Small RNA sequencing analysis of peptide-affinity isolated plasma extracellular vesicles distinguishes pancreatic cancer patients from non-affected individuals

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma.

Emerging Horizons in the Diagnosis of Pancreatic Cancer: The Role of Circulating microRNAs as Early Detection Biomarkers for Pancreatic Ductal Adenocarcinoma

“…We observed that miR-18a and miR-106a in plasma small extracellular vesicles (EVs) were significantly elevated in early-stage PDAC patients compared to healthy controls. This finding aligns with other studies, suggesting these miRNAs as reliable indicators for early PDAC detection [ 3 , 9 , 18 - 20 ]. The consistency across different research efforts reinforces the potential of these miRNAs as non-invasive biomarkers.…”

Extracellular vesicles in pancreatic cancer: a new era in precision medicine

“…EVs from the sample of PC patients and healthy controls and assessed specific miRNA-derived EV content using reverse transcription polymerase chain reaction (RT-PCR) and miRNA-specialised sensors to determine if EVs can be used as diagnostic tool ( 37 - 42 ). MiR-550, miR-21 miR-17-5p and miR-10b expression was found to be elevated in PC patients, implying that they can serve as potential markers for PC detection ( 41 , 43 - 46 ). Madhavan et al examined blood EVs for five PC-initiating cell markers and four combination miRNAs ( 47 ).…”

Plasma extracellular vesicle sampling from high grade gliomas demonstrates a small RNA signature indicative of disease and identifies lncRNA RPPH1 as a high grade glioma biomarker.

“…The Vn96 peptide is a peptide a nity capture method developed for the capture of EVs in clinically relevant bio uids [18-20, 33, 38-41]. Numerous studies have shown captured Vn96-EVs from plasma contain canonical EV markers, and Vn96 has been utilized for the discovery of EV cargobiomarkers in amyotrophic lateral sclerosis [39] and cancers, such as prostate [42], lung [43], and pancreatic ductal adenocarcinoma [44].…”