Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer.
Circulating cell-free DNA
Citrullinated Histone H3
DNA cfDNA fragmentation pattern
DNA size distribution
Liquid biopsy
NETosis
NETs
Neutrophil Elastase
Tumor induced systemic effects
Journal
Journal of experimental & clinical cancer research : CR
ISSN: 1756-9966
Titre abrégé: J Exp Clin Cancer Res
Pays: England
ID NLM: 8308647
Informations de publication
Date de publication:
21 Apr 2022
21 Apr 2022
Historique:
received:
20
10
2021
accepted:
06
04
2022
entrez:
22
4
2022
pubmed:
23
4
2022
medline:
26
4
2022
Statut:
epublish
Résumé
Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC). The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen. Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.
Sections du résumé
BACKGROUND
BACKGROUND
Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).
METHODS
METHODS
The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades.
RESULTS
RESULTS
We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.
CONCLUSION
CONCLUSIONS
Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.
Identifiants
pubmed: 35449078
doi: 10.1186/s13046-022-02359-5
pii: 10.1186/s13046-022-02359-5
pmc: PMC9027343
doi:
Substances chimiques
Biomarkers
0
Cell-Free Nucleic Acids
0
Histones
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
151Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2022. The Author(s).
Références
Borissoff JI, Joosen IA, Versteylen MO, Brill A, Fuchs T, Savchenko AS, et al. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arterioscler Thromb Vasc Biol. 2013;33(8):2032–40. https://doi.org/10.1161/ATVBAHA.113.301627 .
doi: 10.1161/ATVBAHA.113.301627
pubmed: 23818485
pmcid: 3806482
Yang R, Zou X, Tenhunen J, Tønnessen TI. HMGB1 and Extracellular Histones Significantly Contribute to Systemic Inflammation and Multiple Organ Failure in Acute Liver Failure. Mediators Inflamm. 2017;2017:5928078. https://doi.org/10.1155/2017/5928078 .
van Montfoort ML, Stephan F, Lauw MN, Hutten BA, Van Mierlo GJ, Solati S, et al. Circulating nucleosomes and neutrophil activation as risk factors for deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2013;33(1):147–51. https://doi.org/10.1161/ATVBAHA.112.300498 .
doi: 10.1161/ATVBAHA.112.300498
pubmed: 23104849
Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A. 2012;109(32):13076–81. https://doi.org/10.1073/pnas.1200419109 Epub 2012 Jul 23.
doi: 10.1073/pnas.1200419109
pubmed: 22826226
pmcid: 3420209
Cicchillitti L, Corrado G, De Angeli M, Mancini E, Baiocco E, Patrizi L, et al. Circulating cell-free DNA content as blood based biomarker in endometrial cancer. Oncotarget. 2017;8(70):115230–43. https://doi.org/10.18632/oncotarget.23247 .
doi: 10.18632/oncotarget.23247
pubmed: 29383155
pmcid: 5777767
Vano YA, Oudard S, By MA, Têtu P, Thibault C, Aboudagga H, et al. Optimal cut-off for neutrophil-to-lymphocyte ratio: fact or fantasy? a prospective cohort study in metastatic cancer patients. PLoS ONE. 2018;13(4):e0195042. https://doi.org/10.1371/journal.pone.0195042 .
doi: 10.1371/journal.pone.0195042
pubmed: 29624591
pmcid: 5889159
Ethier JL, Desautels DN, Templeton AJ, Oza A, Amir E, Lheureux S. Is the neutrophil-to-lymphocyte ratio prognostic of survival outcomes in gynecologic cancers? a systematic review and meta-analysis. Gynecol Oncol. 2017;145(3):584–94. https://doi.org/10.1016/j.ygyno.2017.02.026.Review .
doi: 10.1016/j.ygyno.2017.02.026.Review
pubmed: 28222899
Olsson AK, Cedervall J. NETosis in cancer - platelet-neutrophil crosstalk promotes tumor-associated pathology. Front Immunol. 2016;7:373 Review.
doi: 10.3389/fimmu.2016.00373
pubmed: 27708646
pmcid: 5030622
Demers M, Wagner DD. Neutrophil extracellular traps: a new link to cancer-associated thrombosis and potential implications for tumor progression. Oncoimmunology. 2013;2(2):e22946.
doi: 10.4161/onci.22946
pubmed: 23526174
pmcid: 3601165
Guglietta S, Chiavelli A, Zagato E, Krieg C, Gandini S, Ravenda PS, et al. Coagulation induced by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis. Nat Commun. 2016;7:11037. https://doi.org/10.1038/ncomms11037 .
doi: 10.1038/ncomms11037
pubmed: 26996437
pmcid: 4802169
Mauracher L-M, Posch F, Martinod K, Grilz E, Däullary T, Hell L, Brostjan C, Zielinski C, Ay C, Wagner DD, Pabinger I, Thaler J. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. J Thromb Haemost. 2018;16:508–18.
doi: 10.1111/jth.13951
pubmed: 29325226
pmcid: 6294121
Thalin C, Lundström S, Seignez C, Daleskog M, Lundström A, Henriksson P, et al. Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer. PLoS One. 2018;13(1):e0191231. https://doi.org/10.1371/journal.pone.0191231 .
doi: 10.1371/journal.pone.0191231
pubmed: 29324871
pmcid: 5764486
Grilz E1, Mauracher LM1, Posch F2, Königsbrügge O1, Zöchbauer-Müller S3, Marosi C3, Lang I4, Pabinger I1, Ay C1. Citrullinated histone H3, a biomarker for neutrophil extracellular trap formation, predicts the risk of mortality in patients with cancer. Br J Haematol. 2019 Apr 9. https://doi.org/10.1111/bjh.15906 .
Ronchetti L, Boubaker NS, Barba M, Vici P, Gurtner A, Piaggio G. Neutrophil extracellular traps in cancer: not only catching microbes. J Exp Clin Cancer Res. 2021;40(1):231. https://doi.org/10.1186/s13046-021-02036-z .
doi: 10.1186/s13046-021-02036-z
pubmed: 34261496
pmcid: 8281578
Boeltz S, Amini P, Anders HJ, Andrade F, Bilyy R, Chatfield S, et al. To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps. Cell Death Differ. 2019;26(3):395–408. https://doi.org/10.1038/s41418-018-0261-x . Epub 2019 Jan 8. Review. PubMed PMID: 30622307; PubMed Central PMCID: PMC6370810.
Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141(1):52–67. https://doi.org/10.1016/j.cell.2010.03.015.Review .
doi: 10.1016/j.cell.2010.03.015.Review
pubmed: 20371345
pmcid: 2862057
Cools-Lartigue J, Spicer J, McDonald B, Gowing S, Chow S, Giannias B, Bourdeau F, Kubes P, Ferri L. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest. 2013;pii: 67484. https://doi.org/10.1172/JCI67484 .
Cedervall J, Zhang Y, Olsson AK. Tumor-Induced NETosis as a risk factor for metastasis and organ failure. Cancer Res. 2016;76(15):4311–5. https://doi.org/10.1158/0008-5472.CAN-15-3051 .
doi: 10.1158/0008-5472.CAN-15-3051
pubmed: 27402078
Park J, Wysocki RW, Amoozgar Z, Maiorino L, Fein MR, Jorns J, et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Sci Transl Med. 2016;8(361):361ra138.
doi: 10.1126/scitranslmed.aag1711
pubmed: 27798263
pmcid: 5550900
Kanamaru R, Ohzawa H, Miyato H, Matsumoto S, Haruta H, Kurashina K, et al. Low density neutrophils (LDN) in postoperative abdominal cavity assist the peritoneal recurrence through the production of neutrophil extracellular traps (NETs). Sci Rep. 2018;8(1):632. https://doi.org/10.1038/s41598-017-19091-2 .
doi: 10.1038/s41598-017-19091-2
pubmed: 29330531
pmcid: 5766579
Rayes RF, Mouhanna JG, Nicolau I, Bourdeau F, Giannias B, Rousseau S, Quail D, Walsh L, Sangwan V, Bertos N, Cools-Lartigue J, Ferri LE, Spicer JD. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects. JCI Insight. 2019;5(16):e128008. https://doi.org/10.1172/jci.insight.128008 PMID:31343990;PMCID:PMC6777835.
doi: 10.1172/jci.insight.128008
pubmed: 31343990
Masucci MT, Minopoli M, Del Vecchio S. Carriero 344 MV the emerging role of Neutrophil Extracellular Traps (NETs) in tumor progression and metastasis. Front Immunol. 2020;16(11):1749.
doi: 10.3389/fimmu.2020.01749
Cedervall J, Dragomir A, Saupe F, Zhang Y, Ärnlöv J, Larsson E, Dimberg A, Larsson A, Olsson AK. Pharmacological targeting of peptidylarginine deiminase 4 prevents cancer-associated kidney injury in mice. Oncoimmunology. 2017;6(8):e1320009.
doi: 10.1080/2162402X.2017.1320009
pubmed: 28919990
pmcid: 5593702
Inoue M, Nakashima R, Enomoto M, Koike Y, Zhao X, Yip K, Huang SH, Waldron JN, Ikura M, Liu FF, Bratman SV. Plasma redox imbalance caused by albumin oxidation promotes lung predominant NETosis and pulmonary cancer metastasis. Nat Commun. 2018;9(1):5116. 480. https://doi.org/10.1038/s41467-018-07550-x .
Teijeira Á, Garasa S, Gato M, Alfaro C, Migueliz I, Cirella A, de Andrea C, Ochoa MC, Otano I, Etxeberria I, Andueza MP, Nieto CP, Resano L, Azpilikueta A, Allegretti M, de Pizzol M, Ponz-Sarvisé M, Rouzaut A, Sanmamed MF, Schalper K, Carleton M, Mellado M, Rodriguez-Ruiz ME, Berraondo P, Perez-Gracia JL, Melero I. CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity. Immunity. 2020;52(5):856-871.e8. https://doi.org/10.1016/j.immuni.2020.03.001 Epub 2020 Apr 13 PMID: 32289253.
doi: 10.1016/j.immuni.2020.03.001
pubmed: 32289253
Margraf S, Logters T, Reipen J, Altrichter J, Scholz M, Windolf J. Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock. 2008;30:352–8. https://doi.org/10.1097/SHK.0b013e31816a6bb1 .
doi: 10.1097/SHK.0b013e31816a6bb1
pubmed: 18317404
Paunel-Görgülü A, Wacker M, El Aita M, Hassan S, Schlachtenberger G, Deppe A, Choi YH, Kuhn E, Mehler TO, Wahlers T. cfDNA correlates with endothelial damage after cardiac surgery with prolonged cardiopulmonary bypass and amplifies NETosis in an intracellular TLR9-independent manner. Sci Rep. 2017;7(1):17421. https://doi.org/10.1038/s41598-017-17561-1 .
doi: 10.1038/s41598-017-17561-1
pubmed: 29234042
pmcid: 5727170
Vizza E, Corrado G, De Angeli M, Carosi M, Mancini E, Baiocco E, et al. Serum DNA integrity index as a potential molecular biomarker in endometrial cancer. J Exp Clin Cancer Res. 2018;37(1):16. https://doi.org/10.1186/s13046-018-0688-4 Erratum.In:JExpClinCancerRes.2018,37(1):35.
doi: 10.1186/s13046-018-0688-4
pubmed: 29382392
pmcid: 5791183
Rivera-Franco MM, Leon-Rodriguez E, Torres-Ruiz JJ, Gómez-Martín D, Angles-Cano E, de la Luz S-G. Neutrophil extracellular traps associate with clinical stages in breast cancer. Pathol Oncol Res. 2020;26(3):1781–5. https://doi.org/10.1007/s12253-019-00763-5 .
doi: 10.1007/s12253-019-00763-5
pubmed: 31656990
Zhang Y, Hu Y, Ma C, Sun H, Wei X, Li M, Wei W, Zhang F, Yang F, Wang H, Gu K. Diagnostic, therapeutic predictive, and prognostic value of neutrophil extracellular traps in patients with gastric adenocarcinoma. Front Oncol. 2020;10:1036. https://doi.org/10.3389/fonc.2020.01036 . eCollection 2020. PMID: 32714865
Bronkhorst AJ, Ungerer V, Holdenrieder S. The emerging role of cell-free DNA as a molecular marker for cancer management. Biomol Detect Quantif. 2019;18(17):100087. https://doi.org/10.1016/j.bdq.2019.100087 PMID:30923679;PMCID:PMC6425120.
doi: 10.1016/j.bdq.2019.100087
Sanchez C, Roch B, Mazard T, Blache P, Dache ZAA, Pastor B, Pisareva E, Tanos R, Thierry AR. Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics. JCI Insight. 2021;6(7):e144561. https://doi.org/10.1172/jci.insight.144561 PMID:33571170;PMCID:PMC8119211.
doi: 10.1172/jci.insight.144561
pubmed: 33571170
pmcid: 8119211
Lamminaho M, Kujala J, Peltonen H, Tengström M, Kosma VM, Mannermaa A. High cell-free DNA integrity is associated with poor breast cancer survival. Cancers (Basel). 2021;13(18):4679. https://doi.org/10.3390/cancers13184679 PMID:34572906;PMCID:PMC8467852.
doi: 10.3390/cancers13184679
pubmed: 34572906
Ivanov M, Baranova A, Butler T, Spellman P, Mileyko V. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation. BMC Genomics. 2015;16 Suppl 13(Suppl 13):S1. https://doi.org/10.1186/1471-2164-16-S13-S1 . Epub 2015 Dec 16. PMID: 26693644; PMCID: PMC4686799.
Ma X, Zhu L, Wu X, Bao H, Wang X, Chang Z, Shao YW, Wang Z. Cell-Free DNA provides a good representation of the tumor genome despite its biased fragmentation patterns. PLoS One. 2017;12(1):e0169231. https://doi.org/10.1371/journal.pone.0169231 PMID:28046008;PMCID:PMC5207727.
doi: 10.1371/journal.pone.0169231
pubmed: 28046008
pmcid: 5207727
Zukowski A, Rao S, Ramachandran S. Phenotypes from cell-free DNA. Open Biol. 2020;10(9):200119. https://doi.org/10.1098/rsob.200119 . Epub 2020 Sep 2. PMID: 32873154; PMCID: PMC7536073.
Ungerer V, Bronkhorst AJ, Van den Ackerveken P, Herzog M, Holdenrieder S. Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures. Sci Rep. 2021;11(1):9460. https://doi.org/10.1038/s41598-021-88866-5 PMID:33947882;PMCID:PMC8096822.
doi: 10.1038/s41598-021-88866-5
pubmed: 33947882
pmcid: 8096822
Fossati G, Moulding DA, Spiller DG, Moots RJ, White MR. Edwards SW The mitochondrial network of human neutrophils: role in chemotaxis, phagocytosis, respiratory burst activation, and commitment to apoptosis. J Immunol. 2003;170(4):1964–72.
doi: 10.4049/jimmunol.170.4.1964
pubmed: 12574365
Leshner M, Wang S, Lewis C, Zheng H, Chen XA, Santy L, Wang Y. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol. 2012;4(3):307. https://doi.org/10.3389/fimmu.2012.00307 PMID:23060885;PMCID:PMC3463874.
doi: 10.3389/fimmu.2012.00307
Allis CD, Jenuwein T. The molecular hallmarks of epigenetic control. Nat Rev Genet. 2016;17(8):487–500. https://doi.org/10.1038/nrg.2016.59 Epub 2016 Jun 27 PMID: 27346641.
doi: 10.1038/nrg.2016.59
pubmed: 27346641
Han DSC, Lo YMD. The Nexus of cfDNA and Nuclease Biology. Trends Genet. 2021;37(8):758–70. https://doi.org/10.1016/j.tig.2021.04.005 Epub 2021 May 15 PMID: 34006390.
doi: 10.1016/j.tig.2021.04.005
pubmed: 34006390
Lazzaretto B, Fadeel B. Intra- and Extracellular Degradation of Neutrophil Extracellular Traps by Macrophages and Dendritic Cells. Journal of Immunology (Baltimore, Md. : 1950). 2019;203(8):2276–2290. https://doi.org/10.4049/jimmunol.1800159 . PMID: 31519860; PMCID: PMC6778307.
Pastor B, Abraham JD, Pisareva E, Sanchez C, Kudriavstev A, Tanos R, Mirandola A, Mihalovičová L, Pezzella V, Adenis A, Ychou M, Mazard T, Thierry AR. Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer. iScience. 2022;25(2):103826. https://doi.org/10.1016/j.isci.2022.103826 . PMID: 35198886; PMCID: PMC8844218.