High-Resolution Secretome Analysis of Chemical Hypoxia Treated Cells Identifies Putative Biomarkers of Chondrosarcoma

Abstract

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

Keywords: GAPDH; biomarkers; chondrosarcoma; extracellular vesicles; hypoxia; proteomics.

Figure 1

Analysis of secretome composition of DMOG-treated SW 1353 cells. (A) Volcano plot showing the −log10 of p-values versus the log2 of protein ratio between DMOG-treated (DMOG) and control SW 1353 cells (CTL) of 856 proteins (n = 3). Proteins significantly regulated are displayed as the filled dots above t-test based p-value < 0.05 (black dashed horizontal line) and fold change DMOG/CTL higher or lower than 50% (black dashed vertical lines). Red dots correspond to more abundant proteins, blue dots to less abundant proteins in the secretome of DMOG-treated cells. (B) Subcellular location of altered proteins detected in the secretome of DMOG-treated SW 1353 cells according to Uniprot annotation.

Figure 2

Characterization of isolated EVs from conditioned media of SW 1353 DMOG-treated or control cells. (A) Representative profiles of size distribution of EVs determined by nanoparticle tracking analysis (NTA), mode of the size distributions is indicated in the figure. Bar graphs show calculation of the mode of the size distribution (B) and total number of EVs per total number of cells (C) from SW 1353 control and DMOG-treated; analyses are displayed as mean values ± standard deviation (*** p < 0.001, Student’s t-test; n = 7).

Figure 3

Analysis of GAPDH levels by immunoblotting. (A) Immunoblots showing GAPDH protein abundance in the conditioned media (CM), in the conditioned media cleared of extracellular vesicles (Cleared CM), in the extracellular vesicles (EVs) and in the cell lysate (LYS) of SW 1353 cells treated with or without DMOG. Calnexin was used as a loading control. (B) Cell viability analysis of SW 1353 cells treated with DMOG or doxorubicin (DOXO). DMSO-treated cells were used as controls (CTL). Data are represented as % of the mean vs. control (n = 6). (C) Immunoblots showing GAPDH protein abundance in the conditioned media (CM) and lysate (LYS) of SW 1353 cells treated with DMSO (CTL), DMOG or doxorubicin (DOXO). Calnexin was used as a loading control (p < 0.001).