Distinct microenvironments define subtypes of neuroblastoma

Originating from the neural crest, neuroblastoma is the most common extracranial solid tumor in children. Infiltrating immune cells contribute to the tumor's growth and treatment response, as patients with high-risk disease, associated with MYCN amplification, benefit from anti-GD2 antibody immunotherapy. However, MYCN -amplified disease remains lethal in more than half of cases and has been associated with immunosuppression in bulk RNA studies. We thus hypothesize that MYCN activation, in conjunction with other molecular and clinical traits, influences the tumor microenvironment (TME), which can either support or suppress the disease. To comprehensively characterize the neuroblastoma TME, we performed CO-Detection by indEXing (CODEX), a multiplex immunohistochemistry technique, on 5 clinically annotated tissue microarrays containing 371 neuroblastic tumors from 179 patients representing all major disease subgroups and treatment protocols. In a subset of specimens, we also applied Visium HD spatial transcriptomics to identify regional malignant programs and their associated immune infiltrates. In parallel, we developed a novel natural language processing approach to detect generalizable spatial cell networks across these tissues. Interrogating more than 40 tumor-, immune-, and stroma-associated proteins revealed that neuroblastomas, despite downregulating MHC class I (MHC-I), harbor rich TMEs composed of about 20 phenotypically distinct cell populations, including multiple lymphoid- and myeloid-derived subsets. MYCN -amplified tumors are profoundly deficient in infiltrating helper, memory, and cytotoxic T cell lineages, whereas they form prominent tertiary structures in non-amplified disease. By contrast, antigen-presenting suppressor-like myeloid cells dominate the MYCN -amplified microenvironment, where they persist in chemotherapy-resistant tumors. These subtype-specific differences prompted functional studies of intrinsic immune and cytokine programs. RNA-seq of multiple human MYCN -amplified cell lines revealed that the differentiation therapy retinoic acid, while suppressing MYCN , drastically upregulated class I antigen presentation and pro-inflammatory cytokines. To further understand MYCN - and treatment-related changes in the inflammatory secretome, we are currently performing extracellular proteomics on these cell lines. Integrative spatial profiling by CODEX and Visium HD reveals that MYCN amplification fosters a T cell-poor, myeloid-rich microenvironment, in contrast to the organized lymphoid structures characteristic of non-amplified neuroblastoma. Together with the finding that retinoic acid restores MHC-I and pro-inflammatory programs in MYCN -amplified tumors, these results suggest that combining retinoic acid with cellular immunotherapies and myeloid-targeting approaches may significantly improve survival in patients with high-risk neuroblastoma.