C. Subramanian1, T. M. Rajendiran1, T. Soni1, V. P. Opipari1, M. S. Cohen1 1University Of Michigan,Genreal Surgery,Ann Arbor, MI, USA
Introduction: Despite improvements in our understanding of the molecular and genetic basis of neuroblastomas (NB), children with aggressive (N-myc amplified) NB still have a low survival rate <30%. Exosomes found in NB cancer patients’ body fluids play an important role in the development, progression and therapy response of their cancer. Exosomes transport cargos of proteins, mRNAs, miRNAs, metabolites, and lipids to target cells. Even though fatty acid dependent oxidative phosphorylation has been identified as the major energy source for N-myc amplified NBs, lipid metabolic biomarkers have yet to be identified for predicting therapy response or progression. Therefore, we hypothesize that exosome lipidomic profiling can be used to identify novel lipid biomarkers for NB disease progression and response.
Methods: Validated NB cells IMR32 and SH-EP were grown in culture. Exosomes were prepared by ultracentrifugation method and characterized using western blot, TEM and nanoparticle analysis. Mass spectrometry based short gun lipidomic analysis was used for untargeted lipid profiling of NB cells and exosomes. Publicly available Kocak data set was used to identify correlation between patient survival and expression of ether lipid generating enzymes.
Results: Principal component analysis (PCA) showed complete separation of IMR32, SH-EP and fibroblast exosomes. Lipidomic profiling of NB cells and exosomes identified around 760 lipids from 26 different classes of which more than 250 were differentially expressed compared to normal fibroblasts. Further evaluation of the top 50 differentially expressed lipids indicated significantly high expression levels of ether lipids in NB cells and exosomes compared to fibroblasts (p<0.001). Heat map and data mining of the top differentially expressed lipid species in exosomes,and cells identified ether lipids such as Plasmeny-PC 34:1, plasmenyl-PC 36:0, Plasmenyl PE 40:1 and Plasmenyl PE 34:2 to be significantly elevated in NB compared to normal fibroblasts (p=0.001) to be highly upregulated in NB compared to normal fibroblasts. Comparison of expression levels of these ether lipids in IMR32 (N-myc amplified) and SH-EP (N-myc non-amplified) NB cells indicated significantly increased expression levels of ether lipids in IMR 32 compared to SH-EP cells (p>0.01). Data mining of 476 primary NB tumors indicated that high expression levels of ether lipid generating enzyme AGPS significantly correlated with poor event-free survival (p=0.0065) as well as poor overall survival (p=0.003) of NB patients.
Conclusion: Lipidomic profiling from N-myc amplified NB exosomes identified significantly high levels of ether lipids compared to less aggressive NBs. Since high levels of AGPS (the enzyme that generates ether lipids) correlates with worse survival with NB, this represents a novel targeting strategy for NB patients that could be utilized for risk assessment, treatment effect, and prognosis.