Here, we present a method for the successful isolation and sequencing of the full mitochondrial genome from whole blood (WB), peripheral blood mononuclear cells (PBMCs), plasma EVs, and tumor tissue from CRC patients as an initial investigation for the potential use of EVs as a source of cell-free mtDNA and their potential as CRC biomarker. In addition, a novel EV population of mitochondrial origin, mitovesicles, was described by D’Acunzo et al.ĭespite the increasing interest in EV mitochondrial components, the characteristics of secreted cell-free mtDNA still remain insufficiently understood. For example, secreted cell-free respiratory-competent mitochondria have been detected in blood and EVs have been shown to contain functional mitochondria and be enriched in mitochondrial proteins. Ĭell-derived mitochondrial components, besides mtDNA, have been found in the extracellular space. Nevertheless, if a mutation is pathogenic, the cell can often tolerate a certain proportion of the mtDNA variant before the biochemical threshold is exceeded with resulting metabolic defects. The mtDNA polymorphisms may alter mitochondrial function, particularly in tissues that are highly dependent on the metabolism. Because the mutation frequency of replicating mtDNA is high, mutant mtDNA copies are often mixed with wild-type copies in the cell (termed heteroplasmy). The mtDNA replication is independent of the cell cycle and also occurs in postmitotic cells. It contains 37 genes that encode 13 protein subunits of the mitochondrial respiratory chain/oxidative phosphorylation system, two rRNAs, and 22 tRNAs for mitochondrial translation. The mitochondrial genome is a 16.5-kilobase circular double-stranded DNA molecule present in multiple copies per cell. Recent reports have demonstrated that the entire mitochondrial genome can be packed inside EVs and restore metabolic activity in cells with impaired metabolism. Variations in the mitochondrial DNA (mtDNA) sequence can act as functional adaptors allowing tumor and immune cells to adjust to the metabolic needs imposed by various tissue environments during cancer progression. Mutations in mitochondrial genes have been reported to have a role in cancer development. Extracellular vesicles (EVs) are known to contribute to tumorigenesis, progression, and drug resistance in CRC and may be important CRC biomarkers. It is a heterogeneous disease in terms of high biological complexity and clinical outcome. Registered 22 March 2013.Ĭolorectal cancer (CRC) is the third most common cancer type worldwide. Taken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. In both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. Pearson correlation analysis was performed. Mann–Whitney U test was used when comparing differences between the cancer types and patient groups. Differences between groups were compared by paired Student’s t-test or ANOVA with Dunnett’s multiple comparison tests when comparing matched samples from patients. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Total DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients. Recent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood.
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