Poster S4 - Detection of pancreatic cancer using mitochondrial abundance from tumor-derived extracellular vesicles

Introduction: Extracellular vesicles (EVs), small membrane-bound structures, are actively secreted by various cells, including tumor cells and they contain biomolecules such as genomic materials reflective of their cellular origin. These materials, including mitochondrial DNA and RNA, play critical roles in intercellular communication essential for tumor development and progression. Analyzing and quantifying EV contents has revealed tumor-specific features that hold promise as tumor biomarkers.
The ”Warburg effect”, characterized by increased mitochondrial activity in tumor cells, has spurred interest in translating EVs and mitochondrial abnormalities into diagnostic biomarkers. Although circulating tumor DNA, microRNA, and single nucleotide polymorphisms have been considered potential markers, they are often subtype-specific and susceptible to variations in sample processing, leading to time-consuming and labor-intensive analyses. Additionally, EVs from nonmalignant sources can mask tumor EVs in the circulatory system, further complicating biomarker identification.
To address these challenges, this study presents an innovative assay that combines immunoprecipitation and quantitative polymerase chain reaction (qPCR) to identify mitochondrial abnormalities (using mitochondrial DNA to nuclear DNA ratio) specifically in tumor-derived EVs isolated from patient serum samples. This approach aims to enhance the specificity and sensitivity of tumor biomarker detection by targeting mitochondrial markers within EVs, providing a potential solution to the limitations associated with existing biomarker detection methodologies.
By developing this assay for tumor-derived EV characterization, this study contributes to advancing the field of liquid biopsy-based diagnostics, offering a promising avenue for noninvasive tumor detection and monitoring.

Materials and

Methods: Nontumor pancreatic cells (hTERT-HPNE) and tumor pancreatic cells (PANC-1, MIA PaCa-2) were obtained from the American Type Culture Collection (ATCC) and cultured at a density of 6 x 105 cells in 10 cm tissue culture dishes. Cells were cultured in medium supplemented with 10% EV-depleted fetal bovine serum (FBS) using a non-starvation method, followed by isolation of extracellular vesicles (EVs) via ultracentrifugation.
DNA extraction from the cultured cells was performed using the AllPrep DNA MiniKit (Qiagen, USA). Quantitative polymerase chain reaction (qPCR) was employed for DNA detection and quantification, utilizing TaqMan assays targeting mitochondrial DNA (MT-ND1 ID: Hs02596873_s1, FAM-MGB) and normalized against a nuclear DNA gene (GAPDH ID: Hs02786624_g1, VICMGB) to ensure high sensitivity and accuracy.
For patient serum samples, 200 µl of serum from healthy individuals, patients with pancreatic ductal adenocarcinoma (PDAC), and pancreatitis were processed. Magnetic beads modified with anti-human epithelial cell adhesion molecule (EpCAM, Invitrogen) were utilized for immunoprecipitation of EVs. Beads were washed and resuspended in phosphate-buffered saline (PBS) before incubation with serum samples for 2 hours at room temperature with gentle agitation. Following PBS washes, beads were dissolved in 10 µl of lysis buffer (Sigma-Aldrich) and incubated for 30 minutes to release EVs. The collected supernatant containing immunoprecipitation-isolated EVs was used for downstream analysis.

Results, Conclusions, and Discussions: Pancreatic cancer cells exhibit increased mitochondrial activity and oxidative stress, altering mitochondrial (mtDNA) to nuclear DNA (nDNA) ratios. Utilizing fluorescent staining and confocal microscopy, we confirmed heightened mitochondrial activity in pancreatic tumor cells compared to nontumor cells, prompting the development of an extracellular vesicle (EV)-based assay to exploit this observation. Analysis of mtDNA/nDNA ratios at both cellular and EV levels demonstrated independence from total DNA content, emphasizing its cell-specific nature and resistance to technical influences of DNA quantification.
For the clinical assay development, we observed higher mtDNA/nDNA ratios in tumor EVs compared to nontumor EVs. We integrated EV immunoprecipitation (IP) targeting EpCAM with duplexed qPCR to measure mtDNA levels, enhancing assay sensitivity. Validation studies in PANC-1 tumor-bearing mice confirmed the assay's ability to differentiate tumor from non-tumor serum.
In a pilot clinical study involving healthy individuals, pancreatitis patients, and pancreatic ductal adenocarcinoma (PDAC) patients, our assay significantly distinguished between groups with high sensitivity and specificity (ROC-AUC: 0.88), outperforming the conventional biomarker CA19-9. Although weakly correlated with CA19-9 levels, our assay presents promise as a complementary non-invasive screening tool.
To streamline the assay, we investigated an isolation-free protocol for evaluating mtDNA/nDNA in tumor EVs. Comparative analysis revealed that the isolation-free method yielded mtDNA/nDNA ratios over three times higher than the DNA-extraction method in mice serum samples, likely due to reduced DNA loss during processing.
The assay’s key attributes, including EV-based mtDNA quantification, isolation-free qPCR, duplex probe, minimal sample volume requirement, user-friendly design, and better discriminatory performance, position it as an effective and convenient non-invasive screening method for pancreatic cancer. These findings highlight the assay's potential to enhance early detection and monitoring of pancreatic cancer, offering a promising avenue for clinical implementation.

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