Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma

Abstract

Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. Methods In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. Results Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient’s exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. Conclusions The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Recent Advances in the Discovery of Biomarkers for Canine Osteosarcoma

“…In relation to outcome, a recent study evaluated exosomes in the sera of 10 canine OSA patients that were defined as poor responders (DFI < 100 days) and good responders (DFI > 300 days). Exosomal tetranectin predicted a good prognosis, while the complement C2 and C3, alpha-2-macroglobulin and protein S predicted a poor prognosis (56). These results are encouraging but preliminary and warrant further investigation in larger patient cohorts.…”

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle

“…The following day, membranes were washed three times (10 min each) with TBST, and incubated with a 1:10,000 dilution of HRP conjugated goat anti-rabbit or HRP conjugated goat anti-mouse secondary antibodies (Sigma Aldrich, St. Louis, MO, USA) diluted in milk for 1 h rocking at room temperature. Membranes were washed three times (10 min each) with TBST, and Immobilon ® Forte Western HRP Substrate (Millipore Sigma, Burlington, MA, USA) was applied and imaged with ImageLab Software (BioRad, Hercules, CA, USA) [ 95 ].…”

Role of extracellular vesicles in osteosarcoma

“…Besides, EVs have be reported to induce chemoresistance of OS by transmitting circular RNAs (circRNAs), indicate exosomal circRNAs as new targets to be used for addressing OS chemoresistance 111 . Researchers also used OS animal models and patient samples to investigate the role of protein cargos, showing that exosomes exhibited unique protein signatures associated with drug resistance 112 . These studies explain part of the mechanism of drug resistance in OS cells and provide a new reference for the selection of drugs in OS patients.…”