Ewing sarcoma family of tumors-derived small extracellular vesicle proteomics identify potential clinical biomarkers

Abstract

Purpose: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs). Materials and Methods: We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT. Results: From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 µl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)]. Conclusions: In this study, we demonstrate utilization of circulating ESFTassociated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

“…Small EVs range from 40-160 nm in size and include exosomes (originating from multivesicular endosome compartments) and heterogeneous populations of plasma membrane-derived microvesicles [13]. EVs are secreted by healthy tissue cells as well as by tumor cells, including EwS [14][15][16]. The EV cargo is protected by a lipid membrane and reflects the physiological or pathological state of the parental cell.…”

Extracellular Vesicles in Reprogramming of the Ewing Sarcoma Tumor Microenvironment

“…Although there are several pieces of evidence of the diversity of transcripts present in EwS EVs, the proteomic content of EwS EVs is only starting to be unraveled. Samuel et al (2020) found that CD99, HINT1, and NGFR can act as biomarkers of small EVs derived from EwS cells. Sorting small EVs derived from EwS cells based on the above protein markers could improve the specificity of EVs derived from heterogeneous cell populations.…”

Liquid Biopsy: A New Translational Diagnostic and Monitoring Tool for Musculoskeletal Tumors

“…The most recent study in 2020 by Samuel et al evaluated the effect of 619 proteins composing the subset of ES derived small extracellular vesicles as biomarkers in monitoring the tumor burden at diagnosis, responsiveness to therapy and recurrence [24]. The CD99/MIC2 and the NGFR membrane proteins were chosen to develop a liquid-based assay in order to detect the mRNA cargo of well-established transcripts in ES, such as the EWS-ETS.…”

“…The CD99/MIC2 and the NGFR membrane proteins were chosen to develop a liquid-based assay in order to detect the mRNA cargo of well-established transcripts in ES, such as the EWS-ETS. The liquid biopsy from ES pediatric patients with localized or metastatic disease showed/revealed that the identification of novel extracellular vesicle proteomics has significant diagnostic value [AUC = 0.92, p value = 0.001 for vesicles numeration, with a Positive predictive value = 1.00, 95% CI = (0.63, 1.00) and a Negative predictive value = 0.67, 95% CI = (0.30, 0.93)] compared with tumor-free donors [24].…”