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

Malkin, Ethan Z; Michino, Steven De; Lambie, Meghan; Gill, Rita G; Zhao, Zhen; Rostami, Ariana; Arruda, Andrea; Minden, Mark D.; Bratman, Scott V.

doi:10.1172/jci.insight.159590

Cited by 7 publications

(7 citation statements)

References 67 publications

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“…Another possible factor in the discrepancy could have been selection of patients with high mutational burden in previous studies and large plasma volumes (up to 12 mL) used to isolate evDNA [ 15 ], which would make mutations in evDNA easier to detect. In line with our results, however, are recent studies suggesting that only a small percentage (<1%) of sEVs from patients with cancer contain DNA [ 25 ], and that DNA is rarely associated with EVs [ 26 ]. Recently, data from a seminal study also showed that exosomes, the EVs thought to carry DNA, do not contain any DNA at all [ 17 ].…”

Section: Discussionsupporting

confidence: 93%

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer

Lapin,

Tjensvoll,

Nedrebø

et al. 2023

PLoS ONE

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Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer.

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Section: Discussionsupporting

confidence: 93%

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer

Lapin,

Tjensvoll,

Nedrebø

et al. 2023

PLoS ONE

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“…This means that most of the cir-mtDNA are associated with structures whose size is over 0.22 µm. Note, our observations point to the existence of particles containing mtDNA in the circulation, as it has been previously indicated by plasma filtrates (Chiu et al, 2003;Malkin et al, 2022). In addition, our data agree with Arance-Criado's observation (Arance-Criado et al, 2020), in HI, of a 99.4% decrease of cir-mtDNA following the HS step using a protocol equivalent to the cirDNA-SPP (Supplementary Table S4A).…”

Section: Discussionsupporting

confidence: 91%

“…Thus, when taking together fragmentomics and plasma fractionation data, we infer that a fraction, at least, of EVs contains mitochondrial full length circular DNA or mitochondria particles that could be internally or externally associated with EVs. Thus, we do not preclude the possibility that mitochondria particle free mtDNA may exist in blood circulation in association with EVs, as reported previously ( Malkin et al, 2022 ). However, its amount corresponds to a minor fraction of the total cir-mtDNA.…”

Section: Discussionmentioning

confidence: 72%

“…Aside from circulating cell-free mitochondria (cir-exMT) origin ( Al Amir Dache et al, 2020 ), plasma-derived mtDNA could originate from other structures: remaining cells, in association with cell debris or membranes, mitochondria, or EVs ( Malkin et al, 2022 ). While exosomes, and apoptotic bodies could be considered as EVs, we arbitrarily differentiated here four types of vesicles: large EVs, small EVs, exosomes, and apoptotic bodies.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA.Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures.Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%).Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

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“…Mitochondrial DNA showed increases over time, indicating that the release of DNA from these organelles by the lysis buffer does not occur as quickly as the cellular genomic DNA. Moreover, a stable contribution from distinct pools of mtDNA might be important for liquid biopsy applications [ 27 – 29 ]. While it is possible that some contribution of non-genomic DNA may also arise from exosomes and other extracellular vesicles, due to the presence of lysis buffer the ability to delineate these sources by standard techniques is not possible [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

Isolation of salivary cell-free DNA for cancer detection

et al. 2023

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Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA– a bona fide cancer biomarker in a subset of head and neck cancers– from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

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

Cited by 7 publications

References 67 publications

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer

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

Isolation of salivary cell-free DNA for cancer detection

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