Abstract 1964: Next-generation sequencing (NGS) reveals higher mitochondrial DNA (mtDNA) diversity in plasma extracellular vesicles (EVs) than in other blood components and tumor tissue from colorectal cancer (CRC) patients
Authors: Bjørnetrø Tonje , Bousquet Paula A. , Redalen Kathrine R. , Trøseid Anne-Marie S. , Lüders Torben , Johansen Christin , Fuglestad Anniken J. , Kersten Christian , Meltzer Sebastian , Ree Anne H. 
Published: June 2022

Abstract
Introduction: EVs released from cells are nanosized vesicles (containing proteins and nucleic acids) that serve as a communication system with other cells or expel cellular waste. Variations in the mtDNA sequence can act as functional adaptors allowing tumor and immune cells to adjust the metabolic needs imposed by various tissue environments during cancer progression. Recent reports have demonstrated that the entire mitochondrial genome can be encapsulated and secreted in EVs. However, the biological attributes of secreted cell-free mtDNA still remain insufficiently understood, mostly because of the difficulty in isolating and characterizing the entire mitochondrial genomes. Here, we present the results from sequencing of mtDNA from various blood compartments, including EVs, and tumor tissue from CRC patients.
Methods: Plasma EVs (median mode size 136.4 nm) were isolated by size exclusion chromatography. We compared the mtDNA in EVs with that in whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor samples from eight rectal cancer patients. Total DNA was isolated from the four tissue types before the mtDNA was enriched by applying two different PCR approaches with either multiple (for FFPE tumor) or only two (for EVs, WB, and PBMCs) primer sets prior to NGS analysis. WB- and fresh-frozen (FF) tumor mtDNA from eight colon cancer patients was sequenced as controls for the two PCR primer approaches. mtDNA sequence mapping and variant analyses were done using the revised Cambridge Reference Sequence, and HaploGrep2- and Variant Effect Predictor softwares.
Results: EV mtDNA presented twice as many variants with significantly more heteroplasmy (mutant mtDNA copies mixed with wild-type copies) than mtDNA from WB and PBMCs. The proportion of EV mtDNA variants that were non-synonymous (i.e. estimated to affect the mitochondrial function) was significantly higher than in WB. FFPE tumors, requiring the multi-primer approach, showed a manyfold increase in detected mtDNA variants, which was not observed for FF tumor mtDNA amplified like WB mtDNA. However, several mtDNA variants detected in FFPE- and FF tumors were not retrieved in WB.
Conclusion: We successfully isolated and sequenced the mtDNA cargo of plasma EVs for comparison with mtDNA in WB, PBMCs, and tumor tissue from CRC patients. The EV mtDNA exhibited higher diversity than in the other blood compartments, with a higher degree of heteroplasmy and more variants with impact on the mitochondrial function. The majority of additional mtDNA variants detected in FFPE tumors were possibly caused by technical biases (the tissue processing or mtDNA amplification procedure). Plasma EVs may be involved in the metabolic homeostasis of tumor and immune cells in CRC patients.
Citation Format: Tonje Bjørnetrø, Paula A. Bousquet, Kathrine R. Redalen, Anne-Marie S. Trøseid, Torben Lüders, Christin Johansen, Anniken J. Fuglestad, Christian Kersten, Sebastian Meltzer, Anne H. Ree. Next-generation sequencing (NGS) reveals higher mitochondrial DNA (mtDNA) diversity in plasma extracellular vesicles (EVs) than in other blood components and tumor tissue from colorectal cancer (CRC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1964.