The Effects of Released Exosomes from NK-92 Cells with IL15 on the Apoptosis of HL-60 Cell Line
,
Abstract
Background: Most cancers are treated through chemotherapy and radiotherapy. However, these methods have limitations due to cancer cells evading immune detection, prompting researchers to explore alternatives such as immunotherapy. Nonetheless, cancer cells can weaken the immune response, necessitating improvements in immunotherapy methods. Exosomes, tiny cell-derived nanoparticles, reflect the traits of their originating cells. Natural Killer NK cells produce exosomes comprising perforin, granzyme, Fas-L, etc. The small size, proximity to tumors, and stability of these exosomes enable easy absorption by cancer cells. This study demonstrates that IL-15 impacts NK-derived exosomes, enhancing their ability to kill cancer cells.
Materials and Methods: With the addition of 100 nanograms per milliliter of IL-15 to NK-92 cell culture, the cells are incubated for 48 hours. Exosomes are then isolated from treated and non-treated NK-92 cell lines through the ultracentrifuge method. After isolation, different concentrations of exosomes from both groups are added to HL-60 cells for treatment. After 24 hours, the apoptosis rate is assessed through the Annexin-V method.
Results: Increased light absorption in the BCA test, along with thicker bands of CD63 and CD81 in the Western blotting test, indicate a higher yield of exosomes after adding IL-15 to the source cells. The low p-value from the t-test demonstrates that exosomes derived from stimulated NK cells are more cytotoxic than those from the control group. Further, two-way ANOVA confirms differences between the control and treatment groups at each concentration, and Welch’s t-test proves that all differences in the ANOVA test are significant.
Conclusion: This article presents evidence that exosomes obtained from IL-15-induced NK cells not only increase in quantity but also demonstrate significant cytotoxicity against leukemic cells compared to exosomes obtained from non-stimulated NK cells.
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6. Cherrier DE, Serafini N, Di Santo JP. Innate Lymphoid Cell Development: A T Cell Perspective. Immunity. 2018;48(6):1091-1103.
7. Zhang Y, Wallace DL, de Lara CM, et al. In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection. Immunology. 2007;121(2):258-65.
8. Zwirner NW, Domaica CI, Fuertes MB. Regulatory functions of NK cells during infections and cancer. J Leukoc Biol. 2021;109(1):185-194.
9. Chen X, Han J, Chu J, et al. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases. Oncotarget. 2016;7(19):27764-27777.
10. Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol. 2021;18(2):85-100.
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13. Batlle E, Massagué J. Transforming growth factor-β signaling in immunity and cancer. Immunity. 2019;50(4):924-940.
14. Groh V, Wu J, Yee C, et al. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature. 2002;419(6908):734-8.
15. Hilpert J, Grosse-Hovest L, Grünebach F, et al. Comprehensive analysis of NKG2D ligand expression and release in leukemia: implications for NKG2D-mediated NK cell responses. J immunol. 2012;189(3):1360-71.
16. Nückel H, Switala M, Sellmann L, et al. The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia. Leukemia. 2010;24(6):1152-9.
17. Jinushi M, Vanneman M, Munshi NC, et al. MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma. Proc Natl Acad Sci U S A. 2008;105(4):1285-1290.
18. Liu G, Lu S, Wang X, et al. Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis. J Clin Invest. 2013;123(10):4410-22.
19. Paschen A, Sucker A, Hill B, et al. Differential clinical significance of individual NKG2D ligands in melanoma: soluble ULBP2 as an indicator of poor prognosis superior to S100B. Clin Cancer Res. 2009;15(16):5208-15.
20. Wang LP, Niu H, Xia YF, et al. Prognostic significance of serum sMICA levels in non-small cell lung cancer. Eur Rev Med Pharmacol Sci. 2015;19(12):2226-30.
21. Liu X, Li L, Si F, et al. NK and NKT cells have distinct properties and functions in cancer. Oncogene. 2021;40(27):4521-4537.
22. Théry C, Amigorena S, Raposo G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006; Chapter 3: Unit 3.22.
23. Valadi H, Ekström K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654-9.
24. Dragomir M, Chen B, Calin GA. Exosomal lncRNAs as new players in cell-to-cell communication. Transl Cancer Res. 2018;7(Suppl 2):S243-S252.
25. Elsharkasy OM, Nordin JZ, Hagey DW, et al. Extracellular vesicles as drug delivery systems: Why and how? Adv Drug Deliv Rev. 2020;159:332-343.
26. Van Niel G, d’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213-228.
27. Igami K, Uchiumi T, Ueda S, et al. Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V. PeerJ Anal Chem. 2020;2:e4.
28. Neviani P, Wise PM, Murtadha M, et al. Natural killer–derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms. Cancer Res. 2019;79(6):1151-1164.
29. Di Pace AL, Pelosi A, Fiore PF, et al. MicroRNA analysis of Natural Killer cell-derived exosomes: the microRNA let-7b-5p is enriched in exosomes and participates in their anti-tumor effects against pancreatic cancer cells. Oncoimmunology. 2023;12(1):2221081.
30. Wu F, Xie M, Hun M, et al. Natural Killer Cell-Derived Extracellular Vesicles: Novel Players in Cancer Immunotherapy. Front Immunol. 2021;12: 658698.
31. Zhu L, Kalimuthu S, Gangadaran P, et al. Exosomes derived from natural killer cells exert therapeutic effect in melanoma. Theranostics. 2017;7(10):2732-2745.
32. Di Pace AL, Tumino N, Besi F, et al. Characterization of human NK cell-derived exosomes: role of DNAM1 receptor in exosome-mediated cytotoxicity against tumor. Cancers (Basel). 2020;12(3):661.
33. Neviani P, Wise PM, Murtadha M, et al. Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms. Cancer Res. 2019;79(6):1151-1164.
34. Federici C, Shahaj E, Cecchetti S, et al. Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors . Front Immunol. 2020:11:262.
35. Parolini I, Federici C, Raggi C, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem. 2009;284(49):34211-22.
36. Samara A, Anbar M, Shapira S, et al. Using natural killer cell-derived exosomes as a cell-free therapy for leukemia. Hematol Oncol. 2023;41(3):487-498.
37. Alvarez-Erviti L, Seow Y, Yin H, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29(4):341-5.
38. Zhu L, Kalimuthu S, Oh JM, et al. Enhancement of antitumor potency of extracellular vesicles derived from natural killer cells by IL-15 priming. Biomaterials. 2019;190–191:38–50.
39. Livshits MA, Khomyakova E, Evtushenko EG, et al. Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep. 2015;5:17319.
40. Cvjetkovic A, Lötvall J, Lässer C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles. 2014;3.
41. Chan AML, Cheah JM, Lokanathan Y, et al. Natural Killer Cell-Derived Extracellular Vesicles as a Promising Immunotherapeutic Strategy for Cancer: A Systematic Review. Int J Mol Sci. 2023;24(4):4026.
42. Hatami Z, Hashemi ZS, Eftekhary M, et al. Natural killer cell-derived exosomes for cancer immunotherapy: innovative therapeutic tools. Cancer Cell Int. 2023;23(1):157.
43. Rezaie J, Feghhi M, Etemadi T. A review on exosomes application in clinical trials: perspective, questions, and challenges. Cell Commun Signal. 2022;20(1):145.
44. Kim I-Y, Kim HY, Song HW, et al. Functional enhancement of exosomes derived from NK cells by IL-15 and IL-21 synergy against hepatocellular carcinoma cells: The cytotoxicity and apoptosis in vitro study. Heliyon. 2023;9: e16962.
45. Khatib ZK, Maleki A, Pourfatollah AA, et al. Antileukemia Activity of Human Natural Killer Cell-Derived Nanomagic Bullets against Acute Myeloid Leukemia (AML). Int J Hematol Oncol Stem Cell Res. 2024;18(2):123-139.
2. Schnittger S, Kohl TM, Haferlach T, et al. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood. 2006;107(5):1791-9.
3. Paschka P, Marcucci G, Ruppert AS, et al. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): A Cancer and Leukemia Group B study. J Clin Oncol. 2006;24(24):3904-11.
4. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol. 2001;19(18):3852-60.
5. Chu J, Gao F, Yan M, et al. Natural killer cells: a promising immunotherapy for cancer. J Transl Med. 2022;20(1):240.
6. Cherrier DE, Serafini N, Di Santo JP. Innate Lymphoid Cell Development: A T Cell Perspective. Immunity. 2018;48(6):1091-1103.
7. Zhang Y, Wallace DL, de Lara CM, et al. In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection. Immunology. 2007;121(2):258-65.
8. Zwirner NW, Domaica CI, Fuertes MB. Regulatory functions of NK cells during infections and cancer. J Leukoc Biol. 2021;109(1):185-194.
9. Chen X, Han J, Chu J, et al. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases. Oncotarget. 2016;7(19):27764-27777.
10. Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol. 2021;18(2):85-100.
11. Hatjiharissi E, Xu L, Santos DD, et al. Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism. Blood. 2007;110(7):2561-4.
12. Smyth MJ, Crowe NY, Pellicci DG, et al. Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide. Blood. 2002;99(4):1259-1266.
13. Batlle E, Massagué J. Transforming growth factor-β signaling in immunity and cancer. Immunity. 2019;50(4):924-940.
14. Groh V, Wu J, Yee C, et al. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature. 2002;419(6908):734-8.
15. Hilpert J, Grosse-Hovest L, Grünebach F, et al. Comprehensive analysis of NKG2D ligand expression and release in leukemia: implications for NKG2D-mediated NK cell responses. J immunol. 2012;189(3):1360-71.
16. Nückel H, Switala M, Sellmann L, et al. The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia. Leukemia. 2010;24(6):1152-9.
17. Jinushi M, Vanneman M, Munshi NC, et al. MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma. Proc Natl Acad Sci U S A. 2008;105(4):1285-1290.
18. Liu G, Lu S, Wang X, et al. Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis. J Clin Invest. 2013;123(10):4410-22.
19. Paschen A, Sucker A, Hill B, et al. Differential clinical significance of individual NKG2D ligands in melanoma: soluble ULBP2 as an indicator of poor prognosis superior to S100B. Clin Cancer Res. 2009;15(16):5208-15.
20. Wang LP, Niu H, Xia YF, et al. Prognostic significance of serum sMICA levels in non-small cell lung cancer. Eur Rev Med Pharmacol Sci. 2015;19(12):2226-30.
21. Liu X, Li L, Si F, et al. NK and NKT cells have distinct properties and functions in cancer. Oncogene. 2021;40(27):4521-4537.
22. Théry C, Amigorena S, Raposo G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006; Chapter 3: Unit 3.22.
23. Valadi H, Ekström K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654-9.
24. Dragomir M, Chen B, Calin GA. Exosomal lncRNAs as new players in cell-to-cell communication. Transl Cancer Res. 2018;7(Suppl 2):S243-S252.
25. Elsharkasy OM, Nordin JZ, Hagey DW, et al. Extracellular vesicles as drug delivery systems: Why and how? Adv Drug Deliv Rev. 2020;159:332-343.
26. Van Niel G, d’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213-228.
27. Igami K, Uchiumi T, Ueda S, et al. Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V. PeerJ Anal Chem. 2020;2:e4.
28. Neviani P, Wise PM, Murtadha M, et al. Natural killer–derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms. Cancer Res. 2019;79(6):1151-1164.
29. Di Pace AL, Pelosi A, Fiore PF, et al. MicroRNA analysis of Natural Killer cell-derived exosomes: the microRNA let-7b-5p is enriched in exosomes and participates in their anti-tumor effects against pancreatic cancer cells. Oncoimmunology. 2023;12(1):2221081.
30. Wu F, Xie M, Hun M, et al. Natural Killer Cell-Derived Extracellular Vesicles: Novel Players in Cancer Immunotherapy. Front Immunol. 2021;12: 658698.
31. Zhu L, Kalimuthu S, Gangadaran P, et al. Exosomes derived from natural killer cells exert therapeutic effect in melanoma. Theranostics. 2017;7(10):2732-2745.
32. Di Pace AL, Tumino N, Besi F, et al. Characterization of human NK cell-derived exosomes: role of DNAM1 receptor in exosome-mediated cytotoxicity against tumor. Cancers (Basel). 2020;12(3):661.
33. Neviani P, Wise PM, Murtadha M, et al. Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms. Cancer Res. 2019;79(6):1151-1164.
34. Federici C, Shahaj E, Cecchetti S, et al. Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors . Front Immunol. 2020:11:262.
35. Parolini I, Federici C, Raggi C, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem. 2009;284(49):34211-22.
36. Samara A, Anbar M, Shapira S, et al. Using natural killer cell-derived exosomes as a cell-free therapy for leukemia. Hematol Oncol. 2023;41(3):487-498.
37. Alvarez-Erviti L, Seow Y, Yin H, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29(4):341-5.
38. Zhu L, Kalimuthu S, Oh JM, et al. Enhancement of antitumor potency of extracellular vesicles derived from natural killer cells by IL-15 priming. Biomaterials. 2019;190–191:38–50.
39. Livshits MA, Khomyakova E, Evtushenko EG, et al. Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep. 2015;5:17319.
40. Cvjetkovic A, Lötvall J, Lässer C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles. 2014;3.
41. Chan AML, Cheah JM, Lokanathan Y, et al. Natural Killer Cell-Derived Extracellular Vesicles as a Promising Immunotherapeutic Strategy for Cancer: A Systematic Review. Int J Mol Sci. 2023;24(4):4026.
42. Hatami Z, Hashemi ZS, Eftekhary M, et al. Natural killer cell-derived exosomes for cancer immunotherapy: innovative therapeutic tools. Cancer Cell Int. 2023;23(1):157.
43. Rezaie J, Feghhi M, Etemadi T. A review on exosomes application in clinical trials: perspective, questions, and challenges. Cell Commun Signal. 2022;20(1):145.
44. Kim I-Y, Kim HY, Song HW, et al. Functional enhancement of exosomes derived from NK cells by IL-15 and IL-21 synergy against hepatocellular carcinoma cells: The cytotoxicity and apoptosis in vitro study. Heliyon. 2023;9: e16962.
45. Khatib ZK, Maleki A, Pourfatollah AA, et al. Antileukemia Activity of Human Natural Killer Cell-Derived Nanomagic Bullets against Acute Myeloid Leukemia (AML). Int J Hematol Oncol Stem Cell Res. 2024;18(2):123-139.
| Files | ||
| Issue | Vol 19 No 2 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Exosome; Natural killer (NK) cells; Immunotherapy; Leukemia | ||
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How to Cite
1.
Abbasi S, Movassaghpour A, Soleimani M, Asghari Molabashi Z. The Effects of Released Exosomes from NK-92 Cells with IL15 on the Apoptosis of HL-60 Cell Line. Int J Hematol Oncol Stem Cell Res. 2025;19(2):126-137.
