Parallel Reaction Monitoring (PRM)-Driven Profiling of Hypoxia-responsive Pathways Associated with Metastasis in Osteosarcoma

In this study, we aimed to refine our understanding of the molecular events associated with the proteomic response to hypoxia in OS cells with different anatomic origins, namely from a primary and metastatic site. There are similarities in biology and treatment of OS in human and canine. We opted to use a canine parental osteosarcoma cell line (POS), which was cultured from a primary canine tumor24; and a highly metastatic POS cell line (HMPOS) that was derived from pulmonary metastatic lesions in mouse POS xenografts. We have chosen a protein-centric study design that combines 1) initially a label-free data dependent acquisition (DDA) method for enabling quantification of larger sets of proteins and global assessment of adaptive responses induced by hypoxia, 2) selection of hypoxia-responsive protein targets and 3) using parallel reaction monitoring (PRM) for quantitative monitoring of signature peptides of hypoxia-responsive protein targets to determine their precise response to hypoxia and to confirm hypoxia-regulated pathways. Functional biochemical assays provide additional validation of the proteomic findings. Taken together, our results describe the hypoxia response of OS cell phenotypes by inducing comprehensive alterations in the proteome biology that are associated with metabolic reprogramming, redox modulation and extra cellular matrix (EMC) remodeling.