A protocol for harvesting and extracting extracellular metabolites from an in vitro model of human renal cell lines was developed to profile the exometabolome by means of a discovery-based metabolomics approach using ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. Metabolic footprints provided by conditioned media (CM) samples (n=66) of two clear cell Renal Cell Carcinoma (ccRCC) cell lines with different genetic background and a non-tumor renal cell line, were compared with the human serum metabolic profile of a pilot cohort (n=10) comprised of stage IV ccRCC patients and healthy individuals. Using a cross-validated orthogonal projection to latent structures-discriminant analysis model, a panel of 21 discriminant features selected by iterative multivariate classification, allowed differentiating control from tumor cell lines with 100% specificity, sensitivity and accuracy. Isoleucine/leucine, phenylalanine, N-lactoyl-leucine, and N-acetyl-phenylalanine, and cysteinegluthatione disulfide (CYSSG) were identified by chemical standards, and hydroxyprolyl-valine was identified with MS and MS/MS experiments. A subset of 9 discriminant features, including the identified metabolites except for CYSSG, produced a fingerprint of classification value that enabled discerning ccRCC patients from healthy individuals. To our knowledge, this is the first time that N-lactoyl-leucine is associated to ccRCC. Results from this study provide a proof of concept that CM can be used as a serum proxy to obtain disease-related metabolic signatures.
Journal of proteome research. 2018 Sep 27 [Epub ahead of print]
María Elena Knott, Malena Manzi, Nicolás Zabalegui, Mario O Salazar, Lydia I Puricelli, Maria Eugenia Monge