Histological and immunohistochemical evaluation of tissues from immunodeficient mice with of tumor xenografts following administration of human NK cells

Immuno-oncology is an actively developing area of applied research that has revolutionized cancer treatment. In particular, NK cells are classified as cytotoxic lymphocytes that play a crucial role in recognizing and eliminating malignant or infected cells. The aim of the study was to perform a histological and immunohistochemical  evaluation  of  human  tumor  xenografts  (human  pancreatic cancer) and internal organs of immunodeficient mice following the administration of a human NK cell suspension. Material and methods. Previously, xenografts were modeled on 4 NBSGW mice by subcutaneous administration of ASPC-1 tumor cells (human pancreatic cancer). On day 24 of the study, the animals were injected with NK cell suspension intravenously in the amount of 8.8 million and 9.2 million to mice №2 and №4, and subcutaneously in the amount of 10 million and 14 million NK cells to mice №1 and №3, respectively. On day 28 of the study, the animals were euthanized and pathomorphologically examined. Routine histological assessment of the internal organs of the mice, immunohistochemistry of xenograft samples for the presence of tumor cells with Cytokeratin 7 and Pan-Cytokeratin antibodies, as well as xenografts and internal organs of mice for the presence of CD56+ cells were performed. Results. The histoarchitecture of organs and tissues in animals was preserved, metastases were found in the lungs, no infiltration of CD56+ cells in this area of the lung tissue was noted. Single weakly positive CD56+ cells were observed in the xenografts, the membrane staining of which was barely visible. In the spleen, CD56+ cells were single, and part of the xenograft and spleen vessels (peripheral nerve fibers, pericytes) were also brightly stained. In the xenograft samples, small-droplet dystrophy of oncocytes, inflammatory infiltration and the cell renewal index were visualized less than or equal to 1.0, which together indicates mild changes in the tumor structure. Conclusion. The therapeutic effect of the NK cell suspension increases tumor pathomorphism, the areas of necrosis increase, while the cell renewal coefficient index decreases, but further studies are needed with an increase in the dose or frequency of administration.

1. Abakushina EV, Kuz'mina EG, Kovalenko EI. Osnovnye svoistva i funktsii NK-kletok cheloveka. Immunologiya. 2012;33 (4):220–224. (In Russ.).

2. Artem'eva AS, Murashkina AA, Rogachev MV. Immunogistokhimiya: osnovy, metodicheskie podkhody, gruppy markerov: Uchebnoe posobie. SPb.: 2020. 76. (In Russ.).

3. Aldushkina YuV, Anisimova NYu, Gritsai AN, Novruzov KM, Dolzhikova YuI, Odaryuk OA, et. al. Klinicheskaya znachimost' obnaruzheniya mikrometastazov v limfaticheskikh uzlakh pri rake yaichnikov. Rossiiskii bioterapevticheskii zhurnal. 2023;22(2):20–26. (In Russ.). doi: 10.17650/1726-9784-2023-22-2-20-26.

4. Boldyshevskaya MA, Tashireva LA, Andryukhova ES, Dronova TA, Vtorushin SV, Perel'muter VM. Nestrukturnaya rol' tsitokeratinov pri zlokachestvennykh novoobrazovaniyakh. Uspekhi molekulyarnoi onkologii. 2023;10:76–84. (In Russ.). doi: 10.17650/2313-805X-2023-10-4-76-85.

5. Borobova EA, Zheravin AA. Natural'nye killery v immunoterapii onkologicheskikh zabolevanii. Sibirskii onkologicheskii zhurnal. 2018;17(6):97-104. (In Russ.). doi: 10.21294/1814-4861-2018-17-6-97-104.

6. Gurevich LE, Kazantseva IA, Korsakova NA, Bobrov MA. Znachenie immunogistokhimii dlya targetnoi terapii i prognozirovaniya v onkologii. Al'manakh klinicheskoi meditsiny. 2012;27:27–34. (In Russ.).

7. Gurevich LE, Korsakova NA, Voronkova IA, Kazantseva IA, Ashevskaya VE, Titov AG, et. al. Prognosticheskoe i differentsial'no- diagnosticheskoe znachenie ekspressii tsitokeratinov 7 i 19 i tireoidnogo faktora transkriptsii-1 v neiroendokrinnykh opukholyakh legkikh raznoi stepeni zlokachestvennosti. Al'manakh klinicheskoi meditsiny. 2016;44(5):613– 23. (In Russ.).

8. Aparicio A, Tzelepi V, Araujo JC, Guo CC, Liang S, Troncoso P, et. al. Neuroendocrine prostate cancer xenografts with large-cell and small-cell features derived from a single patient's tumor: morphological, immunohistochemical, and gene expression profiles. Prostate. 2011 Jun 1;71(8):846-56. doi: 10.1002/pros.21301.

9. Bayrak R, Haltas H, Yenidunya S. The value of CDX2 and cytokeratins 7 and 20 expression in differentiating colorectal adenocarcinomas from extraintestinal gastrointestinal adenocarcinomas: cytokeratin 7-/20+ phenotype is more specific than CDX2 antibody. Diagn Pathol. 2012 Jan 23;7:9. doi: 10.1186/1746-1596-7-9.

10. Biassoni R. Human natural killer receptors, co-receptors, and their ligands. Curr Protoc Immunol. 2009; Chapter 14: 14.10.1-14.10.40. doi: 10.1002/cpim.47.

11. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013 Jul 25;39(1):1-10. doi: 10.1016/j.immuni.2013.07.012.

12. Chiossone L, Dumas PY, Vienne M, Vivier E. Natural killer cells and other innate lymphoid cells in cancer. Nat Rev Immunol. 2018 Nov;18(11):671-688. doi: 10.1038/s41577-018-0061-z. Erratum in: Nat Rev Immunol. 2018 Nov;18(11):726. doi: 10.1038/s41577-018-0077-4.

13. DeLellis RA, Shin SJ, Treaba OD. Chapter 10: Immunohistology of endocrine tumors. In: Dabbs D. J., editor. Diagnostic immunohistochemistry: theranostic and genomic applications. 3rd ed. Philadelphia: Saunders Elsevier; 2010;291–329. doi: 10.1016/B978-1-4160-5766-6.00014-5.

14. Dobiasch S, Kampfer S, Steiger K, Schilling D, Fischer JC, Schmid TE, et. al. Histopathological Tumor and Normal Tissue Responses after 3D-Planned Arc Radiotherapy in an Orthotopic Xenograft Mouse Model of Human Pancreatic Cancer. Cancers (Basel). 2021 Nov 12;13(22):5656. doi: 10.3390/cancers13225656.

15. Dum D, Menz A, Völkel C, De Wispelaere N, Hinsch A, Gorbokon N, et. al. Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers. Exp Mol Pathol. 2022 Jun;126:104762. doi: 10.1016/j.yexmp.2022.104762.

16. Galli F, Rapisarda AS, Stabile H, Malviya G, Manni I, Bonanno E, et. al. In Vivo Imaging of Natural Killer Cell Trafficking in Tumors. J Nucl Med. 2015 Oct;56(10):1575-80. doi: 10.2967/jnumed.114.152918.

17. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018 Mar 23;359(6382):1361-1365. doi: 10.1126/science.aar6711.

18. Kim SW, Roh J, Park CS. Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. J Pathol Transl Med. 2016 Nov;50(6):411-418. doi: 10.4132/jptm.2016.08.08.

19. Maskalenko NA, Zhigarev D, Campbell KS. Harnessing natural killer cells for cancer immunotherapy: dispatching the first responders. Nat Rev Drug Discov. 2022 Aug;21(8):559-577. doi: 10.1038/s41573-022-00413-7.

20. Parodi M, Astigiano S, Carrega P, Pietra G, Vitale C, Damele L, et. al. Murine models to study human NK cells in human solid tumors. Front Immunol. 2023 Jun 14;14:1209237. doi: 10.3389/fimmu.2023.1209237.

21. Pergolini I, Morales-Oyarvide V, Mino-Kenudson M, Honselmann KC, Rosenbaum MW, Nahar S, et. al. Tumor engraftment in patient-derived xenografts of pancreatic ductal adenocarcinoma is associated with adverse clinicopathological features and poor survival. PLoS One. 2017 Aug 30;12(8):e0182855. doi: 10.1371/journal.pone.0182855.

22. Shin E, Bak SH, Park T, Kim JW, Yoon SR, Jung H, et. al. Understanding NK cell biology for harnessing NK cell therapies: targeting cancer and beyond. Front Immunol. 2023 Jul 18;14:1192907. doi: 10.3389/fimmu.2023.1192907.

23. Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007 May;117(5):1137-46. doi: 10.1172/JCI31405.

24. Tarazona R., Lopez-Sejas N., Guerrero B., Hassouneh F., Valhondo I., Pera A., et. al. Current progress in NK cell biology and NK cell-based cancer immunotherapy. Cancer Immunol Immunother. 2020;69(5):879-899. doi: 10.1007/s00262-020-02532-9.

25. Tseng WW, Winer D, Kenkel JA, Choi O, Shain AH, Pollack JR, French R, Lowy AM, Engleman EG. Development of an orthotopic model of invasive pancreatic cancer in an immunocompetent murine host. Clin Cancer Res. 2010 Jul 15;16(14):3684-95. doi: 10.1158/1078-0432.CCR-09-2384.

26. Wolf NK, Kissiov DU, Raulet DH. Roles of natural killer cells in immunity to cancer, and applications to immunotherapy. Nat Rev Immunol. 2023;2:90-105. doi: 10.1038/s41577-022-00732-1