Acoustofluidic Salivary Exosome Isolation

Abstract

Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) have not shown sufficient clinical performance. We hypothesize that salivary exosomes are packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation can enhance diagnostic and prognostic performance for HPV-OPC. In this study, an acoustofluidic (the fusion of acoustics and microfluidics) platform was developed to perform size-based isolation of salivary exosomes. These data showed that this platform is capable of consistently isolating exosomes from saliva samples, regardless of viscosity variation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform is 15 times higher. With this high-yield exosome isolation platform, we show that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16. Overall, these data demonstrated that the acoustofluidic platform can achieve high-purity and high-yield salivary exosome isolation for downstream salivary exosomeebased liquid biopsy applications. Additionally, HPV16 DNA sequences in HPV-OPC patients are packaged in salivary exosomes and their isolation will enhance the detection of HPV16 DNA.

<p>Exosome: A Review of Its Classification, Isolation Techniques, Storage, Diagnostic and Targeted Therapy Applications</p>

“…Salivaderived exosomes are separated and purified based on the size using a newly developed acoustic fluid platform, which combined acoustics with microfluidic technology. The average yield of small RNA in exosomes detected by RT-ddPCR using this technique is 15 times that of differential centrifugation, and studies have found that harvesting exosomes by this technique is beneficial for downstream analysis 62 . In addition, separation techniques such as Tangential Flow Filtration, 63 Flow fieldflow fractionation (FIFFF) 64 and Deterministic lateral displacement array 65 also have good enrichment potential.…”

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods

“…While the problem of specimen has been addressed, we underlined the utility of technical adaptations of classic methods, such as ddPCR or fluorescent-based detection methods as they either provide quicker analysis [127] or feasibility for high-throughput screening [107] and require a smaller screening volume. If these techniques are further improved and the advantages combined, they might make implementing EV isolation in clinical settings possible.…”

“…Pancreatic juice can be very viscous when patients suffer from intraductal papillary mucinous neoplasm due to high mucin concentration, which makes exosome isolation difficult [88]. High viscosity is also a problem reported for whole saliva [107]. These are just a few examples of problems that have to be overcome.…”

“…The method was further improved by combining an acoustic trap and a microfluidic chip. This makes the isolation from EVs from multi-component body fluids like blood or saliva feasible since the flow rate in addition to the acoustic frequency can be adjusted to isolate specific subpopulations of the sample [107,136]. Naturally, every method has its drawbacks such as a high antibody demand for the immunoaffinity isolation, the dependency of sample density of the acoustic trap, or the lower specificity of size-based methods.…”

Extracellular Vesicles in Oncology: from Immune Suppression to Immunotherapy

“…Exosomes from saliva were isolated using an acoustofluidic platform and investigated for small RNA and DNA content using droplet digital RT-PCR. The study demonstrated the enrichment of HPV16 DNA in the exosomes isolated from the saliva of HPV-OPC patients suggesting their putative application in saliva exosome-based liquid biopsies (143). In their review article, Nonaka and Wong have discussed in detail the current status of salivary exosomes and their molecular content in the context of developing diagnostic and prognostic biomarkers and developing saliva exosomes as a novel modality of liquid biopsies.…”