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

Abstract

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 andassess radiation-induced changes. Methods: In this pilot study, we performed targeted exosomal miRNA profiling of 25serum 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 28days 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 200nmin size followed by characterization using electron microscopy, andII) 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, 1ml 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 untreatedsamples; 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 morphologyand 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 thehigh-risk group (p=0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in thehigh-risk group compared tothe intermediate group (p=0.036). Conclusions: Herewith, wedemonstrated a protocol for isolation of serumexosomes and exosomalmiRNAamplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promisingbiomarker potentialthat requires further investigation in larger patient cohorts.