DNA-Containing Extracellular Structures of Tumor Cells Inhibit the Formation of Neutrophil Extracellular Traps in vitro

Aim. To study the parameters of formed DNA-containing extracellular structures during co-cultivation of neutrophils from healthy donors, HСT116 adenocarcinoma cells and K562 myeloblastoma.
Materials and methods. Erythrocyte sedimentation in EDTA-treated blood was carried out using Dextran 500. The neutrophil-enriched layer of blood plasma was collected. The admixture of mononuclear cells was less than 1%. Platelets were removed using differential centrifugation. Isolated neutrophils in RPMI-1640 medium were used in short-term culture experiments. HCT116 adenocarcinoma and K562 myeloblastoma cells were obtained from the American Type Culture Collection. In the experiments, donor neutrophils and tumor cells were co-cultivated for 3 hours. SYBR Green fluorescence microscopy was used to visualize the DNA-containing extracellular structures formation by cells cultured in RPMI-1640 medium.
Results. Neutrophils recognize tumor cells and respond to contact interactions, forming neutrophil extracellular traps in the form of neutrophil networks. Contacts with HCT116 adenocarcinoma cells cause rapid formation of neutrophil web-like structures – within 1 hour. The opening of neutrophil web-like structures induced by contacts with K562 myeloblastoma cells requires a longer incubation (2 hours). HCT116 cells form large bundles of DNAcontaining fibers, which completely inhibit the formation of neutrophil networks. K562 cells suppress neutrophil defense responses by reducing the number and size of neutrophil networks. The effect of inhibition of neutrophil networks by K562 cells is probably due to the action of a soluble factor that suppresses neutrophil functions described earlier.
Conclusion. The study shows that both tumor cell lines are capable of suppressing innate immune cell responses through different mechanisms. Adenocarcinoma cells inhibit neutrophil network formation upon direct contact due to the large size DNA-containing fibers they produce. Myeloblastoma cells produce the same effect, probably acting by secreting humoral factors.

1. Masucci M.T., Minopoli M., Del Vecchio S., Carriero M.V. The emerging role of neutrophil extracellular traps (NETs) in tumor progression and metastasis. Front. Immunol. 2020;11:1749. DOI: 10.3389/fimmu.2020.01749.

2. Rayes R.F., Mouhanna J.G., Nicolau I., Bourdeau F., Giannias B., Rousseau S. et al. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects. JCI Insight. 2019;5(16):e128008. DOI: 10.1172/jci.insight.128008.

3. Munir H., Jones J.O., Janowitz T., Hoffmann M., Euler M., Martins C.P. et al. Stromal-driven and Amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth. Nature Communications. 2021;12(1):683. DOI: 10.1038/s41467-021-20982-2.

4. Dzobo K. Taking a full snapshot of cancer biology: deciphering the tumor microenvironment for effective cancer therapy in the oncology clinic. OMICS. 2020;24(4):175–179. DOI: 10.1089/omi.2020.0019.

5. Khalaf K., Hana D., Chou J.T., Singh C., Mackiewicz A., Kaczmarek M. Aspects of the tumor microenvironment involved in immune resistance and drug resistance. Front. Immunol. 2021;12:656364. DOI: 10.3389/fimmu.2021.656364.

6. Balta E., Wabnitz G.H., Samstag Y. Hijacked immune cells in the tumor microenvironment: molecular mechanisms of immunosuppression and cues to improve t cell-based immunotherapy of solid tumors. Int. J. Mol. Sci. 2021;22(11):5736. DOI: 10.3390/ijms22115736.

7. Gonzalez-Aparicio M., Alfaro C. Influence of interleukin-8 and neutrophil extracellular trap (NET) formation in the tumor microenvironment: is there a pathogenic role? J. Immunol. Res. 2019;2019:6252138. DOI: 10.1155/2019/6252138.

8. Mousset A., Bellone L., Gaggioli C., Albrengues J. NETscape or NEThance: tailoring anti-cancer therapy. Trends Cancer. 2024;10(7):655–667. DOI: 10.1016/j.trecan.2024.03.007.

9. Mousset A., Albrengues J. Neutrophil extracellular traps modulate chemotherapy efficacy and its adverse side effects. Biol. Cell. 2024;116(7):e2400031. DOI: 10.1111/boc.202400031.

10. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И., Рогожина Л.С., Ступин В.А. и др. Нейтрофильные экстраклеточные ловушки при воспалительных заболеваниях брюшной полости. Патологическая физиология и экспериментальная терапия. 2024;68(1):15–25. DOI: 10.25557/0031-2991.2024.01.15-25.

11. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И., Ступин В.А., Ким А.Э. и др. Противоинфекционная защита организма человека с участием нейтрофильных сетей. Бюллетень сибирской медицины. 2024;23(1):56–63. DOI: 10.20538/1682-0363-2024-1-56-63.

12. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И. Новые возможности диагностики и исследования патогенеза различных видов воспаления. Патологическая физиология и экспериментальная терапия. 2022;66(2):34–42. DOI: 10.25557/0031-2991.2022.02.34-42.

13. Yazdani H.O., Roy E., Comerci A.J., van der Windt D.J., Zhang H., Huang H. et al. Neutrophil extracellular traps drive mitochondrial homeostasis in tumors to augment growth. Cancer Research. 2019;79(21):5626–5639. DOI: 10.1158/0008-5472.CAN-19-0800.

14. Wang W.W., Wu L., Lu W., Chen W., Yan W., Qi C. et al. Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to the induction of neutrophil extracellular traps after curative resection. Journal of Cancer Research and Clinical Oncology. 2021;147(9):2609–2619. DOI: 10.1007/s00432-021-03682-8.

15. Amar M., Amit N., Scoazec J.Y., Pasquier C., Babin-Chevaye C., Huu T.P. et al. K562 cells produce an anti-inflammatory factor that inhibits neutrophil functions in vivo. Blood. 1992;80(6):1546–1552.