Phenotype of circulating neutrophils at different stages of cervical neoplasia

At the present time, there is no common point of view to the role of circulating neutrophils (NP) in emergence and development of neoplasia. It is suggested that due to high functional plasticity, the neutrophils may exhibit both pro- and antitumor activity. In order to study the NP phenotype at different stages of cervical neoplasia (CN), we have evaluated their absolute and relative amounts, myeloperoxidase activity, spontaneous and induced NST-test markers, and the level of intracellular cationic proteins. Spontaneous production of elastase and active forms of matrix metalloproteinases, the levels of IL-2, IL-8, IL-18, IFNy, G-CSF were determined in the NP cell lysates and in blood serum. The formation of extracellular traps (NET) was evaluated using 1-day cultures of Saccharomyces cerevisiae as an inducer. We examined 31 patients with cervical intraepithelial neoplasia (CIN) and 21 primary patients with cervical cancer (CC, Ia stage according to FIGO scale), as well as 25 practically healthy women. We revealed increased spontaneous and inducible oxygen-dependent cytolytic and phagocytic activity and spontaneous production of NET if compared to normal values, along with decreased absolute NP numbers in patients with CIN, thus suggesting the antitumor activity of NP. The levels of “pro-tumor” cytokines (MMP-9, IL-2 and G-CSF) become increased over normal levels as early as at the CIN stage, both for the neutrophils and blood plasma. High levels of regulatory IFNy and neutrophil-priming IL-8 in blood plasma do not presume any use of exogenous NP-activating factors at the stage of cervical dysplasia. At the initial stage of cervical cancer, the absolute NP amounts are significantly increased compared to normal counts. However, despite increased spontaneous oxygen-dependent cytolytic activity, the NPs have a significantly reduced activity of phagocytosis and sharply increased spontaneous production of NET, thus, generally, being characteristic to the “pro-tumorous” NP phenotype. IL-2 levels are elevated, and MMP-9 values are still increased in NP and blood plasma of patients with CC (stage Ia). Hence, the obtained results suggest some changes of NP phenotype to a pro-tumorous pattern during transition from intraepithelial dysplasia to cervical cancer. These results allowed us to design an algorithm for examining women with suspected cervical cancer, including IL-2 measurement in blood serum, and MMP-9 amounts in the NP lysates.

1. Berezhnaya N.M. Role of immune system cells in tumor microenvironment. I. Cells and cytokines - the components of inflammation. Oncologiya = Oncology, 2009, no. 1, pp. 6-17. (In Russ.)

2. Gerasimov I.G., Kalutskaya O.A. Kinetics of the reaction of nitroblue tetrazolium reduction by human blood neutrophils. Tsitologiia = Cytology, 2000, Vol. 42, no. 2, pp. 160-165. (In Russ.)

3. Dolgushin I.I., Semenova A.B., Shishkova Yu.S., Kazachkov E.L., Shamanova A.Yu., Vazhenin A.V. Functional activity of neutrophils and processes of formation of extracellular DNA networks after their contact with tumor cells of breast carcinoma. Meditsinskiy vestnik Bashkortostana = Bashkortostan Medical Journal, 2014, Vol. 9, no. 5, pp. 132-135. (In Russ.)

4. Dolgushin I.I., Shishkova Yu.S., Semenova A.B., Kazachkov E.L., Vagenin A.V, Shamanova A.Yu., Dimov G.P. View on the role of neutrophiles extracellular DNA as a component of the microenvironment of tumor in the process of carcinogenesis. Uralskiy meditsinskiy zhurnal = Ural Medical Journal, 2014, no. 2 (116), pp. 19-22. (In Russ.)

5. Maltseva V.N., Avkhacheva N.V., Santalov B.F., Safronova V.G. Dynamic analysis of modification of peripheral neutrophils functional activity and its regulation during tumor growth in vivo. Tsitologiia = Cytology, 2006, Vol. 48, no. 12, pp. 1000-1009. (In Russ.)

6. Medical laboratory diagnostics: programs and algorithms: a guide for doctors. Ed. A.I. Karpischenko. 3rd ed., rev. and enlarged. Moscow: GEOTAR-Media, 2014. 696 p.

7. Nesterova I.V., Kovaleva S.V., Chudilova G.A., Kokov E.A., Lomtatidze L.V., Storozhuk S.V., Uvarov I.B., Kazantseva M.V. The neutrophil phenotype in neoplastic process. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2010, Vol. 4 (13), no. 4, pp. 374-380. (In Russ.)

8. Чердынцева Н.В., Наумов С.А., Удут В.В., Пешкова О.А., Шепеткин И.А. Окислительный метаболизм нейтрофильных гранулоцитов крови при предраке и раке желудка // Биохимия, 1992. № 7. С. 182-187. [Cherdyntseva N.V., Naumov S.A., Udut V.V., Peshkova O.A., Shepetkin I.A. Oxidative metabolism of neutrophilic blood granulocytes in precancer and gastric cancer. Biokhimiya = Biochemistry, 1992, no. 7, pp. 182-187. (In Russ.)]

9. Acuff H.B., Carter K.J., Fingleton B., Gorden D.L., Matrisian L.M. Matrix metalloproteinase-9 from bone marrow-derived cells contributes to survival but not growth of tumor cells in the lung microenvironment. Cancer Res., 2006, Vol. 66, no. 1, pp. 259-266.

10. Albrengues J., Shields M.A., Ng D., Park C.G., Ambrico A., Poindexter M.E., Upadhyay P., Uyeminami D.L., Pommier A., Kuttner V, Bruzas E., Maiorino L., Bautista C., Carmona E.M., Gimotty P.A., Fearon D.T., Chang K., Lyons S.K., Pinkerton K.E., Trotman L.C., Goldberg M.S., Yeh J.T., Egeblad M. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science, 2018, Vol. 361, no. 6409. pii: eaao4227. doi: 10.1126/science.aao4227.

11. Ardi V.C., Kupriyanova T.A., Deryugina E.I., Quigley J.P. Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis. Proc. Natl. Acad. Sci. USA, 2007, Vol. 104, no. 51, pp. 20262-20267.

12. Barth C.R., Funchal G.A., Luft C., de Oliveira J.R., Porto B.N., Donadio M.V. Carrageenan-induced inflammation promotes ROS generation and neutrophil extracellular trap formation in a mouse model of peritonitis. Eur. J. Immunol., 2016, Vol. 46, no. 4, pp. 964-970.

13. Berger-Achituv S., Brinkmann V, Abed U.A., Kuhn L.I., Ben-Ezra J., Elhasid R., Zychlinsky A. A proposed role for neutrophil extracellular traps in cancer immunoediting. Front. Immunol., 2013, Vol. 4, p. 48.

14. Borregaard N., Sorensen O.E., Theilgaard-Mbnch K. Neutrophil granules: a library of innate immunity proteins. Trends Immunol., 2007, Vol. 28, no. 8, pp. 340-345.

15. Bru A., Albertos S., Lopez Garcia-Asenjo J.A., Bru I. Pinning of tumoral growth by enhancement of the immune response. Phys. Rev. Lett., 2004, Vol. 92, no. 23, 238101. doi: 10.1103/PhysRevLett.92.238101.

16. Buckley C.D., Ross E.A., McGettrick H.M., Osborne C.E., Haworth O., Schmutz C., Stone P.C., Salmon M., Matharu N.M., Vohra R.K., Nash G.B., Rainger G.E. Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration. J. Leukoc. Biol., 2006, Vol. 79, no. 2, pp. 303-311.

17. Carmona-Rivera C., Kaplan M.J. Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity. Semin. Immunopathol., 2013, Vol. 35, no. 4, pp. 455-463.

18. Cassatella MA. Neutrophil-derived proteins: selling cytokines by the pound. Adv. Immunol., 1999, Vol. 73, pp. 369-509.

19. Cho H., Hur H.W., Kim S.W., Kim S.H., Kim J.H., Kim Y.T., Lee K. Pre-treatment neutrophil to lymphocyte ratio is elevated in epithelial ovarian cancer and predicts survival after treatment. Cancer Immunol. Immunother., 2009, Vol. 58, no. 1, pp. 15-23.

20. 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. doi: 10.1172/JCI67484.

21. di Carlo E., Forni G., Lollini P., Colombo M.P., Modesti A., Musiani P. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood, 2001, Vol. 97, no. 2, pp. 339-345.

22. di Carlo E., Forni G., Musiani P. Neutrophils in the antitumoral immune response. Chem. Immunol. Allergy, 2003, Vol. 83, pp. 182-203.

23. Donskov F., von der Maase H. Impact of immune parameters on long-term survival in metastatic renal cell carcinoma. J. Clin. Oncol., 2006, Vol. 24, no. 13, pp. 1997-2005.

24. Dumitru C.A., Moses K., Trellakis S., Lang S., Brandau S. Neutrophils and granulocytic myeloid-derived suppressor cells: immunophenotyping, cell biology and clinical relevance in human oncology. Cancer Immunol. Immunother., 2012, Vol. 61, no. 8, pp. 1155-1167.

25. Fridlender Z.G., Sun J., Kim S., Kapoor V., Cheng G., Ling L., Worthen G.S., Albelda S.M. Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus “N2” TAN. Cancer Cell, 2009, Vol. 16, no. 3, pp. 183-194.

26. Fuchs T.A., Abed U., Goosmann C., Hurwitz R., Schulze I., Wahn V., Weinrauch Y., Brinkmann V, Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol., 2007, Vol. 176, no. 2, pp. 231-241.

27. Gregory A.D., Houghton A.M. Tumor-associated neutrophils: new targets for cancer therapy. Cancer Res., 2011, Vol. 71, no. 7, pp. 2411-2416.

28. Gupta A.K., Joshi M.B., Philippova M., Erne P., Hasler P., Hahn S., Resink T.J. Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death. FEBS Lett., 2010, Vol. 584, no. 14, pp. 3193-3197.

29. Ichikawa H., Kokura S., Aw T.Y. Role of endothelial mitochondria in oxidant production and modulation of neutrophil adherence. J. Vasc. Res., 2004, Vol. 41, no. 5, pp. 432-444.

30. Igney F.H., Behrens C.K., Krammer P.H. CD95L mediates tumor counterattack in vitro but induces neutrophil-independent tumor rejection in vivo. Int. J. Cancer, 2005, Vol. 113, no. 1, pp. 78-87.

31. Ishikawa F., Miyazaki S. New biodefense strategies by neutrophils. Arch. Immunol. Ther. Exp. (Warsz), 2005, Vol. 53, no. 3, pp. 226-233.

32. Kim R., Emi M., Tanabe K. Cancer immunoediting from immune surveillance to immune escape. Immunology, 2007, Vol. 121, no. 1, pp. 1-14.

33. Kobayashi A., Greenblatt R.M., Anastos K., Minkoff H., Massad L.S., Young M., Levine A.M., Darragh T.M., Weinberg V., Smith-McCune K.K. Functional attributes of mucosal immunity in cervical intraepithelial neoplasia and effects of HIV infection. Cancer Res., 2004, Vol. 64, no. 18, pp. 6766-6774.

34. Kolaczkowska E., Kubes P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol., 2013, Vol. 13, no. 3, pp. 159-175.

35. Mantovani A., Cassatella M.A., Costantini C., Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol., 2011, Vol. 11, no. 8, pp. 519-531.

36. Masson V, de la Ballina L.R., Munaut C., Wielockx B., Jost M., Maillard C., Blacher S., Bajou K., Itoh T., Itohara S., Werb Z., Libert C., Foidart J.M., Мэё! A. Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes. FASEB J., 2005, Vol. 19, no. 2, pp. 234-236.

37. Mejia S.P., Cano L.E., Lopez J.A., Hernandez O., Gonzalez A. Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis. Microbiology, 2015, Vol. 161, no. 5, pp. 1008-1017.

38. Menegazzi R., Decleva E., Dri P Killing by neutrophil extracellular traps: fact or folklore? Blood, 2012, Vol. 119, no. 5, pp. 1214-1216.

39. Najmeh S., Cools-Lartigue J., Rayes R.F., Gowing S., Vourtzoumis P, Bourdeau F., Giannias B., Berube J., Rousseau S., Ferri L.E., Spicer J.D. Neutrophil extracellular traps sequester circulating tumor cells via p 1-integrin mediated interactions. Int. J. Cancer, 2017, Vol. 140, no. 10, pp. 2321-2330.

40. Nozawa H., Chiu C., Hanahan D. Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proc. Natl. Acad. Sci. USA, 2006, Vol. 103, no. 33, pp. 12493-12498.

41. Ortmann W., Kolaczkowska E. Age is the work of art? Impact of neutrophil and organism age on neutrophil extracellular trap formation. Cell Tissue Res., 2018, Vol. 371, no. 3, pp. 473-488.

42. Otten M.A., Bakema J.E., Tuk C.W., Glennie M.J., Tutt A.L., Beelen R.H., van de Winkel J.G., van Egmond M. Enhanced FcaRI-mediated neutrophil migration towards tumour colonies in the presence of endothelial cells. Eur. J. Immunol., 2012, Vol. 42, no. 7, pp. 1815-1821.

43. Piccard H., Muschel R.J., Opdenakker G. On the dual roles and polarized phenotypes of neutrophils in tumor development and progression. Crit. Rev. Oncol. Hematol., 2012, Vol. 82, no. 3, pp. 296-309.

44. Pillay J., Tak T., Kamp V.M., Koenderman L. Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences. Cell. Mol. Life Sci., 2013, Vol. 70, no. 20, pp. 3813-3827.

45. Saffarzadeh M., Juenemann C., Queisser M.A., Lochnit G., Barreto G., Galuska S.P, Lohmeyer J., Preissner K.T. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS ONE, 2012, Vol. 7, no. 2, e32366. doi: 10.1371/journal.pone.0032366.

46. Sagiv J.Y., Michaeli J., Assi S., Mishalian I., Kisos H., Levy L., Damti P, Lumbroso D., Polyansky L., Sionov R.V, Ariel A., Hovav A.H., Henke E., Fridlender Z.G., Granot Z. Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer. Cell Rep., 2015, Vol. 10, no. 4, pp. 562-573.

47. Schernberg A., Blanchard P, Chargari C., Deutsch E. Neutrophils, a candidate biomarker and target for radiation therapy? Acta Oncol., 2017, Vol. 56, no. 11, pp. 1522-1530.

48. Schmidt H., Suciu S., Punt C.J., Gore M., Kruit W., Patel P, Lienard D., von der Maase H., Eggermont A.M., Keilholz U.; American Joint Committee on Cancer Stage IV Melanoma; EORTC 18951. Pretreatment levels of peripheral neutrophils and leukocytes as independent predictors of overall survival in patients with American Joint Committee on Cancer Stage IV Melanoma: results of the EORTC 18951 Biochemotherapy Trial. J. Clin. Oncol., 2007, Vol. 25, no. 12, pp. 1562-1569.

49. Schmielau J., Finn O.J. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res., 2001, Vol. 61, no. 12, pp. 4756-4760.

50. Sharaiha R.Z., Halazun K.J., Mirza F., Port J.L., Lee PC., Neugut A.I., Altorki N.K., Abrams J.A. Elevated preoperative neutrophil:lymphocyte ratio as a predictor of postoperative disease recurrence in esophageal cancer. Ann. Surg. Oncol., 2011, Vol. 18, no. 12, pp. 3362-3369.

51. Smuda C., Wechsler J.B., Bryce PJ. TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism. Immunol. Lett., 2011, Vol. 141, no. 1, pp. 102-108.

52. Souto J.C., Vila L., Bru A. Polymorphonuclear neutrophils and cancer: intense and sustained neutrophilia as a treatment against solid tumors. Med. Res. Rev., 2011, Vol. 31, no. 3, pp. 311-363.

53. Szuster-Ciesielska A., Hryciuk-Umer E., Stepulak A., Kupisz K., Kandefer-Szerszen M. Reactive oxygen species production by blood neutrophils of patients with laryngeal carcinoma and antioxidative enzyme activity in their blood. Acta Oncol., 2004, Vol. 43, no. 3, pp. 252-258.

54. Tavares-Murta B.M., Mendonqa M.A., Duarte N.L., da Silva J.A., Mutao T.S., Garcia C.B., Murta E.F. Systemic leukocyte alterations are associated with invasive uterine cervical cancer. Int. J. Gynecol. Cancer, 2010, Vol. 20, no. 7, pp. 1154-1159.

55. Tazawa H., Okada F., Kobayashi T., Tada M., Mori Y., Une Y., Sendo F., Kobayashi M., Hosokawa M. Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells: implication of inflammation-associated carcinogenesis and tumor progression. Am. J. Pathol., 2003, Vol. 163, no. 6, pp. 2221-2232.

56. Teramukai S., Kitano T., Kishida Y., Kawahara M., Kubota K., Komuta K., Minato K., Mio T., Fujita Y., Yonei T., Nakano K., Tsuboi M., Shibata K., Furuse K., Fukushima M. Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00-03. Eur. J. Cancer, 2009, Vol. 45, no. 11, pp. 1950-1958.

57. Trellakis S., Farjah H., Bruderek K., Dumitru C.A., Hoffmann T.K., Lang S., Brandau S. Peripheral blood neutrophil granulocytes from patients with head and neck squamous cell carcinoma functionally differ from their counterparts in healthy donors. Int. J. Immunopathol. Pharmacol., 2011, Vol. 24, no. 3, pp. 683-693.

58. Uribe-Querol E., Rosales C. Neutrophils in cancer: two sides of the same coin. J. Immunol. Res., 2015, Vol. 2015, 983698. doi: 10.1155/2015/983698.

59. van Spriel A.B., Leusen J.H., van Egmond M., Dijkman H.B., Assmann K.J., Mayadas T.N., van de Winkel J.G. Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation. Blood, 2001, Vol. 97, no. 8, pp. 2478-2486.

60. Yipp B.G., Petri B., Salina D., Jenne C.N., Scott B.N., Zbytnuik L.D., Pittman K., Asaduzzaman M., Wu K., Meijndert H.C., Malawista S.E., de Boisfleury Chevance A., Zhang K., Conly J., Kubes P Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med., 2012, Vol. 18, no. 9, pp. 1386-1393.

61. Zivkovic M., Poljak-Blazi M., Egger G., Sunjic S.B., Schaur R.J., Zarkovic N. Oxidative burst and anticancer activities of rat neutrophils. Biofactors, 2005, Vol. 24, no. 1-4, pp. 305-312.