Determination of miRNA profile and partial protein analysis in secreting exosomes from chordoma cells

Chordoma is a slow-growing primary bone tumor that develops from embryonic notochord remnants. Although it is a rare disease, it has higher recurrence rates along with its strong resistance to conventional cancer treatments. The recurrence of tumors and the aggressiveness of cancer cells are both driven by cancer stem-like cells (CSCs). Tumorsphere culture method based on sphere formation by anoikis resistance of CSCs is used to enrich CSCs. Exosomes are small extracellular vesicles that carry proteins, lipids, miRNAs, and other cargo to support cell-cell communication. In this study, we have established that enrichment of chordoma CSCs that are generated from chordoma tumor spheres. Following that, exosomes from the the parental and CSC-enriched cell lines were isolated and characterized by exosomal surface tetraspanin determination and nanoparticle size detection using NTA and flow cytometry analyses. The effects of exosome treatment on chordoma cell lines were investigated by using several functional assays including cell viability, invasion/migration, gene quantification for the stem-cell and other prognostic biomarker. Based on these findings, exosomes isolated from two chordoma cell lines, JHC7 and CH22 were found to be the most efficient exosomes and chosen for the omic studies. The miRNA microarray results demonstrated that exosomes secreted from chordoma CSCs are involved in the regulation of cell cycle, growth factor signaling pathways, JAK/Stat pathways and stemness-associated pathways. Furthermore, protein array studies revealed that intrinsic apoptotic pathway proteins were downregulated in chordoma CSCs-driven exosomes. Moreover, exosomes secreted from chordoma CSCs showed to have a role in the regulation of growth factor signaling, particularly EGF pathway. Overall, based on our findings, we can conlcude that the properties of exosomes secreted from chordoma CSCs might be in involved in the progression of chordoma.