Biopolym. Cell. 2018; 34(6):476-486.
Optimization of in vitro model for analysis of tumor cell migration dynamics
1, 2Kravchenko A. O., 1Kosach V. R., 1Shkarina K. A., 1, 2Zaiets I. V., 1Tykhonkova I. O., 1Khoruzhenko A. I.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. Educational and Scientific Center "Institute of Biology and Medicine",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601


Migration ability is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. One of the most perspective and closer to the in vivo conditions is the model of initiation of the cell migration from 3D multicellular spheroids onto growth surface. Aim. Optimization of the model for adequate quantitative characteristics of the tumor cell locomotion during several days. Methods. 2D and 3D MCF-7 cell culture, immunofluorescence analysis, and image analysis using computer software Fiji. Results. Unification of spheroid size allowed avoiding a significant data deviation. The obtained spheroids spread completely for 3 days. The highest migration ratio was observed at the 2nd day. The proliferation level at each of 3-day experiment was the same and did not exceed 3%. The validity of the model was tested after migration inhibition by rapamycin (mTOR signaling inhibitor). Additionally, this model was successfully applied to immunofluorescence analysis, namely investigation of p85S6K1 subcellular localization in moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed unification of their size, which promotes an adequate interpretation of the migration assay. This model enabled the study of tumor cells migration dynamics and can be further used for the development of anticancer drug.
Keywords: Cancer cell migration assay, 2D and 3D culture, p85S6K1, multicellular


[1] Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014;(88).
[2] Aasen TB, Schreiner A. In vitro locomotion of blood monocytes in millipore filters--evaluation of the leading front method. Acta Pathol Microbiol Immunol Scand C. 1986;94(1):45-9.
[3] Aase K, Ernkvist M, Ebarasi L, Jakobsson L, Majumdar A, Yi C, Birot O, Ming Y, Kvanta A, Edholm D, Aspenström P, Kissil J, Claesson-Welsh L, Shimono A, Holmgren L. Angiomotin regulates endothelial cell migration during embryonic angiogenesis. Genes Dev. 2007;21(16):2055-68.
[4] Aarum J, Sandberg K, Haeberlein SL, Persson MA. Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A. 2003;100(26):15983-8.
[5] A Soliman N, Abd-Allah SH, Hussein S, Alaa Eldeen M. Factors enhancing the migration and the homing of mesenchymal stem cells in experimentally induced cardiotoxicity in rats. IUBMB Life. 2017;69(3):162-169.
[6] 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):546-60. PubMed Central PMCID: PMC3606845.
[7] Khoruzhenko AI. 2D- and 3D-culture of cell. Biopolym Cell. 2011;27(1):17–24.
[8] Tavares MR, Pavan IC, Amaral CL, Meneguello L, Luchessi AD, Simabuco FM. The S6K protein family in health and disease. Life Sci. 2015;131:1-10.
[9] Rosner M, Hengstschläger M. Nucleocytoplasmic localization of p70 S6K1, but not of its isoforms p85 and p31, is regulated by TSC2/mTOR. Oncogene. 2011;30(44):4509-22.
[10] Filonenko VV. PI3K/mTOR/S6K signaling pathway – new players and new functional links. Biopolym Cell. 2013; 29(3):207-14.
[11] Kurgan N, Tsakiridis E, Kouvelioti R, Moore J, Klentrou P, Tsiani E. Inhibition of Human Lung Cancer Cell Proliferation and Survival by Post-Exercise Serum Is Associated with the Inhibition of Akt, mTOR, p70 S6K, and Erk1/2. Cancers (Basel). 2017;9(5). pii: E46.
[12] Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012;441(1):1-21.
[13] Filonenko VV, Tytarenko R, Azatjan SK, Savinska LO, Gaydar YA, Gout IT, Usenko VS, Lyzogubov VV. Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors. Exp Oncol. 2004;26(4):294-9.
[14] Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, Unger C, Kramer N, Hengstschläger M, Kenner L, Pfeiffer D, Krupitza G, Dolznig H. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J Cell Sci. 2017;130(1):203-218.
[15] Gotsulyak NYa, Kosach VR, Cherednyk OV, Tykhonkova IO, Khoruzhenko AI. Optimization of cell motility evaluation in scratch assay. Biopolym Cell. 2014;30(3):223–8.
[16] Kosach V, Shkarina K, Kravchenko A, Tereshchenko Y, Kovalchuk E, Skoroda L, Krotevych M, Khoruzhenko A. Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro. F1000Research. 2018;7:1332.
[17] Soule HD, Vazguez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst. 1973;51(5):1409-16.
[18] Savinska LO, Klipa OM, Khoruzenko AI, Shkarina KA, Garifulin OM, Filonenko VV. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1) Biopolym Cell. 2015;31(4):294–300.
[19] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82.
[20] Liu L, Li F, Cardelli JA, Martin KA, Blenis J, Huang S. Rapamycin inhibits cell motility by suppression of mTOR-mediated S6K1 and 4E-BP1 pathways. Oncogene. 2006;25(53):7029-40.