Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria

Abstract

Circulating mitochondrial DNA (cir-mtDNA) could have a potential comparable to circulating nuclear DNA (cir-nDNA), with numerous applications. However, research and development in this area have fallen behind, particularly considering its origin and structural features. To tackle this, we initially combined Q-PCR and low-pass whole genome sequencing in the same analytical strategy previously and successfully used for cir-nDNA. This revealed unexplained structural patterns and led us to correlate these data with observations made during physical examinations such as filtration, and differential centrifugation in various plasma preparations. Both the integrity index and number of reads revealed a very minor proportion of low size-ranged fragments (<1000 bp) in plasma obtained with a standard preparation (0.06%). Filtration and high speed second step centrifugation revealed that 98.7 and 99.4% corresponded to extracellular mitochondria either free or in large extracellular vesicles. When avoiding platelet activation during plasma preparation, the proportion of both types of entities was still preponderant (76-80%), but the amount of detected mitochondrial DNA decreased 67-fold. In correlation with our previous study on the presence of circulating cell-free mitochondria in blood, our differential centrifugation procedure suggested that cir-mtDNA is also associated with approximately 18% small extracellular vesicles, 1.7% exosomes and 4% protein complexes.

Cell-free DNA topology depends on its subcellular and cellular origins in cancer

“…Strikingly, cf-mtDNA abundance from permeabilization/degradation assay samples demonstrated a significant increase in relative DNA abundance after membrane permeabilization versus the untreated control (Figure 3B; MCF10A: +259.69%±6.92%, p<0.0001; B16F10: +109.14%±24.28%, p<0.0001; HCT116: +62.57%±1.41%, p<0.0001). Interestingly, DNase I treatment did not fully degrade cf-mtDNA in either membrane-permeabilized or non-permeabilized samples (Figure 3B), in contrast to our observations of cf-nDNA (Figure 2B); as the DNase-resistant population of cf-mtDNA was in similar abundance despite EV permeabilization, this finding may reflect an alternative or currently unknown protective mechanism (37).…”

Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA

“…This study was carried out from cir-nDNA and cir-mtDNA extracted from plasma preparation avoiding platelet activation. This warrants a proper evaluation of the topological nature and content of cir-mtDNA as demonstrated in our previous report (Roch et al, 2021) while enabling the direct quantitative comparison of their various structural features.…”

“…To do so, various preanalytical conditions might be used with a necessary removal of cells by using a centrifugation step. We herein employed different procedures previously detailed in our previous paper (Roch et al, 2021). Besides, the pre-analytical conditions for all blood samples strictly followed the guidelines we reported (Meddeb et al, 2019a).…”

“…To preclude platelet activation during plasma isolation, we used a protocol previously detailed in our previous paper (Roch et al, 2021). Briefly, we collected fresh blood in dedicated tubes with a subsequent isolation of plasma through differential centrifugations: two consecutive steps at 200 g, a third centrifugation at 300 g, the subsequent addition of an anticoagulant solution containing prostaglandin E1 to the plasma, and three further centrifugations at 1,100, 2,500 and 16,000 g, all centrifugations steps being carried out for 10 min at room temperature.…”

“…The different plasma preparations used here are detailed in our previous paper (Roch et al, 2021). Briefly, we examined different approaches to prepare plasma from each blood sample, with variations considering centrifugation speed and/or F. After an initial 1,200 g centrifugation step, the isolated plasma was split in four equal volumes: (i), the first aliquot (LS) was not submitted to any additional treatment and considered as a control; (ii) the second one was filtered (LS+F) with a 0.22 µm filter; (iii), an additional centrifugation at 16,000 g was performed for the third one (LS+HS); and (iv), the last one was centrifuged at 16,000 g centrifugation after being stored at −20 °C (LS+freezing+HS).…”