Proteomic Profiling of Ectosomes Derived from Paired Urothelial Bladder Cancer and Normal Cells Reveals the Presence of Biologically-Relevant Molecules.

Magdalena Surman,Małgorzata Przybyło,Urszula Jankowska,Sylwia Kędracka-Krok,Ewa Stępień,Anna Drożdż

doi:10.3390/ijms22136816

Abstract

Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on another population of EVs, i.e., ectosomes. In the present study we used a shotgun nanoLC–MS/MS proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC cells and HCV-29 normal ureter epithelial cells. In addition, cancer-promoting effects exerted by UBC-derived ectosomes on non-invasive cells in terms of cell proliferation and migratory properties were assessed. In total, 1158 proteins were identified in T-24-derived ectosomes, while HCV-29-derived ectosomes contained a lower number of 259 identified proteins. Qualitative analysis revealed 938 proteins present uniquely in T-24-derived ectosomes, suggesting their potential applications in bladder cancer management as diagnostic and prognostic biomarkers. In addition, T-24-derived ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. The present study provided a focused identification of biologically relevant proteins in UBC-derived ectosomes, confirming their role in UBC development and progression, and their applicability for further biomarker-oriented studies in preclinical or clinical settings.

Highlights

The purity of T-24- and HCV-29-derived ectosome samples obtained after 18,000× g centrifugation was assessed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)
Approach media was applied to analyze protein of ectosomes in vitro by T-24 urothelial bladder cancer (UBC) and HCV-29 normal ureter culture incontent serum-free media released to avoid the contamination with sera-derived proteins
Ectosomes were isolated from conditioned media collected after 24 h of culture in serum-free media to avoid the contamination with sera-derived proteins and sera-derived extracellular vesicles (EVs)

Summary

Introduction

Urothelial bladder carcinoma (UBC) is one of the most frequent urological malignancies worldwide, with morbidity and mortality rates of 424,000 new cases and 200,000 deaths per year [1]. The main diagnostic approaches in UBC include cystoscopy and urinary cytology. Due to their limited sensitivity and specificity, both methods are considered fully effective only in more advanced stages of UBC [2]. Despite ongoing technological advancements (such as development of white and blue light cystoscopy [3]), alternative, non-invasive diagnostic and surveillance methods for UBC are still needed. Current noninvasive tests for UBC target a variety of urinary proteins or nucleic acids [2,3].

Methods

Results

Conclusion