Abstract 3696: RNAseq of patient-derived cancer stem-like cells and exosomes provides new insights into Ewing's sarcoma
Authors: Bosch Mariona Chicón , Roundhill Elizabeth A. , Droop Alastair P. , Parry Michael , Jeys Lee , Burchill Susan A. 
Published: August 2018

Abstract
Background
Cancer stem-like cells (CSCs) are responsible for disease progression and relapse (Schatton et al, Bioessays 2009, 31:1038-49). Therefore, the eradication of Ewing's sarcoma (ES)-CSCs is expected to improve outcome for patients. Since the transfer of exosome cargo between cells drives ES progression (Ventura et al, Oncogene, 2016, 35:3944-54) or can be exploited as biomarker (Miller et al, Biology of the Cell 2013, 105:289-303; Tsugita et al, PLoS ONE 2013, 8:e77416) we have characterized primary patient-derived ES, paired ES-CSCs, and the exosomal cargo from these cultures to identify potential prognostic biomarkers of risk and targets for the development of new treatments.
Methods
ES taken at diagnosis and resection were collected following patient consent (GenoEwing; IRAS-167880). ES-CSCs were isolated using a functional self-renewing assay. EWSR1 translocations were confirmed by FISH using a break-apart probe and RT-PCR. Exosomes were collected after incubation of cultures in serum-free media for 48h, isolated using exoEasy Maxi kit (Qiagen) and characterized by Nanoparticle tracking, Western blot and flow cytometry. The expression of the ES-marker CD99 was investigated by immunocytochemistry, Western blot and flow cytometry. RNA was extracted from ES cells and paired exosomes using the miRNeasy Micro kit (Qiagen) and RNA quality confirmed employing the Agilent Bioanalyser. Total RNA libraries were prepared and sequenced using the HiSeq3000 (Illumina®). Reads were processed using fqtools (Droop, Bioinformatics 2016, 32:1883-4), aligned by STAR (Dobin et al, Bioinformatics 2013, 29:15-21) and differential expression was determined by DESeq2 (Anders et al, Genome Biology 2010, 11:R106). Genes were ranked on adjusted p value and log2 fold change; candidates were validated by RTqPCR.
Results
ES patient-derived cells expressed CD99 and contained an EWSR1 gene translocation (23/23). The median size of isolated ES exosomes was 84nm (range 32-132nm). Exosomes were enriched for small RNA (53±5% in exosomes vs 12±3% in cells). The optimal protein panel to identify ES exosomes is CD81, CD63 and TSG-101; CD9 was not detected. Consistent with the exosomal cell of origin, expression of CD99 was detected in 71-79% of isolated vesicles.
The RNA profile of parental cultures and daughter ES-CSCs was compared and two genes with increased expression in ES-CSCs were validated by RTqPCR (p&lt;0.0001). Using a novel experimental approach in ES, we have sequenced the total RNA profile of paired exosomes from patient-derived ES cultures and confirmed the expression of CD99 and one putative ES-CSC biomarker in exosomes.
Conclusions
For the first time, we have sequenced paired exosomes and patient-derived ES cells to identify a potential prognostic biomarker and novel target for therapy.
Work funded by University of Leeds, Ewing's Sarcoma Research Trust (ESRT) and Bone Cancer Research Trust (BCRT).
Citation Format: Mariona Chicón Bosch, Elizabeth A. Roundhill, Alastair P. Droop, Michael Parry, Lee Jeys, Susan A. Burchill. RNAseq of patient-derived cancer stem-like cells and exosomes provides new insights into Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3696.