Proteomic Exploration of Plasma Exosomes and Other Small Extracellular Vesicles in Pediatric Hodgkin Lymphoma: A Potential Source of Biomarkers for Relapse Occurrence.

Ombretta Repetto,Maurizio Mascarin,Agostino Steffan,Filippo Romanato,Valli De Re,Salvatore Buffardi,Caterina Elia,Daniel Enderle,Marta Pillon,Alessandra Sala,Roberta Burnelli,Federica Lovisa,Lara Mussolin

doi:10.3390/diagnostics11060917

Abstract

Exosomes and other small extracellular vesicles (EVs) are potential sources of cancer biomarkers. Plasma-derived EVs have not yet been studied in pediatric Hodgkin lymphoma (HL), for which predictive biomarkers of relapse are greatly needed. In this two-part proteomic study, we used two-dimensional difference gel electrophoresis (2D-DIGE) followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze EV proteins of plasma collected at diagnosis from children with nodular sclerosis HL, relapsed or not. EVs isolated using membrane affinity had radii ranging from 20 to 130 nm and contained the programmed cell death 6-interacting (ALIX) and the tumor susceptibility gene 101 (TSG101) proteins, whereas calnexin (CANX) was not detected. 2D-DIGE identified 16 spots as differentially abundant between non-relapsed and relapsed HL (|fold change| ≥ 1.5, p < 0.05). LC–MS/MS identified these spots as 11 unique proteins, including five more abundant in non-relapsed HL (e.g., complement C4b, C4B; fibrinogen γ chain, FGG) and six more abundant in relapsed HL (e.g., transthyretin, TTR). Shotgun LC–MS/MS on pooled EV proteins from non-relapsed HL identified 161 proteins, including 127 already identified in human exosomes (ExoCarta data). This EV cargo included 89 proteins not yet identified in exosomes from healthy plasma. Functional interrogation by the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that the EV proteins participate in platelet degranulation and serine-type endopeptidase activity as the most significant Gene Ontology (GO) biological process and molecular function (p < 0.01).

Highlights

Hodgkin lymphoma (HL) is a malignant lymphoma accounting for around 5% of childhood and 15% adolescent cancers [1,2]
extracellular vesicles (EVs) preparations were concentrated in Vivaspin concentrators with a 100,000 molecular weight cut-off, and the concentrate and flow-through were subjected to protein extraction (Figure 1a)
In plasma EVs of pediatric HL patients, our study identified a differential protein display as potentially predictive of relapse: a decrease in FGG and C4B abundances together with an increase in TTR content occurred at diagnosis in relapsed HL

Summary

Introduction

Hodgkin lymphoma (HL) is a malignant lymphoma accounting for around 5% of childhood (ages 0–14) and 15% adolescent (ages 15–19) cancers [1,2] It is considered as a curable pediatric cancer, since most patients are cured with first-line treatment and the 5year survival rate is 90–95% [3]. Exosomes carry heat shock proteins (Hsp) (e.g., Hsp, Hsp20) [15], membrane transport and fusion proteins (e.g., Ras analog in brain guanosine triphosphatases (Rab GTPases), annexins, flotillins), proteins involved in sorting of cargo into exosomes (e.g., programmed cell death 6-interacting protein, ALIX; tumor susceptibility gene 101 protein, TSG101) [16], some cell-specific proteins (e.g., major histocompatibility complex (MHC) class II and cluster of differentiation 86 (CD86)on exosomes from mature dendritic cells [17]), lipids [18], and mRNAs and non-coding RNAs [19]. They are produced by almost all kind of cells, including hematopoietic cells (B and T lymphocytes [20,21,22], platelets [23], natural-killer cells [24] and non-hematopoietic cells (intestinal epithelial cells [25], adipocytes [26], fibroblasts [27])

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Conclusion