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. 2018 Mar 2;11(1):20.
doi: 10.1186/s13048-018-0391-2.

Comparison of benign peritoneal fluid- and ovarian cancer ascites-derived extracellular vesicle RNA biomarkers

Cindy M Yamamoto  1 , Melanie L Oakes  2 , Taku Murakami  2 , Michael G Muto  3 , Ross S Berkowitz  3 , Shu-Wing Ng  3   4
Affiliations

Comparison of benign peritoneal fluid- and ovarian cancer ascites-derived extracellular vesicle RNA biomarkers

Cindy M Yamamoto et al. J Ovarian Res. .

Abstract

Background: Extracellular vesicles (EVs) are considered as a new class of resources for potential biomarkers. We analyzed expression of specific mRNA and miRNA in EVs derived from ovarian cancer ascites and the ideal controls, peritoneal fluids from benign patients for potential early detection and prognostic biomarkers.

Methods: Fluids were collected from subjects with benign cysts or endometrioma (n = 10), or low/high grade serous ovarian carcinoma (n = 8). EV particles were captured using primarily ExoComplete filterplate or ultracentrifugation and analyzed by nanoparticle tracking analysis, ELISA, and scanning electron microscopy. EV RNAs extracted from two ascites and three peritoneal fluids were submitted for next-generation sequencing. The expression of 34 mRNA and 18 miRNAs in the EVs isolated from patient fluids and cell line media was determined using qPCR.

Results: EVs isolated from patient samples had concentrations greater than 1010 EV particles/mL and 30% were EpCAM-positive based on ELISA. EV particle sizes averaged 113 ± 11.5 nm. The qPCR studies identified five mRNA (CA11, MEDAG, LAMA4, SPINT2, NANOG) and six miRNA (let-7b, miR23b, miR29a, miR30d, miR205, miR720) that were significantly differentially expressed between cancer ascites and peritoneal fluids. In addition, CA11 mRNA was decreased to 0.5-fold and SPINT2 and NANOG mRNA were significantly increased up to 100-fold in conditioned media of cancer cells compared to immortalized ovarian surface and fallopian tube epithelial cell lines, the hypothesized cells of origin for ovarian cancer development.

Conclusions: This study indicates that EV mRNA profiles can reflect the disease stage and may provide a potentially novel source for discovery of biomarkers in ovarian cancer.

Keywords: Ascites; Biomarkers; Extracellular vesicles; Ovarian cancer; Peritoneal fluids.

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Conflict of interest statement

Ethics approval and consent to participate

Human samples were collected at Brigham and Women’s hospital under informed consent and Internal Review Board approval.

Consent for publication

N/A

Competing interests

CMY, MO, and TM are employees of Hitachi Chemical Co. America, Ltd. R&D Center and the two authors, CMY and SWN have submitted a provisional patent (Appl. No. 62/507,091) entitled, “Methods for detecting ovarian cancer using extracellular vesicles for molecular analysis”.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
a,b Scanning electron microscopy of ovarian cancer ascites EVs captured on the membrane of the ExoComplete filterplate. Ovarian cancer ascites was applied to the filterplate, fixed, labeled with anti-CD63 primary monoclonal antibody and anti-mouse IgG colloidal gold with silver enhancement. Black arrows indicate selected extracellular vesicles with diameters < 200 nm captured on the membrane. Images were obtained with accelerating voltages of 2.0 kV in detection mode
Fig. 2
Fig. 2
Relative gene expression in peritoneal fluids (n = 10) and ovarian cancer ascites (n = 8) EV samples. a-e CA11, MEDAG, LAMA4, SPINT2, NANOG normalized to ACTB are shown as 2^-ΔCT gene expression levels with average and SD indicated by horizontal lines. Maximum gene expression was set at a cut-off of 10. Gene expression values for each individual subject are represented as solid circle and square symbols for ascites and peritoneal fluids, respectively. Statistical significance for (a-d) is p < 0.05 and (e) is p < 0.005 using Student’s t-test
Fig. 3
Fig. 3
Relative miRNA expression levels in peritoneal fluids (n = 10) and ovarian cancer ascites (n = 8) EV samples. a-f Let7b, miR205, miR23b, miR29a, miR30d, miR720 normalized to SNORD61 are shown as 2^-ΔCT expression levels with average and SD indicated by horizontal lines. Gene expression values for each individual subject are represented as solid circle and square symbols for ascites and peritoneal fluids, respectively. Statistical significance for (a-d) is p < 0.05 and (e) is p < 0.005 using Student’s t-test
Fig. 4
Fig. 4
Specific mRNA expression normalized to ACTB from immortalized fallopian (FTSEC194), ovarian surface epithelial (OSE7, HOSE1–15) and ovarian cancer cell lines (SKOV3, OVCA3) EVs released in conditioned media. a SPINT2, (b) NANOG, (c) CA11, (d) LAMA4 mRNA quantitation (2^-ΔCT) is shown as column graph with average and SD (n = 2) and statistical significance indicated by a bar representing p < 0.05 by Student’s t-test
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