for its clinical applicability as it share the same mechanism of action. Confirmed Sorafenib’s potent HMGB1 induction despite needing sequential dosing to spare NK viability. Validated Sorafenib’s superior efficacy over Sulfasalazine across organoid models.
In vivo proof of concept
Established that NK92MI monotherapy delays tumor progression in PDX models, with greater effect in chemo‐resistant tumor models. Mapped expression of activating (e.g. DR4) and inhibitory (HLA-Class I) ligands post‐NK treatment, informing resistance mechanisms.
Correlative and predictive insights
Revealed that low expression of key NK‐activating genes associates with poorer survival in neuroblastoma patients. Identified neuroblastoma among the top three most sensitive cancers to Erastin and Sorafenib in large‐scale cell‐line screens.
These findings establish a robust preclinical foundation for combining ferroptosis inducers with NK cell therapy in refractory neuroblastoma and inform patient stratification strategies for upcoming clinical trials.
The project IronKiller has demonstrated, for the first time in this context, that pre-treatment with ferroptosis-inducing agents can significantly enhance NK cell infiltration in HR-NB models, a major hurdle for immunotherapy in solid tumors. We identified a clinically approved drug, Sorafenib, that can be repurposed for this combination, accelerating potential clinical translation. Additionally, we provided new insights into the immunogenic mechanisms of ferroptosis and its interplay with NK cell activity.
Potential Impacts and Next Steps:
These results pave the way for clinical trials using ferroptosis inducers to boost NK cell therapy in refractory neuroblastoma. Nevertheless, further research is needed to complete in vivo validation and to complete the mathematical modelling for personalized treatment prediction.
Key needs for further uptake and success include:
Completion of confirmatory preclinical studies; continued collaboration with clinical, academic, and patient advocacy partners; regulatory engagement for academic trial approval; open access dissemination and protocol sharing to facilitate reproducibility and uptake.
Potential Impacts and Next Steps:
These results pave the way for clinical trials using ferroptosis inducers to boost NK cell therapy in refractory neuroblastoma. Nevertheless, further research is needed to complete in vivo validation and to complete the mathematical modelling for personalized treatment prediction.
Key needs for further uptake and success include:
Completion of confirmatory preclinical studies; continued collaboration with clinical, academic, and patient advocacy partners; regulatory engagement for academic trial approval; open access dissemination and protocol sharing to facilitate reproducibility and uptake.