High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer

Abstract

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

On-chip miRNA extraction platforms: recent technological advances and implications for next generation point-of-care nucleic acid tests

“…Besides successfully enriching EVs, the miRNAs were subsequently extracted 55 . This method of EV isolation was also used later as part of a high-throughput screening to identify miRNA biomarkers for prostate cancer 56 incorporated thermoelectric lysis onto a microfluidic device in order to extract miRNAs directly from cells which was followed by gel electrophoresis to isolate them. When compared to the mirVana isolation kit (Ambion), the chip's performance was miRNA-dependent, performing 110-220 times better for miR-16 and 8.5-14 times better for let-7a (in terms of Ct values obtained).…”

“…55 This method of EV isolation was also used later as part of a high-throughput screening to identify miRNA biomarkers for prostate cancer. 56 Lee et al used electrophoresis to extract miRNAs from serum samples through a nanofilter membrane and demonstrated comparable miRNA Cq values using RT-qPCR to a commercially available miRNA extraction kit, with the whole process of on-chip extraction lasting around 30 min. 57 Behrmann et al incorporated thermoelectric lysis onto a microfluidic device in order to extract miRNAs directly from cells which was followed by gel electrophoresis to isolate them.…”

Potential of miRNAs in urinary extracellular vesicles for management of active surveillance in prostate cancer patients

“…In this study, NGS was used to identify novel miRNAs in small urinary EVs that could help to distinguish these grades. miRNAs derived from EVs have been used to stratify prostate cancer patients before [ 41 , 42 , 59 ], but previous studies have included patients with ISUP Grades 2 and 3 in the same group. Our results showed that 9 miRNAs had a significantly different expression in at least one of the possible comparisons between those three grades.…”

“…Finally, miRNAs from urinary EVs have also been used to discriminate between patients with different GS. When comparing patients with GS 6, 7 and 8, the level of miR-2909 was shown to be higher in more aggressive prostate tumours [ 41 ], and 6 miRNAs have been reported to be differentially expressed in patients with GS ≥ 9 [ 42 ].…”

Regulation of Neuroendocrine-like Differentiation in Prostate Cancer by Non-Coding RNAs

“…Downregulation of miR-27a caused by aberrant AR signalling and PI3K/Akt signalling after ADT was proposed to promote the progression of castration-resistant prostate cancer [352]. Although reports of miR-27a expression in tumour samples are conflicting [352,[354][355][356][357], high serum levels of miR-27a correlate with poor survival [356] and indicate presence of metastases [358].…”