Metabolic Profiling of Neuroblastomas

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in children and accounts for a disproportionate amount (~15%) of childhood cancer deaths 1. Afflicted children are risk-stratified based upon age, stage, and biologic characteristics including MYCN-amplification 2. Children with high-risk NBs have dismal 5-year survival despite aggressive chemotherapy, radiation, and surgery 3. These outcomes highlight the need to develop new approaches and improve our understanding of this challenging childhood cancer. Despite much attention being devoted to understanding the cancer genome, little progress has been made in our understanding of how the glycome and glycoproteome impacts cancer progression. Cancers frequently display different carbohydrate chains (glycans) on their surface compared to normal cell counterparts. Glycosylation is the addition of sugars to proteins and lipids 4. These post-translational modifications alter macromolecule trafficking, stability, and function. N-linked glycosylation is a major subtype of protein glycosylation where glycans are added to the asparagine residues 4,5. The majority of cell surface proteins (~90%) are glycosylated 5. Glycosylation has been shown to alter growth factor signaling, cell growth, intercellular and cell- to-extracellular matrix interactions that underpin adult cancer angiogenesis, invasion, immune evasion, and metastasis 5. Little is known about the N-linked glycome of pediatric solid tumors or its role in the progression of pediatric solid tumors 6. We believe that deciphering the N-linked glycome of NBs will reveal critical aberrancies that can be exploited for therapeutic benefit. NBs are heterogeneous featuring neuroblastic, stromal, and immune cell subpopulations, along with a non-cellular tumor microenvironment. Matrix-assisted laser desorption ionization- mass spectroscopy imaging (MALDI-MSI) is a state-of-the-art technique that permits in situ detection of analytes with spatial distribution at a near single cell level and preservation of tissue morphology 7. We are utilizing this technology to characterize the n-linked glycome of pediatric solid tumors. Of importance, formalin fixation and parrafin embedding (FFPE) preserves glycoproteins, allowing of many existing tumor repositories. We have developed a workflow for enzyme-assisted N-linked glycan liberation in human NB samples and retrieval with the MALDI- MSI platform. Our pilot study in human FFPE NB samples demonstrates enrichment of core fucosylated n-linked glycans within the neuroblastic subpopulations of stage IV NBs. These findings demonstrate our capacity to perform an unbiased screen of the n-linked glycome of NB tumor cell subpopulations and provide preliminary evidence that fucosylated glycans are enriched within neuroblast-rich areas of advanced NBs. Fucosylation plays critical roles in modulating growth factor signaling, inflammation, and cell/cell and cell matrix interactions that are critical for metastasis in adult cancers 9. Fucosylation is a specific type of glycosylation whereby fucose is added to proteins and lipids 8. GDP-fucose is the key substrate for fucosylation and is shuttled into the golgi apparatus where it can be attached to proteins. GDP-fucose can either be 1) synthesized de novo from GDP-mannose or 2) salvaged from the cytosol or extracellular space (Fig. 1) 8. Fucosidases play a critical role in salvaging fucose from existing glycan sources. Observation of increased fucosylated glycans in neuroblastic regions of advanced stage NBs led us to our central hypothesis that aberrant accumulation of fucosylated glycans is a critical mediator of NB metastasis. Kaplan-meier analysis of NB tumor datasets suggest that high expression of key drivers of de novo GDP-fucose production, such as GDP-mannose 4,6-dehydratase (GMDS), are associated with poor overall survival in NB. Conversely, low expression of α-L fucosidase (FUCA1) is also associated with poor overall survival in NB patients. This preliminary data led us to hypothesize that fucosylated glycans may accumulate 1) due to increased de novo fucose synthesis and/or 2) decreased fucosylated glycan breakdown via downregulation of fucosidases.