Chimeric antigen receptor (CAR) T-cell therapy is a promising and innovative cancer therapy. However, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable efficacy. Here, we aimed to identify and target immunosuppressive factors in the TME of neuroblastoma, a pediatric extracranial solid tumor, to improve CAR-T efficacy. Immunosuppressive factors were identified using a multi-omics approach, including single-cell RNA sequencing (scRNA-seq) of 24 neuroblastoma tumors, published bulk-RNA sequencing datasets, and mass-spectrometry of patient-derived tumoroid models. Candidate targets were validated with functional assays in vitro and in vivo. Protein degradation of the top immunosuppressive target by PROTAC technology was used to evaluate the effect on CAR T-cell activity. ScRNA-seq revealed 13 immunosuppressive interactions in the TME of neuroblastoma, two effectors of which, Midkine (MDK) and Macrophage Migration Inhibitory Factor (MIF), were validated as candidate targets across multiple published datasets. Both factors were among the top 6 % of most abundantly secreted factors by patient-derived tumoroid models, substantiating their potential relevance in the TME. In vitro and in vivo functional assays confirmed MIF to be a potent inhibitor of CAR T-cell activation and killing capacity. To translate these findings into a potentially clinically applicable treatment, we explored MIF targeting by PROTAC technology, which significantly enhanced activation of CAR T-cells targeting GPC2 and B7-H3. By defining the immunosuppressive effects of neuroblastoma's TME on CAR T-cell efficacy, revealing the pivotal role of MIF, we provide an analytic pipeline and therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy and potentially other solid tumors.

Despite druggable events to be present in 80 % of neuroblastomapatients within the Princess Máxima Center precision medicine program 'iTHER', clinical uptake of treatment recommendations has been low, and the clinical impact for individual patients remains hard to predict. This stresses the need for a method integrating genomics and transcriptomics with functional approaches into therapeutic decision making. We aimed to launch an online repository integrating genomics and transcriptomics with high-throughput drug screening (HTS) of nineteen commonly used neuroblastoma cell lines and fifteen neuroblastoma patient-derived organoids (NBL-PDOs). Cell lines, NBL-PDOs and their parental tumors were characterized utilizing (lc)WGS, WES and RNAseq. Cells were exposed to ∼200 compounds. Results were transferred to the R2 visualization platform. A powerful reference set of cell lines is available, reflecting distinct known pharmacologic vulnerabilities. HTS identified additional therapeutic vulnerabilities, such as a striking correlation between a positive mesenchymal signature and sensitivity to BCL2-inhibitor venetoclax. Finally, we explored personalized drug sensitivities within iTHER, demonstrating HTS can support genomic and transcriptomic results, thereby strengthening the rationale for clinical uptake. We established a dynamic publicly available dataset with detailed genomic, transcriptomic, and pharmacological annotation of classical neuroblastoma cell lines as well as novel sharable NBL-PDOs, representing the heterogeneous landscape of neuroblastoma. We anticipate that in vitro drug screening will be complementary to genomic-guided precision medicine by supporting clinical decision making, thereby improving prognosis for all neuroblastoma patients in the future.

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.

Immunotherapy development for solid tumors remains challenging, partially due to a lack of reproducible, cost-effective in vitro three-dimensional (3D) models to mimic the heterogeneous and complex tumor microenvironment. Here, we investigate the cellular anti-tumor reactivity of αβ T cells engineered to express a defined γδ TCR (TEG A3). For that purpose, we developed a 3D cytotoxicity assay targeting cell line–derived spheroids or patient-derived tumor organoids formed in serum-free media. Tumor cell lysis by TEG A3 was monitored using the Incucyte S3 live-cell imaging system with the apoptosis marker caspase 3/7 green and endpoint readouts of IFN-γ secretion in the supernatant. The 3D cytotoxicity assay model system was able to adequately demonstrate TEG A3 reactivity toward targets expressing an isoform of CD277 (CD277J). To obtain a more complex heterogeneous tumor microenvironment, patient-derived organoids were mixed with unmatched patient-derived fibroblasts or matched cancer-associated fibroblasts. In all assays, we demonstrated the tumor target specificity of TEG A3, lysing tumor cells within 48 h. Our study demonstrates the utility of complex 3D cytotoxicity assay model systems incorporating the tumor microenvironment in the functional evaluation of T cell–based adoptive immunotherapy, providing a useful platform for early-stage preclinical development of immunotherapies.

Immunosuppressive tumor microenvironments in solid tumors hamper the efficacy of immunotherapies, including CAR T-cell therapy. To identify immunosuppressive secreted factors in high-risk neuroblastoma, a childhood solid tumor with a 5-year overall survival of below 60%, we mapped secreted factors of 16 genetically diverse patient-derived neuroblastoma tumoroids by LC-MS. The secretomes suppressed T cell activation, proliferation and cytotoxicity to varying extents, suggesting that this panel constituted a valuable discovery foundation to identify clinically relevant immunosuppressive factors. 29 proteins were significantly enriched in highly suppressive secretomes, of which Neuropeptide Y (NPY) had the highest correlation with functionally-determined immunosuppression. No direct or indirect effect of NPY on (CAR) T cell activation could be validated. Instead, NPY proved to be a biomarker for extracellular vesicle (EV) secretion. Finally, we demonstrated that neuroblastoma-derived EVs potently suppress T cell activation. Altogether, this study provides an atlas of the neuroblastoma secretome and reveals immunosuppressive effects of EVs on T cells. These insights contribute to the understanding of the immunosuppressive neuroblastoma TME.

While chimeric antigen receptor (CAR) T-cell therapies are showing highly promising first results in neuroblastoma, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable clinical efficacy. To improve CAR T-cell efficacy further, we applied a multi-omics approach including single-cell RNA sequencing and proteomics, which identified 13 targetable immunosuppressive factors in neuroblastoma. Of these, macrophage migration inhibitory factor (MIF) and midkine (MDK) were validated across multiple published RNA datasets. Moreover, they were secreted in high abundance by neuroblastoma tumoroids. Functional validation experiments revealed MIF as a potent inhibitor of CAR T-cells, in vitro and in vivo. Degradation of MIF by PROTAC technology significantly enhanced CAR T-cell activation targeting GPC2 and B7-H3, providing a potential intervention against MIF. By defining the immunosuppressive effects of neuroblastoma’s TME on CAR T-cell efficacy, particularly the pivotal role of MIF, we provide a therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy.