Rabia Bilge ÖZGÜL ÖZDEMİR, Alper Tunga ÖZDEMİR, Cengiz KIRMAZ, Mehmet İbrahim TUĞLU, Özgür ŞENOL, Cenk Serhan ÖZVEREL, Afig BERDELİ

Mezenkimal kök hücrelerin, meme tümörü hücreleri MDA-MB-231 ve MCF-7’nin IDO, HLA-G ve PD-L1 ifadeleri üzerine etkileri

Amaç: Mezenkimal kök hücreler (MKH) güçlü immünomodülatör hücreleridir ve ayrıca tümör mikroçevresinin bir bileşenidir. Bu çalışmada meme tümör hücre hatları MDA-MB-231 ve MCF-7’nin immün evazif moleküller olan Indoleamine 2,3-dioxygenase (IDO), Human Leukocyte Antigen G (HLA-G) and Programmed Death-Ligand 1 (PD-L1) ifadelerine yağ dokusu kökenli mezenkimal kök hücrelerin etkilerini araştırmayı amaçladık. Yöntemler: Bu amaçla MKH, MDA-MB-231 ve MCF-7 hücrelerini artan dozlarda interferon gama (IFN-g) ile kültüre edildi. Başka bir kültür kabında MKH’ler ile tümör hücreleri trans-well insertler ve aynı IFN-g uyarımı ile kültür edildi. Kültür süresinin bitiminde HLA-G ve PD-L1 ifadeleri flow-sitmotetri yöntemi ile IDO ifadeleri Luminex yöntemi ile analiz edildi. Bulgular: Düşük dozlu IFN-g uyarımında (10 ng/mL), MSC'lerin MCF-7 hücrelerinin HLA-G ve PD-L1 ekspresyonunda önemli bir artışa yol açtığını bulduk. Aksine, yüksek doz IFN-g ile (50 ng/mL) bu ifadelerin her iki tümör hücresinde de önemli ölçüde azaldığını gördük. Ek olarak, MDA-MB-231 hücrelerinin IDO ekspresyonunun MSC'lerin varlığında anlamlı şekilde arttığını, ancak MCF-7 hücrelerinin etkilenmediği gördük. Sonuç: Sonuç olarak, MDA-MB-231 hücreleri için MSC'ler, sitotoksik hücrelerin baskılanmasında ve T hücrelerinin tükenmesinde önemli bir rol oynayan HLA-G ve PD-L1 ekspresyonunu azalttığı için koruyucu bir rol oynayabilir. Öte yandan, MSC'ler yüksek IDO seviyeleri için önemli bir kaynak olabilir ve bu nedenle anti-tümör immün yanıtı olumsuz yönde etkileyebilir. Bununla birlikte, verilerimiz diğer çalışmalarla desteklenmelidir.

Anahtar Kelimeler:

Mezenkimal kök hücreler, immün kaçınma, HLA-G, PD-L1, IDO

The effects of mesenchymal stem cells on the IDO, HLA-G and PD-L1 expression of breast tumor cells MDA-MB-231 and MCF-7

Aim: Mesenchymal stem cells (MSCs) are strong immunomodulatory cells and a component of the tumor microenvironment. In this study, we aimed to investigate the effects of MSCs derived from adipose tissue on the expressions of immune evasive molecules indoleamine 2,3-dioxygenase (IDO), human leukocyte antigen G (HLA-G) and programmed death-ligand 1 (PD-L1) of breast tumor cell lines MDA-MB-231 and MCF-7. Methods: For this purpose, MSCs, MDA-MB-231 and MCF-7 cells were cultured with increased doses of interferon gamma (IFN-g). In another plate, tumor cells were cultured in transwell inserts using the same IFN-g stimulation to evaluate the effect of MSCs. At the end of the culture period, the HLA-G and PD-L1 expression was detected by flow cytometry, and IDO expression by the Luminex method. Results: We found that in low-dose IFN-g stimulation (10 ng/mL), MSCs led to a significant increase in the HLA-G and PD-L1 expression of MCF-7 cells. On the contrary, at a high dose of IFN-g (50 ng/mL), their expression significantly decreased in both tumor cells. In addition, we observed that the IDO expression of MDA-MB-231 cells was significantly increased in the presence of MSCs, but MCF-7 cells were not affected. Conclusion: In conclusion, for MDA-MB-231 cells, MSCs may play a protective role because they reduce the expression of HLA-G and PD-L1 that are involved in the suppression of cytotoxic cells and exhaustion of T cells. On the other hand, MSCs may be an important source of high IDO levels, and therefore may negatively affect the antitumor immune response. However, our data should be supported by further studies.

Keywords:

Mesenchymal stem cells, immune evasion, HLA-G, PD-L1, IDO,

PDF

___

1. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–7.
2. Sangiorgi B, Panepucci RA. Modulation of Immunoregulatory Properties of Mesenchymal Stromal Cells by Toll-Like Receptors: Potential Applications on GVHD. Stem Cells Int. 2016;2016:e9434250.
3. Chen P-M, Yen M-L, Liu K-J, Sytwu H-K, Yen B-L. Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells. J Biomed Sci. 2011;18:49.
4. Gao F, Chiu SM, Motan D a. L, Zhang Z, Chen L, Ji H-L, et al. Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell Death Dis. 2016;7:e2062.
5. Özdemir RBÖ, Özdemir AT, Sarıboyacı AE, Uysal O, Tuğlu Mİ, Kırmaz C. The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells. Immunobiology. 2019;224:585–94.
6. Castro-Manrreza ME, Montesinos JJ. Immunoregulation by Mesenchymal Stem Cells: Biological Aspects and Clinical Applications. J Immunol Res. 2015; 2015:e394917.
7. Erbey F, Atay D, Akcay A, Ovali E, Ozturk G. Mesenchymal Stem Cell Treatment for Steroid Refractory Graft-versus-Host Disease in Children: A Pilot and First Study from Turkey. Stem Cells Int. 2016;2016:1641402.
8. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RA. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557–63.
9. Ridge SM, Sullivan FJ, Glynn SA. Mesenchymal stem cells: key players in cancer progression. Mol Cancer. 2017;16:31.
10. Wang Y, Liu J, Jiang Q, Deng J, Xu F, Chen X, et al. Human adipose-derived mesenchymal stem cell-secreted CXCL1 and CXCL8 facilitate breast tumor growth by promoting angiogenesis. Stem Cells. 2017;35:2060-70.
11. Brown SD, Warren RL, Gibb EA, Martin SD, Spinelli JJ, Nelson BH, et al. Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival. Genome Res. 2014;24:743–50.
12. Dahlberg CIM, Sarhan D, Chrobok M, Duru AD, Alici E. Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity. NK Cell Biol. 2015;6:605.
13. Eskicioğlu F, Özdemir AT, Özdemir RB, Turan GA, Akan Z, Hasdemir SP. The association of HLA-G and immune markers in recurrent miscarriages. J Matern Fetal Neonatal Med. 2016;29:3056-60.
14. Özgül Özdemir RB, Özdemir AT, Oltulu F, Kurt K, Yiğittürk G, Kırmaz C. A comparison of cancer stem cell markers and nonclassical major histocompatibility complex antigens in colorectal tumor and noncancerous tissues. Ann Diagn Pathol. 2016;25:60–3.
15. Amiot L, Ferrone S, Grosse-Wilde H, Seliger B. Biology of HLA-G in cancer: a candidate molecule for therapeutic intervention? Cell Mol Life Sci. 2011;68:417–31.
16. Goldman-Wohl DS, Ariel I, Greenfield C, Hanoch J, Yagel S. HLA-G expression in extravillous trophoblasts is an intrinsic property of cell differentiation: a lesson learned from ectopic pregnancies. Mol Hum Reprod. 2000;6:535–40.
17. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, et al. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Front Pharmacol. 2017;8:561.
18. Buchbinder EI, Desai A. CTLA-4 and PD-1 Pathways. Am J Clin Oncol. 2016;39:98–106.
19. Francisco LM, Sage PT, Sharpe AH. The PD-1 Pathway in Tolerance and Autoimmunity. Immunol Rev. 2010;236:219–42.
20. Soliman H, Mediavilla-Varela M, Antonia S. Indoleamine 2,3-dioxygenase: is it an immune suppressor? Cancer J Sudbury Mass. 2010;16:354–9.
21. Routy J-P, Routy B, Graziani GM, Mehraj V. The Kynurenine Pathway Is a Double-Edged Sword in Immune-Privileged Sites and in Cancer: Implications for Immunotherapy. Int J Tryptophan Res. 2016;9:67–77.
22. Takikawa O, Tagawa Y, Iwakura Y, Yoshida R, Truscott RJW. Interferon-Gamma-Dependent/Independent Expression of Indoleamine 2,3-Dioxygenase. Adv Exp Med Biol. 1999;467:553-7.
23. Bukur J, Jasinski S, Seliger B. The role of classical and non-classical HLA class I antigens in human tumors. Semin Cancer Biol. 2012;22:350–8.
24. He X, Dong D, Yie S, Yang H, Cao M, Ye S, et al. HLA-G expression in human breast cancer: implications for diagnosis and prognosis, and effect on allocytotoxic lymphocyte response after hormone treatment in vitro. Ann Surg Oncol. 2010;17:1459–69.
25. Ozgul Ozdemir RB, Ozdemir AT, Oltulu F, Kurt K, Yigitturk G, Kirmaz C. The Expressions of Cancer Stem Cell Markers and Nonclassical HLA antigens in Breast Tumors. Istanb Med J. 2017;18:128–34.
26. Elliott RL, Jiang XP, Phillips JT, Barnett BG, Head JF. Human leukocyte antigen G expression in breast cancer: role in immunosuppression. Cancer Biother Radiopharm. 2011;26:153–7.
27. Pangault C, Amiot L, Caulet‐Maugendre S, Brasseur F, Burtin F, Guilloux V, Drenou B, Fauchet R, Onno M. HLA‐G protein expression is not induced during malignant transformation. Tissue Antigens. 2002;53:335–46.
28. Mazel M, Jacot W, Pantel K, Bartkowiak K, Topart D, Cayrefourcq L, et al. Frequent expression of PD-L1 on circulating breast cancer cells. Mol Oncol. 2015;9:1773–82.
29. Rom-Jurek E-M, Kirchhammer N, Ugocsai P, Ortmann O, Wege AK, Brockhoff G. Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice. Int J Mol Sci. 2018;13:19.
30. Castro F, Cardoso AP, Gonçalves RM, Serre K, Oliveira MJ. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front Immunol. 2018;4;9.
31. Osum KC, Burrack AL, Martinov T, Sahli NL, Mitchell JS, Tucker CG, et al. Interferon-gamma drives programmed death-ligand 1 expression on islet β cells to limit T cell function during autoimmune diabetes. Sci Rep. 2018;8:1–12.
32. Holmgaard RB, Zamarin D, Munn DH, Wolchok JD, Allison JP. Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4. J Exp Med. 2013;210:1389–402.
33. Munn DH, Mellor AL. IDO in the Tumor Microenvironment: Inflammation, Counter-Regulation, and Tolerance. Trends Immunol. 2016;37:193–207.
34. Larrain MTI, Rabassa ME, Lacunza E, Barbera A, Cretón A, Segal-Eiras A, et al. IDO is highly expressed in breast cancer and breast cancer-derived circulating microvesicles and associated to aggressive types of tumors by in silico analysis. Tumor Biol. 2014;35:6511–9.
35. Wei L, Zhu S, Li M, Li F, Wei F, Liu J, Ren X. High Indoleamine 2,3-Dioxygenase Is Correlated With Microvessel Density and Worse Prognosis in Breast Cancer. Front Immunol. 2018;9:724.