Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer

Pastor, Brice; Abraham, Jean-Daniel; Pisareva, Ekaterina; Sanchez, Colline; Kudriavstev, Andrei; Tanos, Rita; Mirandola, Alexia; Mihalovičová, Lucia; Pezzella, Veronica; Adenis, Antoine; Ychou, Marc; Mazard, Thibault; Thierry, Alain R.

doi:10.1016/j.isci.2022.103826

Cited by 18 publications

(23 citation statements)

References 72 publications

(104 reference statements)

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“…S3). In previous studies, we have already described the decrease of MNR [ 26 , 35 , 47 ] and the increase of NE and MPO in cancer plasma, as compared to healthy individuals [ 26 ]. We believe this to be linked to the activation of neutrophils and the release of NET and could be explained by the structural differences between nuclear and mitochondrial DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Specifically, we believe that chromatin fragments, oligonucleosomes, and nucleosomes can be liberated by the process of NET degradation which occurs during inflammation and that these contribute to the pool of cirDNA and histones in blood [ 25 ]. We have already demonstrated the association of NET formation with cirDNA production in metastatic colorectal cancer [ 26 ], and we also postulate that NET have a key role in COVID-19 pathogenesis [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

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Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

et al. 2022

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Background As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. Methods We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). Results Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); “ex vivo” NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1–10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. Conclusions Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

et al. 2022

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“…S4). In previous studies, we have already described the decrease of MNR [26,35,45] and the increase of NE and MPO in cancer plasma, as compared to healthy individuals [26]. We believe this to be linked to the activation of neutrophils and the release of NET, and could be explained by the structural differences between nuclear and mitochondrial DNA.…”

Section: Discussionmentioning

confidence: 87%

“…Some authors even postulate that NET are a seedbed for metastasis [86]. We recently demonstrated the association of MPO and NE concentrations with the amounts of cirDNA and anti-cardiolipin auto-antibodies in a large cohort (N=217) of newly diagnosed metastatic colorectal cancer (mCRC) patients [26]. In this current study, we clearly confirmed this observation in a small group of mCRC patients, in whom we found a statistically significant higher level of NE, MPO and cirDNA plasma concentration, as compared to healthy individuals.…”

Section: Discussionmentioning

confidence: 99%

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

Pisareva

Mihalovičová

Pastor

et al. 2022

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Background: Because circulating DNA (cirDNA) are mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA Methods: We used the synergistic analytical information provided by specifically quantifying DNA by qPCR, and analyzing fragment size analysis by shallow WGS, and capillary electrophoresis to unequivocally study the following: the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; in vitro NET degradation in serum. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N= 28; systemic lupus erythematosus, N= 10; metastatic colorectal cancer, N= 10; and from healthy individuals, N= 114). Results: Our studies reveal that: gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24H incubation in serum corresponded to mono-N DNA); ex vivo NET formation, as demonstrated by a concurrent 5-, 5- and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2% and 41% of the remaining DNA after 2 hours in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO and cirDNA was detected in plasma samples from lupus, COVID-19 and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. Conclusions: Our work thus describes the mechanisms by which NET and cirDNA are linked, by demonstrating that NET are a major source of mono-N cirDNA independent of apoptosis, and thus establishing a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions, as well as demonstrating a link between immune response and cirDNA.

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“…In a recent study, researchers were able to distinguish between patients with metastatic colorectal cancer and healthy individuals based on cfDNA, MPO, and neutrophil elastase (NE) concentrations. It was further suggested that cfDNA levels, in combination with MPO-DNA and NE, could serve as potential biomarkers for disease severity, prognosis or treatment guidance of metastatic colorectal cancer and cancer in general [ 395 ]. Furthermore, correlation of the markers mentioned above with levels of autoantibodies, e.g., anti-dsDNA, may give insight into autoimmune activity [ 396 ].…”

Section: Potential Pathological Effects and Biological Functions Of C...mentioning

confidence: 99%

New Perspectives on the Importance of Cell-Free DNA Biology

et al. 2022

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Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research.

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Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer

Cited by 18 publications

References 72 publications

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

New Perspectives on the Importance of Cell-Free DNA Biology

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