Supplementary Materials and Methods CLEAN version

Supplementary Figure 3 Neuroblastoma conditioning upregulates IL-1ï¢ï€ and TNF-ï¡ï€ expression in macrophages

Supplementary Figure Legends and Tables

Supplementary Figure 5 : IL-1ï¢ï€ and TNF-ï¡ï€ drive neuroblastoma cell proliferation

Supplementary Figure 4 Macrophage infiltration and downstream effects on neuroblastoma tumours

Supplementary Figure 1: M1-Macrophage infiltrate neuroblastoma

Supplementary Figure 2 Cytokine profile of tumour induced macrophages or granulocytes

Abstract Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)–dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)–dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest–derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. Significance: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.