Spatial transcriptomics exploration of the primary neuroblastoma microenvironment unveils novel paracrine interactions

High-risk neuroblastomas exhibit a high degree of intratumoral heterogeneity. Single-cell RNA sequencing has greatly improved our understanding of these tumors, but the method lacks cellular tissue context and spatial information about local signaling dynamics. To address this gap, we profiled untreated and chemotherapy-treated high-risk neuroblastomas from 2 patients using spatial transcriptomics. We confirmed the transcriptional and cellular heterogeneous nature of the neuroblastoma microenvironment and identified several unique spatial niches and patterns. In one of the treated tumors, a spatially constrained cluster of undifferentiated and 11p-gained cancer cells was identified, surrounded by a rim of macrophages. A signaling interaction between the chemokine CCL18 and its receptor PITPNM3 was predicted between these cells and we experimentally demonstrated that CCL18 increases neuroblastoma cell migration. In the other tumor, we identified a stromal cluster with high transcriptional similarity to the adrenal cortex. These adrenocortical-like cells expressed the ALK ligand ALKAL2 and were predicted to communicate with neighboring ALK expressing cancer cells. We demonstrated a unique developmental pattern of adrenal medulla-specific expression of ALK and adrenocortical-specific expression of ALKAL2, suggesting a role of these signaling interactions in neuroblastoma carcinogenesis.