Francisca J Bergsma

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

<h2>Abstract</h2><h3>Background</h3> Immunotherapies achieved remarkable success in adult cancers, yet their efficacy in pediatric brain and extracranial solid tumors remains limited. Insights into the unique immune landscape of pediatric tumors are crucial to improve immunotherapies for pediatric patients. <h3>Methods</h3> We performed a systematic search for studies reporting immunohistochemistry (IHC), single-cell- or single-nucleus RNA-sequencing (sc/snSeq) data on the immune landscape of pediatric tumors. For IHC studies, data on macrophages, T cells, T helper cells, regulatory T cells, NK cells and B cells were extracted. For sc/snSeq studies, cell cluster counts were extracted. 47 IHC studies (2187 tumor samples) and 26 sc/snSeq studies (272 tumor samples) were included in the analysis. <h3>Results</h3> Our integrative in-depth analysis of 73 studies covered 35 unique pediatric tumor types with 17 tumor types analyzed by IHC, 4 by sc/snSeq, and 13 by both techniques. Regardless of variability in analysis methods, both IHC and sc/snSeq showed that peripheral nerve tumors and soft tissue sarcomas had relatively immune-infiltrated, T cell-rich tumor microenvironments (TME). Brain tumors exhibited a macrophage/microglia-rich, NK cell-infiltrated and T cell-depleted TME. Sc/snSeq data confirmed these observations, showing a macrophage/microglia-rich brain TME. Compared to adult tumors, (CD8⁺) T cell infiltration and macrophage infiltration was low for all pediatric tumor types. Integrated IHC data was visualized in interactive heatmaps, publicly available on R2 as a comprehensive atlas [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1; map Immune_landscape_mm2(by patient)_v4]. <h3>Conclusion</h3> We provide a comprehensive, integrated overview of the immune landscape of pediatric (extra)cranial solid tumors. These insights can aid the development and selection of immunotherapeutic strategies for specific pediatric cancers, tailored to their unique immune characteristics.

BACKGROUND: Immunotherapy has transformed cancer treatment, highlighting the importance of effective antitumor immunity to fight cancer. However, its success in pediatric cancer remains limited, underscoring the urgent need to identify new immunotherapeutic targets. In this study, we explored the clinical relevance of B cells and tertiary lymphoid structures (TLS) in neuroblastoma (NB), a pediatric tumor with a heterogeneous immune landscape. METHODS: We analyzed 87 treatment-naïve NB specimens, spanning both localized and metastatic disease previously characterized for T-cell and dendritic cell (DC) infiltration. B cells were detected by immunohistochemistry, and plasma cells were quantified using multiple immunofluorescence. Spatial organization and functional status of immune cells within TLSs were assessed by imaging mass cytometry using a 29-antibody panel. In parallel, gene expression profiles were obtained through NanoString PanCancer Immune Profiling and further validated using publicly available bulk and single-cell RNA-sequencing data from untreated and treated NB samples. These transcriptomic datasets were used to support protein-level findings and to identify prognostic gene signatures. RESULTS: B-cell infiltration in NB tumors strongly correlated with the presence of T cells and DCs at both protein and transcriptomic levels, and was associated with improved prognosis. Similar to other solid tumors, B cells in NB were either scattered throughout the tumor or organized into TLSs of varying maturity. Spatial proteomic and transcriptomic analyses revealed that localized tumors often contain mature TLSs, with functional B cells able to antigen presentation and immunoglobulin expression, alongside high cytotoxic T cells. In contrast, metastatic tumors primarily exhibited immature TLSs, with evidence of B-cell and T-cell dysfunction. Importantly, we identified gene signatures associated with B cells and TLSs that not only predicted survival in NB but were also prognostic in multiple adult cancers. CONCLUSIONS: Our findings highlight a central role for B cells and TLSs in shaping the immune microenvironment of NB. Their presence and maturation status are linked to clinical outcome, suggesting their potential as prognostic biomarkers and targets for novel immunotherapeutic strategies in pediatric oncology.

Abstract Background Immunotherapies achieved remarkable success in adult cancers, yet their efficacy in pediatric brain and solid tumors remains limited. Insights into the unique immune landscape of pediatric tumors are crucial to improve immunotherapies for pediatric patients. Methods We performed a systematic search for studies reporting immunohistochemistry (IHC), single-cell- or single-nucleus RNA-sequencing (sc/snSeq) data on the immune landscape of pediatric tumors. For IHC studies, data on macrophages, T cells, T helper cells, regulatory T cells, NK cells and B cells were extracted. For sc/snSeq studies, cell cluster counts were extracted. 47 IHC studies and 26 sc/snSeq studies were included in the analysis. Results Our integrative in-depth analysis of 73 studies covered 35 unique pediatric tumor types with 17 tumor types analyzed by IHC, 4 by sc/snSeq, and 14 by both techniques. Regardless of variability in analysis methods, both IHC and sc/snSeq showed that peripheral nerve tumors and soft tissue sarcomas had relatively immune-infiltrated, T cell-rich tumor microenvironments (TME). Brain tumors exhibited a macrophage/microglia-rich, NK cell- infiltrated and T cell-depleted TME. Sc/snSeq data confirmed these observations, showing a macrophage/microglia-rich brain TME. Compared to adult tumors, (CD8 + ) T cell infiltration and macrophage infiltration was low for all pediatric tumor types. Integrated IHC and sc/snSeq data were visualized in interactive heatmaps, publicly available on R2 as a comprehensive atlas [ https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1 ; map Immune_landscape_mm2(by patient)_v4]. Conclusion We provide a comprehensive, integrated overview of the immune landscape of pediatric brain and solid tumors. These insights can aid the development and selection of immunotherapeutic strategies for specific pediatric cancers, tailored to their unique immune characteristics.