Thesis defense Josephine Strijker

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Neuroblastoma, a childhood cancer affecting ~30 children in The Netherlands yearly, is one of the most challenging pediatric malignancies. Despite an intense treatment protocol, including chemotherapy, surgery and radiotherapy, outcomes for patients with refractory or relapsed disease remain poor. Immunotherapy, especially adoptive cell therapy, has emerged as a promising novel therapy, yet its efficacy is often limited in bulkier tumors due to an immunosuppressive tumor microenvironment (TME). This thesis explored the mechanisms in which neuroblastoma restrains immune function and investigated strategies to release those brakes, thereby moving towards unlocking the full potential of immunotherapy for neuroblastoma. First, αβ-T cells engineered to express a γδ-T cell receptor (TEG) were explored as a novel adoptive cell therapy for neuroblastoma. TEGs combine cytotoxic and durable capacities of αβ-T cells with tumor recognition capacities of the γδ-T cell receptor. This receptor can recognize tumor cells independent of Major Histocompatibility Complex I (MHC-I) expression, which is often absent on neuroblastoma cells. Using patient-derived tumor organoid models, we showed that TEGs indeed recognized neuroblastoma independent of MHC-I expression. Only half the tumoroids were killed by TEGs, suggesting more is needed to effectively activate T cell therapies for neuroblastoma In order to be able to effectively activate T cell therapies, we explored the immunosuppressive TME of neuroblastoma, with a focus on all factors secreted by the tumor, the secretome. With a multi-omics approach, we identified several proteins secreted by neuroblastoma with a predicted suppressive effect on T cell activation and cytotoxicity. With functional experimentation, we confirmed that Macrophage Migration Inhibitory Factor (MIF), which is highly secreted by neuroblastoma, suppresses chimeric antigen receptor (CAR) T cell activation and cytotoxicity. Knockdown of MIF in the tumor cells could increase the efficacy of CAR T cells both in vitro and in vivo. Pharmacological blockade of MIF, using innovative PROTAC technology, could also increase the efficacy of several flavors of CAR T cells, bringing us a step closer to a clinical solution for the full potential of immunotherapy for neuroblastoma. Thirdly, we used an unbiased approach to further explore the immunosuppressive TME. Here, we mapped the full proteome of the neuroblastoma secretome across a wide spectrum of patient-derived neuroblastoma tumoroid cultures, with varying immunosuppressive capacity. This approach yielded a comprehensive catalogue of secreted proteins potentially contributing to immunosuppression. Comparative analysis identified specific factors enriched in highly suppressive tumoroids, several of which were implicated in immune regulation, extracellular matrix remodeling, and vesicle-mediated communication. We indeed found that more suppressive tumoroids had an increased secretion of extracellular vesicles (EVs). EVs seem to play a crucial role in the suppression of T cells in the TME of neuroblastoma. Further exploration of the mechanism through which EVs can suppress T cell activation in needed. Together, these studies show a dual approach is needed: by combining therapies to block immunosuppressive factors such as MIF or EVs with adoptive cell therapy such as TEG or CAR T cells, we are working towards unlocking the full potential of immunotherapy for neuroblastoma.

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