Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy.

Malla, Bijaya; Aebersold, Daniel; Dal Pra, Alan (2018). Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy. Journal of translational medicine, 16(1), p. 223. BioMed Central 10.1186/s12967-018-1592-6

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BACKGROUND Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes. METHODS In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtained from PCa patients with intermediate- and high-risk disease treated with curative radiotherapy (RT), and controls. Post-treatment blood samples were collected at least 28 days after radiation therapy as a paired follow-up sample. The complete workflow consisted of two phases: I) filtration and polyethylene glycol salt precipitation phase which enriched particles below 200 nm in size followed by characterization using electron microscopy, and II) flow cytometry. Finally, miRNA expression analysis between untreated and treated patient samples was performed using RNA extraction kit, and qRT-PCR. RESULTS In our preliminary data, 1 ml of serum from PCa patients showed higher exosomal concentration (3.68E+10) compared to controls (6.07E+08). The overall expression of exosomes after RT was found to be higher compared to untreated samples; the median value changed from 3.68E+10 to 5.40E+10; p = 0.52. Using electron microscopy, we were able to visualize cup-shaped vesicles with morphology and size compatible with exosomes. The bead-based flow cytometry showed positivity for exosomal tetraspanins surface markers CD63 and CD9. All five miRNAs (hsa-let-7a-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-99b-5p) have been identified in exosomes. Despite overall changes in hsa-let-7a-5p expression after radiation, the difference was significant only in the high-risk group (p = 0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in the high-risk group compared to the intermediate group (p = 0.036). CONCLUSIONS Herewith, we demonstrated a protocol for isolation of serum exosomes and exosomal miRNA amplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promising biomarker potential that requires further investigation in larger patient cohorts.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Radiation Oncology

UniBE Contributor:

Malla, Bijaya; Aebersold, Daniel and Dal Pra, Alan

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1479-5876

Publisher:

BioMed Central

Language:

English

Submitter:

Beatrice Scheidegger

Date Deposited:

07 Nov 2018 10:01

Last Modified:

23 Oct 2019 19:10

Publisher DOI:

10.1186/s12967-018-1592-6

PubMed ID:

30103771

Uncontrolled Keywords:

Biomarker Exosomes Prostate cancer Radiation oncology miRNA

BORIS DOI:

10.7892/boris.120956

URI:

https://boris.unibe.ch/id/eprint/120956

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