Assistant Professor North Dakota State University, United States
Introduction:: Pancreatic cancer patients are predominantly present with advanced disease at diagnosis, contributing to its high mortality. Current screening methods are invasive, time-consuming, and expensive, highlighting the need for noninvasive fast-screening and efficient techniques. Tumor-derived extracellular vesicles (tdEVs) bearing information from parental cells have emerged as a promising cancer diagnostic biomarker. However, most tdEV-based assays have impractical sample volumes, time-consuming, complex, and costly techniques. To overcome these limitations, we developed a novel diagnostic method for pancreatic cancer screening termed EvIPqPCR.
Materials and Methods:: We developed a high-throughput noninvasive assay by utilizing the mitochondrial DNA to nuclear DNA ratio (mtDNA/nDNA) of the tumor-derived EVs as a collective cell-specific characteristic to target EVs originating from malignant and nonmalignant cells. We introduce EvIPqPCR, a fast method that combines immunoprecipitation (IP) and qPCR quantification to detect tumor-derived EVs directly from patient serum. Importantly, our method employs DNA isolation-free and duplexing probes for qPCR, saving at least three hours.
Results, Conclusions, and Discussions:: We introduce an assay called EvIPqPCR that combines immunoprecipitation (IP) and qPCR to identify a mitochondrial abnormality in tumor-derived extracellular vesicles (tdEVs) in serum samples. Extracellular vesicles (EVs) bearing information from parental cells play important roles in cell−cell signaling, immune response, and metastasis. The EvIPqPCR assay introduced in this study offers several advantages over existing EV analysis methods. Specifically, it includes both DNA isolation-free and duplexing probes methods. The DNA isolation-free protocol reduces the time-consuming and labor-intensive DNA purification procedures required by most other DNA analysis methods. The duplex probes used in the assay can quantify both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in a single run. This not only helps to minimize interferences for using multiple fluorescent probes in qPCR measurement, but also introduces a collective attribute mtDNA/nDNA ratio as a relative marker to reduce the individual bias effectively. Due to their membrane structure, EVs protect DNA from degradation in the bloodstream, minimizing the risk of false indications when using circulating tumor DNA (ctDNA) as a target. Additionally, by targeting tumor EV membrane markers before qPCR detection, this method avoids time-consuming EV isolation steps. This technique has the potential to serve as a translational assay for cancer screening with a weak correlation to prognosis biomarkers and sufficient discriminatory power between healthy control, pancreatitis, and pancreatic cancer cases compared to most standard EV analysis methods.
