The present study aims at identifying the structures containing mitochondrial DNA in peripheral blood. By examining McfDNA integrity, and associated structure size and density, we revealed the presence of stable particles with full‐length mitochondrial genomes. We characterized the structures by fluorescence and electron microscopy (EM), flow cytometry, and we identified the presence of intact mitochondria in the circulation. Oxygen consumption assays suggested the functional viability of at least some of these extracellular mitochondria. Our work demonstrates for the first time the presence in blood of circulating cell‐free respiratory competent mitochondria. Overall, in view of the potential roles of mitochondria in cell to cell communication, immune response, and inflammation, our discovery has broad implications in homeostasis and disease, and paves the way for new paths toward the treatment and prevention of diseases.

Cell‐derived mitochondrial components, including mitochondrial DNA, have been found in the extracellular space. 9 , 10 Those deoxyribonucleic acid (DNA) fragments were found in the physiological circulating fluid of healthy subjects and patients with various diseases. 11 Lately, mitochondrial cell‐free DNA (McfDNA) has emerged as an attractive circulating biomarker due to its potential role in diagnostic applications in multiple diseases (eg, Diabetes, acute myocardial infarction, cancer…), 12 - 14 and in physio‐pathological conditions (eg, trauma). 15 Despite the promising future of McfDNA in clinical applications, knowledge regarding its origin, composition, and function is still lacking. In addition, the structure of McfDNA is currently unknown. In contrast, the structure of circulating DNA of nuclear origin is being characterized 16 and mono and di‐ nucleosomes and, to a lesser extent, transcription factors are found as stabilized cell‐free DNA (cfDNA)‐associated structures in the blood‐stream. 17 , 18 It is expected that there are important configuration differences between nuclear and mitochondrial circulating DNA, since mitochondrial DNA is a small circular genome, without protective histones, and thus is more sensitive to degradation in the circulation. However, by revealing recently that there are approximately 50 000‐fold more copies of the mitochondrial genome than the nuclear genome in the plasma of healthy individuals, 19 we confirmed that McfDNA is sufficiently stable to be detected and quantified, 12 , 20 implying the presence of stable structures protecting these DNA molecules.

The presence of mitochondria in unicellular to mammalian organisms originates from an ancient symbiosis between primitive eukaryotic cells and free‐living aerobic prokaryotes. 1 , 2 Mitochondria are crucial organelles for central cell functions, 3 and they are the principal nutrient‐up‐taking and energy‐producing cell organelle; they also take part in calcium signaling, reactive oxygen species (ROS) production, cell death, and diverse cell signaling events. 4 - 7 Mitochondria have retained many of their ancestral bacterial features including length, proteome, double membrane, and circular genome. 8

2 MATERIALS AND METHODS

2.1 Plasma isolation Blood samples from healthy volunteers were provided by the “Etablissement Français du Sang (E.F.S),” the blood transfusion center of Montpellier, France (Convention EFS‐PM N° 21PLER2015‐0013). Blood samples from 50 mCRC patients were provided by a clinical study comparing the detection of KRAS exon 2 and BRAF V600E mutations by cfDNA analysis to conventional detection by tumor tissue analysis. All blood samples were processed within 4 hours after collection. Plasma was extracted by various protocols, depending on the experiments. 2.1.1 For healthy individuals Plasma isolation using Ficoll Fresh blood was collected in EDTA tubes. Plasma was isolated by a Ficoll density‐gradient centrifugation, performed at 400 g for 30 minutes at 18°C (Ficoll paque plus GE Healthcare, Fisher Scientific, Illkirch, France). Plasma isolation by centrifugation Fresh blood was collected in EDTA tubes. Plasma was isolated by a single centrifugation, performed at 1200 g for 10 minutes at 4°C.21 Plasma isolation without platelet activation Fresh blood was collected in a BD Vacutainer citrate–theophylline–adenosine–dipyridamole (CTAD) tubes (Ozyme, Montigny‐le‐Bretonneux, France). Plasma was isolated via differential centrifugations, all performed for 10 minutes at room temperature without a break: two successive centrifugations were first performed at 200 g and were followed by a third centrifugation at 300 g. Preheated (37°C) anticoagulant Citrate Dextrose Solution A (ACD‐A) buffer (0.1 M Trisodium citrate, 0.11 M Glucose and 0.08 M citric acid) and Prostaglandin E1 (1 µM) (Sigma‐Aldrich, St. Quentin Fallavier Cedex) were then added to the plasma, which was further centrifuged at 1100 g and finally at 2500 g. 2.1.2 For mCRC patients Blood was collected in Ethylenediaminetetraacetic acid (EDTA) tubes, and plasma was isolated by a single centrifugation, performed at 1200 g for 10 minutes at 4°C.

2.2 Cell lines Human colon cancer cell lines (DLD‐1/SW620) were obtained from the American type culture collection (ATCC) and a normal immortalized cell line (CCD‐18Co) was obtained from Andrei Turtoi’s laboratory (IRCM, Montpellier, France). SW620 and CCD‐18Co cells were grown in RPMI 1640 and DLD‐1 cells in DMEM (Gibco, Fisher Scientific, Illkirch, France), both supplemented with 10% fetal bovine serum (Eurobio, les Ulis, France) and 1X streptomycin/penicillin (Gibco, Fisher Scientific, Illkirch, France) . Cell culture, for all cell lines, was performed at 37°C in 5% CO 2. 1.5 million cells were seeded in a T‐75 flask with 10 mL of appropriate supplemented medium. Cells were incubated for either 24 hours or 60 hours. Culture media were replaced with fresh medium 24 hours before experiments. Collected media from cultured cells were centrifuged at 600 g for 10 minutes at 4°C, to precipitate both floating cells and cells debris, and were further processed by various protocols, depending on the experiments.

2.3 Quantification of nuclear cell‐free DNA (NcfDNA) and mitochondrial cell free DNA (McfDNA) in healhy individuals and cancer patients NcfDNA and McfDNA in healthy individuals (Figures 1A,B and S2A), and in mCRC patients (Figures S1 and S2B) were quantified from a plasma supernatant obtained following the first centrifugation step mentioned before at 1200 g for 10 minutes at 4°C and a second centrifugation step at 16 000 g for 10 minutes at 4°C. Following this centrifugation, total cfDNA was extracted from supernatant and analyzed with quantitative polymerase chain reaction (q‐PCR). Figure 1 Open in figure viewer Plasma of healthy individuals contain a population of cell‐free mitochondrial DNA that is well preserved and not fragmented. A, NcfDNA and McfDNA quantification in plasma samples of 99 healthy individuals. McfDNA copy number is significantly higher than that of NcfDNA (P < .0001). The lines inside the boxes and the upper and lower limits of the boxes indicate the median, 75th and 25th percentiles, respectively. The upper and lower horizontal bars indicate the maximum and minimum values, respectively. B, NcfDNA and McfDNA integrity index calculated for plasma samples of 13 healthy individuals, demonstrating that almost all the quantified McfDNA have a main size above 300 bp, and are less fragmented than NcfDNA (P < .0001). C, Size fractionation following agarose gel electrophoresis of a pool of cfDNA extracts obtained from 80 healthy individuals’ plasma. McfDNA fragments were quantified by q‐PCR from extracts of the excised agarose gel slices above (Long) and below (Short) 500 bp. D, DNA fragment size profile at single base resolution obtained by paired‐end massively parallel WGS of cfDNA extracted from a healthy individual’s plasma. (Blue for NcfDNA and red for McfDNA)

2.4 Kinetic study of cfDNA stability First, SW620 and DLD‐1 culture medium was removed after 24 hours of culture, centrifuged at 1200 g, and then at 16 000 g for 10 minutes, both at 4°C, and further incubated at 37°C in 5% CO 2 for 4 days. An aliquot of cell culture supernatant (400 µL) was withdrawn every day for DNA extraction. NcfDNA and McfDNA were quantified by q‐PCR using specific primers (KRAS F2 and KRAS R1 for NcfDNA, MIT MT‐CO3 F, and MIT MT‐CO3 R2 for McfDNA) (Table S1).

2.5 cfDNA extraction Cell‐free DNA was extracted with a Qiagen Blood Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s recommendations, except that extraction was performed with 1mL of plasma sequentially loaded on a single column and that the cfDNA was eluted with 130 μL of elution buffer. The cfDNA was stored at −20°C for further analysis. Freeze‐thawing was avoided to reduce cfDNA fragmentation.

2.6 Q‐PCR analysis Cell‐free DNA was quantified by q‐PCR according to an innovative design of short (60–100 bp ± 10 bp) and long (300 bp ± 10 bp) amplicons targeting the wild‐type sequences of specific genes: the KRAS nuclear gene and the mitochondrial Cytochrome oxidase III gene, MT‐CO3 (Table S1). Quantification of the short and long amplicons provides an estimation of the concentrations of the total NcfDNA and McfDNA. Q‐PCR amplifications were performed in triplicate in a 25 μL final reaction volume controlled by the CFX manager software of a CFX96 touch Real‐Time PCR detection system (Bio‐Rad). Each polymerase chain reaction (PCR) reaction mixture was composed of 12.5 μL of SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad, Marnes‐la‐Coquette, France), 2.5 μL of nuclease‐free water (Qiagen), 2.5 μL of forward and 2.5 μL reverse primers (3 pmoL/µL), and 5 μL of template. Thermal cycling conditions were as follows: 95°C (3:00) + [95°C (0:10) + 60°C (0:30)] × 40 cycles. Melting curves were investigated by increasing the temperature from 60°C to 90°C, reading the plate every 0.2°C. Each q‐PCR run was performed with 1.8 ng/μL of genomic DNA extracted from the DiFi cell line (ATCC) for the standard curve and without DNA as the control condition. Q‐PCR amplification was validated by melt curve differentiation.

2.7 cfDNA calibration assay and copy number calculation 2.7.1 NcfDNA quantification KRAS colorectal cells was used for the NcfDNA calibration assay. Initial genomic DNA solution concentration and purity were determined by measuring optic density at λ = 260, 230, and 280 nm, with an Eppendorf BioPhotometer D30. Starting genomic DNA concentration was adjusted to 1800 pg/µL for the first dilution point, according to optical density measurement at λ = 260 nm. A q‐PCR standard curve was obtained by six successive dilutions of the vector solution (to 1800, 180, 45, 20, 10, and 5 pg/µL). The standard curve was used to determine the NcfDNA concentration per milliliter of plasma and cell media supernatant. The NcfDNA copy number was calculated as follows: Q nuclear = c 3.3 × V elution V plasma A genomic DNA extract from human wild‐typecolorectal cells was used for the NcfDNA calibration assay. Initial genomic DNA solution concentration and purity were determined by measuring optic density at λ = 260, 230, and 280 nm, with an Eppendorf BioPhotometer D30. Starting genomic DNA concentration was adjusted to 1800 pg/µL for the first dilution point, according to optical density measurement at λ = 260 nm. A q‐PCR standard curve was obtained by six successive dilutions of the vector solution (to 1800, 180, 45, 20, 10, and 5 pg/µL). The standard curve was used to determine the NcfDNA concentration per milliliter of plasma and cell media supernatant. The NcfDNA copy number was calculated as follows: Q nuclear is the NcfDNA copy number per milliliter; c is the NcfDNA mass concentration (pg/µL) determined by a q‐PCR targeting the nuclear KRAS gene sequence and 3.3 pg is the human haploid genome mass. V elution is the volume of cfDNA extract (µL) and V plasma is the starting volume of plasma used for the extraction (mL). 2.7.2 McfDNA quantification A 3382‐bp human ORF vector with a 786‐bp MT‐CO3 insert was obtained from ABM (accession no.YP_003024032) and used for the McfDNA calibration assay. Initial vector solution testing, starting concentration adjustments and q‐PCR standard curves were performed as for NcfDNA above. Q mito = c × Na 2 × MW × Lvector × V elution V plasma The standard curve was used to determine the McfDNA concentration per milliliter of plasma and of cell media supernatant. The McfDNA copy number was calculated as follows: Q mitochondrial is the McfDNA copy number per milliliter, “c” is the McfDNA mass concentration (g/µL) determined by q‐PCR targeting the mitochondrial MT‐CO3 gene sequence. N A is Avogadro’s number (6.02 × 1023 molecules per mol), L vector is the plasmid length (nucleotides), and MW is the molecular weight of one nucleotide (g/moL); V elution is the elution volume of cfDNA extract (µL) and V plasma is the starting volume of plasma used for the extraction (mL).

2.8 DNA integrity index calculation The degree of cfDNA fragmentation was assessed simultaneously by targeting KRAS and MT‐CO3 sequences from each plasma DNA sample and calculating the DII (DNA integrity index). The DII was determined by calculating the ratio of the concentration determined using the primer set amplifying a large target (KRAS F2/R1 and MT‐CO3 F/R2) to the concentration determined using the primer set amplifying a short target (KRAS F1/R1 and MT‐CO3 F/R1) (Table S1).

2.9 Examination of the percentage of McfDNA short and long fragments Cell‐free DNA was extracted from a pool of 80 healthy individual’s plasma, with the Maxwell RSC ccfDNA plasma kit (Promega, Charbonnières‐les‐Bains, France). To obtain highly concentrated cfDNA, extracts were subjected to a second extraction with the same method followed by a Qiamp DNA blood Mini Kit extraction resulting in a final volume of 30 µL. The samples were then electrophoresed on a 2% agarose gel and DNA fractions of short (<500 bp) and long (>500 bp) fragments were extracted from the gel with a QIAquick Gel extraction Kit (Qiagen). The quantity of mitochondrial DNA fragments was then assessed with q‐PCR by using mitochondrial specific primer (MT‐CO3 F/R1) (Table S1).

2.10 Library preparation for whole genome sequencing Dual‐indexed single‐stranded libraries were prepared from 1 to 11 ng human DNA using TL137 as a linker oligo.22 To allow sequencing with single‐stranded libraries, custom double‐stranded adapters were generated, identical to those used in the single‐stranded method, by annealing a short oligo SLP4.23 Ligations were performed with an NEBNext Quick Ligation Module kit (New England Biolabs) with 0.04 µM of each annealed adapter in a 50 µL total volume, incubated for 30 minutes at 20°C. Adapters were elongated by adding 50 µL OneTaq 1× Master Mix (New England Biolabs) to the ligation mixture and incubating for 20 minutes at 60°C. All oligos were purchased from Eurogentec (Kaneka Eurogentec, Seraing, Belgium). Single‐stranded libraries were quantified via q‐PCR, with a diluted fraction, in a LightCycler 2 (Roche Applied Science, Mannheim, Germany). Samples were then amplified using Taq polymerase and an optimal number of cycles for each sample to avoid plateau phase, as calculated from the Ct of the diluted q‐PCR. Amplified samples were then purified by two rounds of Macherey‐Nagel NGS beads at 1.3× volume, to retain shorter inserts, and eluted in 30 µL EBT. Purified libraries were then visualized on a Bioanalyzer 2100 (Agilent, Santa Clara, CA) and quantified via q‐PCR, Qubit 2 Fluorometer (Life Technologies, Grand Island, NY), and Bioanalyzer 2100. Libraries were then pooled in equimolar amounts and sequenced on an Illumina MiSeq platform using two MiSeq v3 150 (2 × 75) kits, with sequencing primer CL72 replacing the first read sequencing primer.

2.11 Sequence analysis Adapter sequences were removed with cutadapt 1.324 and reads were aligned to the human genome reference (hg19) with BWA aln25 using the default parameters and filtered for mapping quality 20 with SAMtools 1.5.26 Duplicate removal and histogram generation was performed with MarkDuplicates and CollectInsertSizeMetrics, respectively, from Picard tools 1.88.

2.12 Differential centrifugation and filtration of plasma and cell media Schematic view and details are presented in Figure S6.

2.13 Isolation of intracellular mitochondria Mitochondria from cultured cells were isolated as a positive control. Cells were scraped from culture dishes, washed with phosphate‐buffered saline (PBS) and centrifuged at 1300 rpm for 5 minutes at room temperature. Pelleted cells were resuspended in 1 mL of mitochondrial isolation buffer (MIB) (0.2 M sucrose, 0.01 M tris, 0.001 M EGTA, 1X protease inhibitor) and gently lysed using an IKA T18 Basic Dispersers Homogenizer (Ultraturrax) at speed 2 for 10 seconds until obtaining 80%‐90% intact nuclei. Lysed cells were then centrifuged at 600 g at 4°C for 10 minutes to remove cell debris and nuclei, to recover supernatant containing both cytoplasm and mitochondria. Pelleted intact nuclei were washed twice in 1ml MIB and centrifuged again at 600 g for 10 minutes at 4°C, to be used as a negative control in experiments with flow cytometry, while the resulting supernatants were pooled together with the previous supernatants to further isolate mitochondria. For this, the 3 mL of supernatant were centrifuged at 600 g at 4°C for 10 minutes to eliminate contaminating nuclei and cell debris. The resulting supernatant was then further centrifuged at 8000 g for 10 minutes at 4°C to pellet mitochondria. The supernatant was collected again and centrifuged again at 8000 g for 10 minutes at 4°C. These pellets were pooled together and gently resuspended in 500 µL MIB, transferred to 1.5 mL tubes and centrifuged again at 8000 g for 10 minutes at 4°C. The final supernatant was carefully discarded to collect the pelleted mitochondria.

2.14 Isolation of extracellular mitochondria Mitochondria in the plasma, extracted by Ficoll gradient, were isolated by sequential centrifugations at 600 g, then 1200 g and finally 2000 g for 10 minutes at 4°C to remove any contaminating blood cells or platelets. The resulting supernatant was centrifuged at 16 000 g to collect the extracellular mitochondria pellet. Mitochondria in the plasma obtained without platelet activation were pelleted by a one‐step centrifugation at 16 000 g for 10 minutes at 4°C. Mitochondria in the cell media supernatant were isolated by sequential centrifugations at 600 g, then 1200 g for 10 minutes at 4°C to remove any contaminating cells. The pellet of extracellular mitochondria was collected from a subsequent centrifugation at 16 000 g. Note that an extracellular mitochondrial pellet was also recovered from a centrifugation at 8000 g instead of 16 000 g to protect the mitochondrial membrane.

2.15 Amplification of the mitochondrial genome The DNA extracted from cell media pellets (40 flask T‐75/cell line), and from two different plasma pools (without platelet activation) pellets was selectively amplified with the Repli‐g mitochondrial DNA kit (Qiagen), according to the manufacturer’s recommendations. The amplified mitochondrial genome was then amplified by long range (LR) PCR performed in a 50 μL total volume in a Mastercycle nexus eco thermal cycler (Eppendorf). Each PCR reaction mixture was composed of 30.5 μL of free water (Qiagen), 5 μL of 10× LA PCR Buffer II (Mg2+ plus), 8 μL of dNTPs (2.5 mM each), 5 μL of mixed forward and reverse primers (10 µM each), 1 μL of template (DNA mass between 10 and 100 ng, calculated with a Qubit broad range kit), and 0.5 µL of TAKARA LA Taq (5 U/µL) (Ozyme, Saint Quentin Yvelines, France). Thermal cycling conditions were as follows: 95°C (2:00) + [95°C (0:15) + 68°C (10:00)] × 30 cycles + 68°C (20:00) + 4°C (∞). PCR amplifications were performed with five pairs of overlapping primers (Mito1/Mito2/Mito3/hmt1/hmt2) (Integrated DNA Technologies, Leuven, Belgium). All PCR amplified products were loaded on a 0.8% agarose gel to check the size of amplicons with reference to the 1Kb plus DNA ladder (Thermo Fischer).

2.16 Mitochondrion staining The 16 000 g pellets (16gP) from both, the plasma without platelet activation (pool from 5 healthy individuals) and from the cell media (2 flask T‐75/ cell line), were stained with 200 nM MitoTracker Green FM (Thermofisher) for 45 minutes, washed twice with PBS, and resuspended in 10 µL of PBS to be visualized by ApoTome microscopy (ZEISS Axio Imager 2). Images were edited with ZEN Software. In parallel, cell culture media pellets (8 T‐75 flask/cell line), Ficoll‐isolated plasma pellet and positive and negative controls were resuspended in 150 µL of PBS and analyzed by Gallios flow cytometry (Beckman Coulter). Mitochondria isolated from cultured cells were used as a positive control for the MitoTracker specificity and as a standard to delineate both the appropriate gate and voltages for the flow cytometry. All sample plots were collected according to size (Forward scatter FSC) and granularity (side scatter SSC) in logarithmic mode. Voltages were adjusted for submicron particles and the flow rate was set at “low.” Doublets were excluded by plotting FSC‐Area vs FSC‐Height, both in logarithmic mode. Collected events in the newly created gate were gated as singlet. Following confirmation that all events recorded in this gate were MitoTracker Green positive, the gate was applied to all samples. Plot histograms were created for both unstained and stained samples. Results were analyzed with Kaluza analysis 1.5 software.

2.17 Western blot Pellets obtained at 8000 g from both the plasma (pool of 5 healthy individuals) and the cell media supernatant (100 flask T‐75/cell line) were resuspended in 50 µL of 1X Laemli buffer, loaded on a 12% Acrylamide/Bis‐Acrylamide gel and transferred onto nitrocellulose membranes (GE Healthcare). Membranes were blocked in 10% milk in 1X PBS‐tween for 1h. Blotting was performed with either 1/500 diluted primary mouse anti‐TOM22 (Sigma, St. Quentin Fallavier Cedex) or with 1/1000 diluted primary mouse anti‐TIM23 (BD Transduction Laboratorie, France) antibodies over‐night at 4°C and further with 1/10 000 diluted HRP‐conjugated secondary Rabbit anti‐mouse antibodies (Merck, Île‐de‐France, France) for 1h at room temperature. Immunoblots were visualized by ECL (Perkin Elmer, Villebon sur Yvette, France).

2.18 Electron microscopy The 8000 g pellets from either cell media (30 flask T‐75/cell line) or plasma prepared without platelet activation (pool from 3 healthy individuals) were immersed in a solution of 2.5% glutaraldehyde in PHEM buffer (1X, pH 7.4) overnight at 4°C, washed in PHEM and post‐fixed in 0.5% osmic acid for 2 hours in the dark at room temperature. Samples were then washed twice in PHEM buffer, dehydrated in a graded series from 30% to 100% ethanol solutions and finally embedded in EmBed 812 using an Automated Microwave Tissue Processor for Electronic Microscopy (Leica EM AMW). 70nm sections (Leica‐Reichert Ultracut E), collected at different levels of each block, were counterstained, with 1.5% uranyl acetate in 70% Ethanol and lead citrate, and observed with a Tecnai F20 transmission electron microscope at 200 kV.

2.19 Metabolic activity Mitochondrial bioenergetics function was determined by oxygen consumption rate (OCR), with a Seahorse XF‐96 extracellular flux analyzer (Agilent). We performed the electron flow assay that allows the functional assessment of selected mitochondrial complexes together in the same period. After isolation, 64 µg of freshly isolated DLD‐1 mitochondria (positive control), and 64 µg of DLD‐1 cell media pellet and plasma pool pellet prepared without platelets activation, were plated in each well in a volume of 25 µL containing MAS 1× (70 mM sucrose, 220 mM mannitol, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM HEPES, 1 mM EGTA, and 0.2% fatty acid‐free BSA, pH 7.2) supplemented with 10 mM pyruvate, 2 mM malate and 4 µM Carbonyl cyanide‐4‐(trifluoromethoxy) phenylhydrazone (FCCP). Note, pyruvate and malate drive respiration via complex I, and FCCP uncouples the mitochondrial function. The XF plate was then centrifuged for 20 minutes at 2000 g at 4°C. After centrifugation, 155 µL of electron flow substrate‐containing 1×MAS was added to each well, and the plate was warmed up in a 37°C non‐CO 2 incubator for 5‐10 minutes. About 10‐fold concentrated compounds were loaded in the ports of the cartridge, using the “loading helper” plate: port A, 20 µL of 20 µM rotenone, a complex I inhibitor (2 µM final); port B, 22 µL of 100 mM succinate, a complex II activator (10 mM final); port C, 24 µL of 40 µM Antimycin A, a complex III inhibitor (4 µM final); port D, 26 µL of 100 mM ascorbate plus 1 mM TMPD, complex IV activator (10 mM and 100 µM final, respectively). After calibration of the cartridge by the XF machine, the XF plate was introduced and the assay continued using Agilent’s protocol for isolated mitochondria (IM).