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Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas

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Bulletin of Experimental Biology and Medicine Aims and scope

The use of relevant, accessible, and easily reproducible preclinical models of diffuse gliomas is a prerequisite for the development of successful therapeutic approaches to their treatment. Here we studied the gene expression profile of 3D spheroids in a comparison with 2D cell cultures and tissue strains of diffuse high-grade gliomas. Using real time PCR, we evaluated the expression of Gfap, Cd44, Pten, S100b, Vegfa, Hif1a, Sox2, Melk, Gdnf, and Mgmt genes playing an important role in the progression of gliomas and regulating tumor cell proliferation, adhesion, invasion, plasticity, apoptosis, DNA repair, and recruitment of tumor-associated cells. Gene expression analysis showed that 3D spheroids are more similar to tumor tissue strains by the expression levels of Gfap, Cd44, and Pten, while the expression levels of Hif1a and Sox2 in 3D spheroids did not differ from those of 2D cell cultures, the expression levels S100b and Vegfa in 3D spheroids was higher than in other models, and the expression levels of Melk, Gdnf, and Mgmt genes changed diversely. Thus, 3D spheroid model more closely mimics the tumor tissue than 2D cell culture, but still is not the most relevant, probably due to too small size of spheroids, which does not allow reproducing hypoxia and apoptotic and necrotic processes in the tumor tissue.

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References

  1. Whitfield BT, Huse JT. Classification of adult-type diffuse gliomas: Impact of the World Health Organization 2021 update. Brain Pathol. 2022;32(4):e13062. https://doi.org/10.1111/bpa.13062

    Article  PubMed  PubMed Central  Google Scholar 

  2. Jiang T, Nam DH, Ram Z, Poon WS, Wang J, Boldbaatar D, Mao Y, Ma W, Mao Q, You Y, Jiang C, Yang X, Kang C, Qiu X, Li W, Li S, Chen L, Li X, Liu Z, Wang W, Bai H, Yao Y, Li S, Wu A, Sai K, Li G, Yao K, Wei X, Liu X, Zhang Z, Dai Y, Lv S, Wang L, Lin Z, Dong J, Xu G, Ma X, Zhang W, Zhang C, Chen B, You G, Wang Y, Wang Y, Bao Z, Yang P, Fan X, Liu X, Zhao Z, Wang Z, Li Y, Wang Z, Li G, Fang S, Li L, Liu Y, Liu S, Shan X, Liu Y, Chai R, Hu H, Chen J, Yan W, Cai J, Wang H, Chen L, Yang Y, Wang Y, Han L, Wang Q; Chinese Glioma Cooperative Group (CGCG); Society for Neuro‐Oncology of China (SNO-China); Chinese Brain Cancer Association (CBCA); Chinese Glioma Genome Atlas (CGGA); Asian Glioma Genome Atlas (AGGA) network. Clinical practice guidelines for the management of adult diffuse gliomas. Cancer Lett. 2021;499:60-72. https://doi.org/10.1016/j.canlet.2020.10.050

  3. Barthel FP, Johnson KC, Varn FS, Moskalik AD, Tanner G, Kocakavuk E, Anderson KJ, Abiola O, Aldape K, Alfaro KD, Alpar D, Amin SB, Ashley DM, Bandopadhayay P, Barnholtz-Sloan JS, Beroukhim R, Bock C, Brastianos PK, Brat DJ, Brodbelt AR, Bruns AF, Bulsara KR, Chakrabarty A, Chakravarti A, Chuang JH, Claus EB, Cochran EJ, Connelly J, Costello JF, Finocchiaro G, Fletcher MN, French PJ, Gan HK, Gilbert MR, Gould PV, Grimmer MR, Iavarone A, Ismail A, Jenkinson MD, Khasraw M, Kim H, Kouwenhoven MCM, LaViolette PS, Li M, Lichter P, Ligon KL, Lowman AK, Malta TM, Mazor T, McDonald KL, Molinaro AM, Nam DH, Nayyar N, Ng HK, Ngan CY, Niclou SP, Niers JM, Noushmehr H, Noorbakhsh J, Ormond DR, Park CK, Poisson LM, Rabadan R, Radlwimmer B, Rao G, Reifenberger G, Sa JK, Schuster M, Shaw BL, Short SC, Smitt PAS, Sloan AE, Smits M, Suzuki H, Tabatabai G, Van Meir EG, Watts C, Weller M, Wesseling P, Westerman BA, Widhalm G, Woehrer A, Yung WKA, Zadeh G, Huse JT, De Groot JF, Stead LF, Verhaak RGW; GLASS Consortium. Longitudinal molecular trajectories of diffuse glioma in adults. Nature. 2019;576:112-120. https://doi.org/10.1038/s41586-019-1775-1

  4. Fogel DB. Factors associated with clinical trials that fail and opportunities for improving the likelihood of success: A review. Contemp. Clin. Trials Commun. 2018;11:156-164. https://doi.org/10.1016/j.conctc.2018.08.001

    Article  PubMed  PubMed Central  Google Scholar 

  5. Lenting K, Verhaak R, Ter Laan M, Wesseling P, Leenders W. Glioma: experimental models and reality. Acta Neuropathol. 2017;133(2):263-282. https://doi.org/10.1007/s00401-017-1671-4

    Article  PubMed  PubMed Central  Google Scholar 

  6. Barth RF, Kaur B. Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J. Neurooncol. 2009;94(3):299-312. https://doi.org/10.1007/s11060-009-9875-7

    Article  PubMed  PubMed Central  Google Scholar 

  7. Oraiopoulou ME, Tampakaki M, Tzamali E, Tamiolakis T, Makatounakis V, Vakis AF, Zacharakis G, Sakkalis V, Papamatheakis J. A 3D tumor spheroid model for the T98G Glioblastoma cell line phenotypic characterization. Tissue Cell. 2019;59:39-43. https://doi.org/10.1016/j.tice.2019.05.007

    Article  CAS  PubMed  Google Scholar 

  8. Roy SM, Garg V, Barman S, Ghosh C, Maity AR, Ghosh SK. Kinetics of nanomedicine in tumor spheroid as an in vitro model system for efficient tumor-targeted drug delivery with insights from mathematical models. Front. Bioeng. Biotechnol. 2021;9:785937. https://doi.org/10.3389/fbioe.2021.785937

    Article  PubMed  PubMed Central  Google Scholar 

  9. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45. https://doi.org/10.1093/nar/29.9.e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034. https://doi.org/10.1186/gb-2002-3-7-research0034

  11. Däster S, Amatruda N, Calabrese D, Ivanek R, Turrini E, Droeser RA, Zajac P, Fimognari C, Spagnoli GC, Iezzi G, Mele V, Muraro MG. Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. Oncotarget. 2017;8(1):1725-1736. https://doi.org/10.18632/oncotarget.13857

  12. Restrepo A, Smith CA, Agnihotri S, Shekarforoush M, Kongkham PN, Seol HJ, Northcott P, Rutka JT. Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas. Neuro Oncol. 2011;13(1):42-50. https://doi.org/10.1093/neuonc/noq145

    Article  CAS  PubMed  Google Scholar 

  13. Sereika M, Urbanaviciute R, Tamasauskas A, Skiriute D, Vaitkiene P. GFAP expression is influenced by astrocytoma grade and rs2070935 polymorphism. J. Cancer. 2018;9(23):4496-4502. https://doi.org/10.7150/jca.26769

    Article  PubMed  PubMed Central  Google Scholar 

  14. Yang Z, Wang KK. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci. 2015;38(6):364-374. https://doi.org/10.1016/j.tins.2015.04.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jia W, Jiang X, Liu W, Wang L, Zhu B, Zhu H, Liu X, Zhong M, Xie D, Huang W, Jia W, Li S, Liu X, Zuo X, Cheng D, Dai J., Ren C. Effects of three-dimensional collagen scaffolds on the expression profiles and biological functions of glioma cells. Int. J. Oncol. 2018;52(6):1787-1800. https://doi.org/10.3892/ijo.2018.4330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pibuel MA, Poodts D, Díaz M, Hajos SE, Lompardía SL. The scrambled story between hyaluronan and glioblastoma. J. Biol. Chem. 2021;296:100549. https://doi.org/10.1016/j.jbc.2021.100549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Maherally Z, Smith JR, Ghoneim MK, Dickson L, An Q, Fillmore HL, Pilkington GJ. Silencing of CD44 in glioma leads to changes in cytoskeletal protein expression and cellular biomechanical deformation properties as measured by AFM nanoindentation. BioNanoSci. 2016;6:54-64. https://doi.org/10.1007/s12668-015-0189-2

    Article  Google Scholar 

  18. Kolliopoulos C, Ali MM, Castillejo-Lopez C, Heldin CH, Heldin P. CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence. Cancers (Basel). 2022;14(15):3747. https://doi.org/10.3390/cancers14153747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu G, Song X, Liu J, Li S, Gao W, Qiu M, Yang C, Ma Y, Chen Y. Expression of CD44 and the survival in glioma: a meta-analysis. Biosci. Rep. 2020;40(4):BSR20200520. 10.1042/BSR20200520

  20. Wang HH, Liao CC, Chow NH, Huang LL, Chuang JI, Wei KC, Shin JW. Whether CD44 is an applicable marker for glioma stem cells. Am. J. Transl. Res. 2017;9(11):4785-4806.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Wiranowska M, Ladd S, Moscinski LC, Hill B, Haller E, Mikecz K, Plaas A. Modulation of hyaluronan production by CD44 positive glioma cells. Int. J. Cancer. 2010;127(3):532-542. https://doi.org/10.1002/ijc.25085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Han F, Hu R, Yang H, Liu J, Sui J, Xiang X, Wang F, Chu L, Song S. PTEN gene mutations correlate to poor prognosis in glioma patients: a meta-analysis. Onco Targets Ther. 2016;9:3485-3492. https://doi.org/10.2147/OTT.S99942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee JJ, Kim BC, Park MJ, Lee YS, Kim YN, Lee BL, Lee JS. PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation. Cell Death Differ. 2011;18(4):666-677. https://doi.org/10.1038/cdd.2010.139

    Article  CAS  PubMed  Google Scholar 

  24. Pinto B, Henriques AC, Silva PMA, Bousbaa H. Three-dimensional spheroids as in vitro preclinical models for cancer research. Pharmaceutics. 2020;12(12):1186. https://doi.org/10.3390/pharmaceutics12121186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kochanek SJ, Close DA, Camarco DP, Johnston PA. Maximizing the value of cancer drug screening in multicellular tumor spheroid cultures: a case study in five head and neck squamous cell carcinoma cell lines. SLAS Discov. 2020;25(4):329-349. https://doi.org/10.1177/2472555219896999

    Article  PubMed  PubMed Central  Google Scholar 

  26. Berezovsky AD, Poisson LM, Cherba D, Webb CP, Transou AD, Lemke NW, Hong X, Hasselbach LA, Irtenkauf SM, Mikkelsen T, deCarvalho AC. Sox2 promotes malignancy in glioblastoma by regulating plasticity and astrocytic differentiation. Neoplasia. 2014;16(3):193-206, 206.e19-25. https://doi.org/10.1016/j.neo.2014.03.006

  27. Wuebben EL, Rizzino A. The dark side of SOX2: cancer — a comprehensive overview. Oncotarget. 2017;8(27):44 917-44 943. https://doi.org/10.18632/oncotarget.16570

  28. Annovazzi L, Mellai M, Caldera V, Valente G, Schiffer D. SOX2 expression and amplification in gliomas and glioma cell lines. Cancer Genomics Proteomics. 2011;8(3):139-147.

    CAS  PubMed  Google Scholar 

  29. Aaberg-Jessen C, Nørregaard A, Christensen K, Pedersen CB, Andersen C, Kristensen BW. Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures. Int. J. Clin. Exp. Pathol. 2013;6(4):546-560.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Ayanlaja AA, Zhang B, Ji G, Gao Y, Wang J, Kanwore K, Gao D. The reversible effects of glial cell line-derived neurotrophic factor (GDNF) in the human brain. Semin. Cancer Biol. 2018;53:212-222. https://doi.org/10.1016/j.semcancer.2018.07.005

    Article  CAS  PubMed  Google Scholar 

  31. Gu C, Banasavadi-Siddegowda YK, Joshi K, Nakamura Y, Kurt H, Gupta S, Nakano I. Tumor-specific activation of the C-JUN/MELK pathway regulates glioma stem cell growth in a p53-dependent manner. Stem Cells. 2013;31(5):870-881. https://doi.org/10.1002/stem.1322

    Article  CAS  PubMed  Google Scholar 

  32. Hardeman AA, Han YJ, Grushko TA, Mueller J, Gomez MJ, Zheng Y, Olopade OI. Subtype-specific expression of MELK is partly due to copy number alterations in breast cancer. PLoS One. 2022;17(6):e0268693. https://doi.org/10.1371/journal.pone.0268693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Feldheim J, Kessler AF, Monoranu CM, Ernestus RI, Löhr M, Hagemann C. Changes of O6-methylguanine DNA methyltransferase (MGMT) promoter methylation in glioblastoma relapse — a meta-analysis type literature review. Cancers (Basel). 2019;11(12):1837. https://doi.org/10.3390/cancers11121837

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Stanković T, Ranđelović T, Dragoj M, Stojković Burić S, Fernández L, Ochoa I, Pérez-García VM, Pešić M. In vitro biomimetic models for glioblastoma-a promising tool for drug response studies. Drug Resist. Updat. 2021;55:100753. https://doi.org/10.1016/j.drup.2021.100753

    Article  CAS  PubMed  Google Scholar 

  35. Musah-Eroje A, Watson S. A novel 3D in vitro model of glioblastoma reveals resistance to temozolomide which was potentiated by hypoxia. J. Neurooncol. 2019;142(2):231-240. https://doi.org/10.1007/s11060-019-03107-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to I. V. Arutyunyan.

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Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 126-135, June, 2023

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Arutyunyan, I.V., Soboleva, A.G., Kovtunov, E.A. et al. Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas. Bull Exp Biol Med 175, 576–584 (2023). https://doi.org/10.1007/s10517-023-05906-y

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