Defining Optimal Conditions for Tumor Extracellular Vesicle DNA Extraction for Mutation Profiling

Abstract

(1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients.

Extracellular Vesicles in Diffuse Large B Cell Lymphoma: Characterization and Diagnostic Potential

“…The present study’s primary aim is to compare the proteome of extracellular vesicles isolated from the plasma of DLBCL patients and HDs. For mass spectrometry analysis, we have isolated extracellular vesicles from 32 newly diagnosed DLBCL patients and 15 aged matched HDs using sequential centrifugation, as previously described [ 18 ]. The particles have been analyzed by nanoparticle tracking analysis ( Figure 1 a,b).…”

“…As previously described [ 18 ], frozen plasma specimens were thawed and centrifuged at 3000× g for 20 min at 4 °C and then at 12,000× g for 60 min at 4 °C. Clarified plasma was ultracentrifuged in an Optima TM L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 100,000× g at 4 °C for 120 min with a Type 50.2 Ti rotor (k-factor: 157.7) to pellet EVs.…”

“…As previously described [18], frozen plasma specimens were thawed and centrifuged at 3000× g for 20 min at 4 • C and then at 12,000× g for 60 min at 4 • C. Clarified plasma was ultracentrifuged in an Optima TM L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 100,000× g at 4 • C for 120 min with a Type 50.2 Ti rotor (k-factor: 157.7) to pellet EVs. The supernatant was carefully removed, and crude EV-containing pellets were resuspended in ice-cold PBS, followed by floatation on a sucrose cushion (30%, D 2 O) (Sigma-Aldrich, Saint Louis, MO, USA) for 60 min at 100,000× g at 4 • C to remove non-EV protein complexes.…”

Canine diffuse large B-cell lymphoma downregulates the activity of CD8 + T-cells through tumor-derived extracellular vesicles

“…NTU-107-EL-00200). Extracellular vesicles (EVs) were isolated from the plasma according to the previous publication [ 25 ]. Frozen plasma specimens were thawed and subjected to centrifugation at 3000 × g for 20 min at 4 °C, followed by centrifugation at 12,000 × g for 60 min at 4 °C.…”

Methods to Evaluate Changes in Mitochondrial Structure and Function in Cancer

“…gov have incorporated EV-DNA as a cancer biomarker, including those for soft tissue sarcoma [179], colorectal cancer [180], and non-small cell lung cancer (NSCLC) [181,182]. Many previous studies using EV-DNA to detect cancer-related mutations face the challenge of ensuring EV purity [183]. A recent study [184] confirmed the contamination of cell culture EV samples isolated using ultracentrifugation with naked DNA (i.e., non-EV DNA).…”