The optimization of methods for the establishment of heterogeneous three-dimensional cellular models of breast cancer

Abstract

BACKGROUND: Spheroids are self-assembled clusters of cells mimicking a tissue-like architecture. Since the structure of complex three-dimensional cellular models is not stable, the formation of core spheroids and further maintenance are crucial stages within the cultivation process. There are a lot of options described for the establishment of 3D cell models. A wide range of reagents is presented from simple hydrogels to complex natural and synthetic composites. However, cultivation of 3D models is still a technically challenging task requiring adaptations of protocols for particular purposes.

AIM: To compare methods of the formation of gomogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and/or stromal cells of breast cancer using hydrogels such as agarose, gelatin and Matrigel™, as well as using ultra-low-adherent plates.

MATERIALS AND METHODS: Breast cancer cell lines MCF7, MDA-MB-231 SK-BR-3 and stromal fibroblasts BrC4f, BrC120f, BN120f were used as a models for 3D and 3D-2 cultures. Spheroids were obtained on a substrate of simple hydrogels or when cultured on a low-adhesive plastic. The processes of formation and growth of spheroids, as well as "crushed preparations" were visualized using a Nikon Eclipse Ti-S series fluorescent inverted microscope (Nikon, Japan).

RESULTS: We demonstrated, that gelatin-based hydrogel is not suitable as a substrate for obtaining 3D and 3D-2 spheroids for any of the cell lines used in the work. The use of only one type of hydrogels does not allow to obtain the entire repertoire of tumor, stromal, and heterogeneous 3D models. Agarose exhibited high output for stromal spheroids and Matrigel™ for tumor cells, and the use of ultra-low-adherent Nunclon™ Sphera™ plates was preferable for 3D-2 models combining both cell types. We also revealed that the application of cooled cultivation plastic and solutions is a technological advantage for handling spheroids growing in low-adherent plates.

CONCLUSION: The proposed approaches for the formation of both homogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and (or) stromal breast cancer cells are a generalized guide to the most effective production of spheroids of various cellular composition.

Keywords

hydrogel / preclinical models / breast cancer / tumor cells / stromal cells / primary cell cultures / 3D cultures / spheroids

References

[1] Segeritz C-P, Vallier L. Cell culture: growing cells as model systems in vitro. In: Jalali M, Saldanha F, Jalali M, editors. Basic science methods for clinical researchers. Elsevier: Academic Press; 2017. P. 151–172. doi: 10.1016/B978-0-12-803077-6.00009-6

[2] Segeritz C.-P., Vallier L. Cell culture: growing cells as model systems in vitro. In: Jalali M., Saldanha F., Jalali M., editors. Basic science methods for clinical researchers. Elsevier: Academic Press, 2017. P. 151–172. doi: 10.1016/B978-0-12-803077-6.00009-6

[3] Lin A, Sved Skottvoll F, Rayner S, et al. 3D cell culture models and organ-on-a-chip: meet separation science and mass spectrometry. Electrophoresis. 2020;41(1-2):56–64. doi: 10.1002/elps.201900170

[4] Lin A., Sved Skottvoll F., Rayner S., et al. 3D cell culture models and organ-on-a-chip: meet separation science and mass spectrometry // Electrophoresis. 2020. Vol. 41, N 1-2. P. 56–64. doi: 10.1002/elps.201900170

[5] Saji Joseph J, Tebogo Malindisa S, Ntwasa M. Two-dimensional (2d) and three-dimensional (3d) cell culturing in drug discovery. In: Ali Mehanna R, editor. Cell culture. IntechOpen; 2019. doi: 10.5772/intechopen.81552

[6] Saji Joseph J., Tebogo Malindisa S., Ntwasa M. Two-dimensional (2D) and three-dimensional (3D) cell culturing in drug discovery. In: Ali Mehanna R., editor. Cell culture. London : IntechOpen, 2019. P. 21–42. doi: 10.5772/intechopen.81552

[7] Ravi M, Kaviya SR, Paramesh V. Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures. Cytotechnology. 2016;68(3):429–441. doi: 10.1007/s10616-014-9795-z

[8] Ravi M., Kaviya S.R., Paramesh V. Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures // Cytotechnology. 2016. Vol. 68, N 3. P. 429–441. doi: 10.1007/s10616-014-9795-z

[9] Badea MA, Balas M, Hermenean A, et al. Influence of matrigel on single- and multiple-spheroid cultures in breast cancer research. SLAS Discov. 2019;24(5):563–578. doi: 10.1177/2472555219834698

[10] Badea M.A., Balas M., Hermenean A., et al. Influence of Matrigel on single- and multiple-spheroid cultures in breast cancer research // SLAS Discov. 2019. Vol. 24, N 5. P. 563–578. doi: 10.1177/2472555219834698

[11] Zhao Y, Yao R, Ouyang L, et al. Three-dimensional printing of Hela cells for cervical tumor model in vitro. Biofabrication. 2014;6(3):035001. doi: 10.1088/1758-5082/6/3/035001

[12] Zhao Y., Yao R., Ouyang L., et al. Three-dimensional printing of Hela cells for cervical tumor model in vitro // Biofabrication. 2014. Vol. 6, N 3. P. 035001. doi: 10.1088/1758-5082/6/3/035001

[13] Hoarau-Véchot J, Rafii A, Touboul C, Pasquier J. Halfway between 2d and animal models: are 3d cultures the ideal tool to study cancer-microenvironment interactions? Int J Mol Sci. 2018;19(1):181. doi: 10.3390/ijms19010181

[14] Hoarau-Véchot J., Rafii A., Touboul C., Pasquier J. Halfway between 2d and animal models: are 3d cultures the ideal tool to study cancer-microenvironment interactions? // Int J Mol Sci. 2018. Vol. 19, N 1. P. 181. doi: 10.3390/ijms19010181

[15] Park Y, Huh KM, Kang SW. Applications of biomaterials in 3d cell culture and contributions of 3d cell culture to drug development and basic biomedical research. Int J Mol Sci. 2021;22(5):2491. doi: 10.3390/ijms22052491

[16] Park Y., Huh K.M., Kang S.-W. Applications of biomaterials in 3D cell culture and contributions of 3D cell culture to drug development and basic biomedical research // Int J Mol Sci. 2021. Vol. 22, N 5. P. 2491. doi: 10.3390/ijms22052491

[17] Nushtaeva AA, Karpushina AA, Ermakov MS, et al. Establishment of primary human breast cancer cell lines using “pulsed hypoxia” method and development of metastatic tumor model in immunodeficient mice. Cancer Cell Int. 2019;19(1):46. doi: 10.1186/s12935-019-0766-5

[18] Nushtaeva A.A., Karpushina A.A., Ermakov M.S., et al. Establishment of primary human breast cancer cell lines using “pulsed hypoxia” method and development of metastatic tumor model in immunodeficient mice // Cancer Cell Int. 2019. Vol. 19, N 1. P. 46. doi: 10.1186/s12935-019-0766-5

[19] Nushtaeva AA, Karpushina AA, Ermakov MS, et al. Breast cancer cells in 3D models changes their response to hormonal and growth factors. Tsitologyia. 2022;64(4):353–365. (In Russ). doi: 10.31857/S0041377122040046

[20] Нуштаева А.А., Савинкова М.М., Ермаков М.С., и др. Клетки рака молочной железы изменяют чувствительность к гормональным и ростовым стимулам при 3D-культивировании // Цитология. 2022. T. 64, № 4. C. 353–365. doi: 10.31857/S0041377122040046

[21] Neeley C, Gaarn L, Marwood T, et al. Abstract 208: a novel cell culture surface supports effective formation of three dimensional cancer spheroids in suspension. Cancer Res. 2013;73(8 suppl.):208. doi: 10.1158/1538-7445.AM2013-208

[22] Neeley C., Gaarn L., Marwood T., et al. Abstract 208: a novel cell culture surface supports effective formation of three dimensional cancer spheroids in suspension // Cancer Res. 2013. Vol. 73, N 8 (suppl.). P. 208. doi: 10.1158/1538-7445.AM2013-208

[23] Van Der Valk J, Brunner D, De Smet K, et al. Optimization of chemically defined cell culture media — replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro. 2010;24(4):1053–1063. doi: 10.1016/j.tiv.2010.03.016

[24] Van Der Valk J., Brunner D., De Smet K., et al. Optimization of chemically defined cell culture media — replacing fetal bovine serum in mammalian in vitro methods // Toxicol In Vitro. 2010. Vol. 24, N 4. P. 1053–1063. doi: 10.1016/j.tiv.2010.03.016

[25] Bielecka ZF, Maliszewska-Olejniczak K, Safir IJ, et al. Three-dimensional cell culture model utilization in cancer stem cell research: 3D cell culture models in CSCs research. Biol Rev Camb Philos Soc. 2017;92(3):1505–1520. doi: 10.1111/brv.12293

[26] Bielecka Z.F., Maliszewska-Olejniczak K., Safir I.J., et al. Three-dimensional cell culture model utilization in cancer stem cell research: 3D cell culture models in CSCs research // Biol Rev Camb Philos Soc. 2017. Vol. 92, N 3. P. 1505–1520. doi: 10.1111/brv.12293

[27] Xie G, Ji A, Yuan Q, et al. Tumour-initiating capacity is independent of epithelial–mesenchymal transition status in breast cancer cell lines. Br J Cancer. 2014;110(10):2514–2523. doi: 10.1038/bjc.2014.153

[28] Xie G., Ji A., Yuan Q., et al. Tumour-initiating capacity is independent of epithelial–mesenchymal transition status in breast cancer cell lines // Br J Cancer. 2014. Vol. 110, N 10. P. 2514–2523. doi: 10.1038/bjc.2014.153

[29] Johnson S, Chen H, Lo PK. In vitro tumorsphere formation assays. Bio Protoc. 2013;3(3):e325. doi: 10.21769/BioProtoc.325

[30] Johnson S., Chen H., Lo P.-K. In vitro tumorsphere formation assays // Bio Protoc. 2013. Vol. 3, N 3. P. e325. doi: 10.21769/bioprotoc.325

[31] Lo PK, Kanojia D, Liu X, et al. CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin–TGFβ signaling. Oncogene. 2012;31(21):2614–2626. doi: 10.1038/onc.2011.439

[32] Lo P.K., Kanojia D., Liu X., et al. CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin–TGFβ signaling // Oncogene. 2012. Vol. 31, N 21. P. 2614–2626. doi: 10.1038/onc.2011.439

[33] Yang Z, Xiong HR. Culture conditions and types of growth media for mammalian cells. In: Ceccherini-Nelli L, editor. Biomedical tissue culture. InTech; 2012. doi: 10.5772/52301

[34] Yang Z., Xiong H.R. Culture conditions and types of growth media for mammalian cells. In: Ceccherini-Nelli L, Matteolo B., editors. Biomedical tissue culture. Rijeka : InTech, 2012. P. 3–18. doi: 10.5772/52301

[35] Jorissen R. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res. 2003;284(1):31–53. doi: 10.1016/S0014-4827(02)00098-8

[36] Jorissen R. Epidermal growth factor receptor: mechanisms of activation and signalling // Exp Cell Res. 2003. Vol. 284, N 1. P. 31–53. doi: 10.1016/s0014-4827(02)00098-8

[37] Yun YR, Won JE, Jeon E, et al. Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng. 2010;1(1):218142. doi: 10.4061/2010/218142

[38] Yun Y.R., Won J.E., Jeon E., et al. Fibroblast growth factors: biology, function, and application for tissue regeneration // J Tissue Eng. 2010. Vol. 1, N 1. P. 218142. doi: 10.4061/2010/218142

[39] Dallas M, Balhouse B, Navarro D, et al. Improving cell culture outcomes through stabilized bfgf. Genetic Engineering & Biotechnology News. 2018;38(19):22–23. doi: 10.1089/gen.38.19.07

[40] Dallas M., Balhouse B., Navarro D., et al. Improving cell culture outcomes through stabilized bFGF // Genetic Engineering & Biotechnology News. 2018. Vol. 38, N 19. P. 22–23. doi: 10.1089/gen.38.19.07

[41] Devireddy LR, Myers M, Screven R, et al. A serum-free medium formulation efficiently supports isolation and propagation of canine adipose-derived mesenchymal stem/stromal cells. PLoS One. 2019;14(2):e0210250. doi: 10.1371/journal.pone.0210250

[42] Devireddy L.R., Myers M., Screven R., et al. A serum-free medium formulation efficiently supports isolation and propagation of canine adipose-derived mesenchymal stem/stromal cells // PLoS One. 2019. Vol. 14, N 2. P. e0210250. doi: 10.1371/journal.pone.0210250

[43] Francis GL. Albumin and mammalian cell culture: implications for biotechnology applications. Cytotechnology. 2010;62(1):1–16. doi: 10.1007/s10616-010-9263-3

[44] Francis G.L. Albumin and mammalian cell culture: implications for biotechnology applications // Cytotechnology. 2010. Vol. 62, N 1. P. 1–16. doi: 23.10.1007/s10616-010-9263-3

[45] Mokhtari RB, Qorri B, Sambi M, et al. 3d multicellular stem-like human breast tumor spheroids enhance tumorigenicity of orthotopic xenografts in athymic nude rat model. Cancers (Basel). 2021;13(11):2784. doi: 10.3390/cancers13112784

[46] Mokhtari R.B., Qorri B., Sambi M., et al. 3d multicellular stem-like human breast tumor spheroids enhance tumorigenicity of orthotopic xenografts in athymic nude rat model // Cancers (Basel). 2021. Vol. 13, N 11. P. 2784. doi: 10.3390/cancers13112784

[47] Troitskaya O, Novak D, Nushtaeva A, et al. Egfr transgene stimulates spontaneous formation of mcf7 breast cancer cells spheroids with partly loss of her3 receptor. Int J Mol Sci. 2021;22(23):12937. doi: 10.3390/ijms222312937

[48] Troitskaya O., Novak D., Nushtaeva A., et al. EGFR Transgene stimulates spontaneous formation of MCF7 breast cancer cells spheroids with partly loss of HER3 receptor // Int J Mol Sci. 2021. Vol. 22, N 23. P. 15. doi: 10.3390/ijms222312937

[49] Azamjah N, Soltan-Zadeh Y, Zayeri F. Global trend of breast cancer mortality rate: a 25-year study. Asian Pac J Cancer Prev. 2019;20(7):2015–2020. doi: 10.31557/APJCP.2019.20.7.2015

[50] Azamjah N., Soltan-Zadeh Y., Zayeri F. Global trend of breast cancer mortality rate: a 25-year study // Asian Pac J Cancer Prev. 2019. Vol. 20, N 7. P. 2015–2020. doi: 10.31557/APJCP.2019.20.7.2015

[51] Jo Y, Choi N, Kim K, et al. Chemoresistance of cancer cells: requirements of tumor microenvironment-mimicking in vitro models in anti-cancer drug development. Theranostics. 2018;8(19):5259–5275. doi: 10.7150/thno.29098

[52] Jo Y., Choi N., Kim K., et al. Chemoresistance of cancer cells: requirements of tumor microenvironment-mimicking in vitro models in anti-cancer drug development // Theranostics. 2018. Vol. 8, N 19. P. 5259–5275. doi: 10.7150/thno.29098

[53] Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumor biology. Neoplasia. 2015;17(1):1–15. doi: 10.1016/j.neo.2014.12.004

[54] Weiswald L.-B., Bellet D., Dangles-Marie V. Spherical cancer models in tumor biology // Neoplasia. 2015. Vol. 17, N 1. P. 1–15. doi: 10.1016/j.neo.2014.12.004

[55] Echave MC, Burgo LS, Pedraz JL, Orive G. Gelatin as biomaterial for tissue engineering. Curr Pharm Des. 2017;23(24):3567–3584. doi: 10.2174/0929867324666170511123101

[56] Echave M.C., Burgo L.S., Pedraz J.L., Orive G. Gelatin as biomaterial for tissue engineering // Curr Pharm Des. 2017. Vol. 23, N 24. P. 3567–358. doi: 10.2174/0929867324666170511123101

[57] Bello AB, Kim D, Kim D, et al. Engineering and functionalization of gelatin biomaterials: from cell culture to medical applications. Tissue Eng Part B Rev. 2020;26(2):164–180. doi: 10.1089/ten.teb.2019.0256

[58] Bello A.B., Kim D., Kim D., et al. Engineering and functionalization of gelatin biomaterials: from cell culture to medical applications // Tissue Eng Part B Rev. 2020. Vol. 26, N 2. P. 164–180. doi: 10.1089/ten.TEB.2019.0256

[59] Saotome T, Shimada N, Matsuno K, et al. Gelatin hydrogel nonwoven fabrics of a cell culture scaffold to formulate 3-dimensional cell constructs. Regen Ther. 2021;18:418–429. doi: 10.1016/j.reth.2021.09.008

[60] Saotome T., Shimada N., Matsuno K., et al. Gelatin hydrogel nonwoven fabrics of a cell culture scaffold to formulate 3-dimensional cell constructs // Regen Ther. 2021. Vol. 18, P. 418–429. doi: 10.1016/j.reth.2021.09.008

[61] Shim K, Kim SH, Lee D, et al. Fabrication of micrometer-scale porous gelatin scaffolds for 3D cell culture. Journal of Industrial and Engineering Chemistry. 2017;50:183–189. doi: 10.1016/j.jiec.2017.02.012

[62] Shim K., Kim S.H., Lee D., et al. Fabrication of micrometer-scale porous gelatin scaffolds for 3D cell culture // Journal of Industrial and Engineering Chemistry. 2017. Vol. 50. P. 183–189. doi: 10.1016/j.jiec.2017.02.012

[63] Shin S, Ikram M, Subhan F, et al. Alginate–marine collagen–agarose composite hydrogels as matrices for biomimetic 3D cell spheroid formation. RSC Adv. 2016;6(52):46952–46965. doi: 10.1039/C6RA01937D

[64] Shin S., Ikram M., Subhan F., et al. Alginate–marine collagen–agarose composite hydrogels as matrices for biomimetic 3D cell spheroid formation // RSC advances. 2016. Vol. 6, N 52. P. 46952–46965. doi: 10.1039/C6RA01937D

[65] Tang Y, Liu J, Chen Y. Agarose multi-wells for tumour spheroid formation and anti-cancer drug test. Microelectronic Engineering. 2016;158:41–45. doi: 10.1016/j.mee.2016.03.009

[66] Tang Y., Liu J., Chen Y. Agarose multi-wells for tumour spheroid formation and anti-cancer drug test // Microelectronic Engineering. 2016. Vol. 158, P. 41–45. doi: 10.1016/j.mee.2016.03.009

[67] Kanda N. Fibroblast spheroids: a useful assay for drug screening in idiopathic pulmonary fibrosis? [dissertation]. London (UK); 2015.

[68] Kanda N. Fibroblast spheroids: a useful assay for drug screening in idiopathic pulmonary fibrosis? [dissertation]. London (UK), 2015.

[69] Bizik J, Kankuri E, Ristimäki A, et al. Cell–cell contacts trigger programmed necrosis and induce cyclooxygenase-2 expression. Cell Death Differ. 2004;11(2):183–195. doi: 10.1038/sj.cdd.4401317

[70] Bizik J., Kankuri E., Ristimäki A., et al. Cell–cell contacts trigger programmed necrosis and induce cyclooxygenase-2 expression // Cell Death Differ. 2004. Vol. 11, N 2. P. 183–195. doi: 10.1038/sj.cdd.4401317

[71] Tan Y, Suarez A, Garza M, et al. Human fibroblast-macrophage tissue spheroids demonstrate ratio-dependent fibrotic activity for in vitro fibrogenesis model development. Biomater Sci. 2020;8(7):1951–1960. doi: 10.1039/C9BM00900K

[72] Tan Y., Suarez A., Garza M., et al. Human fibroblast-macrophage tissue spheroids demonstrate ratio-dependent fibrotic activity for in vitro fibrogenesis model development // Biomater Sci. 2020. Vol. 8, N 7. P. 1951–1960. doi: 10.1039/C9BM00900K

[73] Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol. 2005;15(5):378–386. doi: 10.1016/j.semcancer.2005.05.004

[74] Kleinman H.K., Martin G.R. Matrigel: basement membrane matrix with biological activity // Semin Cancer Biol. 2005. Vol. 15, N 5. P. 378–386. doi: 10.1016/j.semcancer.2005.05.004

[75] Lerman MJ, Lembong J, Muramoto S, et al. The evolution of polystyrene as a cell culture material. Tissue Eng Part B Rev. 2018;24(5):359–372. doi: 10.1089/ten.teb.2018.0056

[76] Lerman M.J., Lembong J., Muramoto S., et al. The evolution of polystyrene as a cell culture material // Tissue Eng Part B Rev. 2018. Vol. 24, N 5. P. 359–372. doi: 10.1089/ten.TEB.2018.0056

[77] Mei J, Böhland C, Geiger A, et al. Development of a model for fibroblast-led collective migration from breast cancer cell spheroids to study radiation effects on invasiveness. Radiat Oncol. 2021;16(1):159. doi: 10.1186/s13014-021-01883-6

[78] Mei J., Böhland C., Geiger A., et al. Development of a model for fibroblast-led collective migration from breast cancer cell spheroids to study radiation effects on invasiveness // Radiat Oncol. 2021. Vol. 16, N 1. P. 159. doi: 10.1186/s13014-021-01883-6

[79] Xie G., Yao Q., Liu Y., et al. IL-6-induced epithelial-mesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures. Int J Oncol. 2012;40(4):1171–1179. doi: 10.3892/ijo.2011.1275

[80] Xie G., Yao Q., Liu Y., et al. IL-6-induced epithelial-mesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures // Int J Oncol. 2012. Vol. 40, N 4. P. 1171–1179. doi: 10.3892/ijo.2011.1275

[81] Karnieli O, Friedner OM, Allickson JG, et al. A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy. 2017;19(2):155–169. doi: 10.1016/j.jcyt.2016.11.011

[82] Karnieli O., Friedner O.M., Allickson J.G., et al. A consensus introduction to serum replacements and serum-free media for cellular therapies // Cytotherapy. 2017. Vol. 19, N 2. P. 155–169. doi: 10.1016/j.jcyt.2016.11.011

[83] Han C, Takayama S, Park J. Formation and manipulation of cell spheroids using a density adjusted PEG/DEX aqueous two phase system. Sci Rep. 2015;5(1):11891. doi: 10.1038/srep11891

[84] Han C., Takayama S., Park J. Formation and manipulation of cell spheroids using a density adjusted PEG/DEX aqueous two phase system // Sci Rep. 2015. Vol. 5, N 1. P. 11891. doi: 10.1038/srep11891

[85] De Hoogt R, Estrada MF, Vidic S, et al. Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project. Sci Data. 2017;4(1):170. doi: 10.1038/sdata.2017.170

[86] De Hoogt R., Estrada M.F., Vidic S., et al. Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project // Sci Data. 2017. Vol. 4, N 1. P. 170. doi: 10.1038/sdata.2017.170

[87] Vinci M, Box C, Eccles SA. Three-dimensional (3d) tumor spheroid invasion assay. J Vis Exp. 2015;(99):52686. doi: 10.3791/52686

[88] Vinci M., Box C., Eccles S.A. Three-dimensional (3D) tumor spheroid invasion assay // J Vis Exp. 2015. N 99. P. 52686. doi: 10.3791/52686

[89] Summerbell ER, Mouw JK, Bell JSK, et al. Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning. Sci Adv. 2020;6(30):eaaz6197. doi: 10.1126/sciadv.aaz6197

[90] Summerbell E.R., Mouw J.K., Bell J.S.K., et al. Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning // Sci Adv. 2020. Vol. 6, N 30. P. eaaz6197. doi: 10.1126/sciadv.aaz6197

[91] Rolver MG, Elingaard-Larsen LO, Pedersen SF. Assessing cell viability and death in 3d spheroid cultures of cancer cells. J Vis Exp. 2019;(148):59714. doi: 10.3791/59714

[92] Rolver M.G., Elingaard-Larsen L.O., Pedersen S.F. Assessing cell viability and death in 3D spheroid cultures of cancer cells // J Vis Exp. 2019. N 148. P. 59714. doi: 10.3791/59714

[93] Nagelkerke A, Bussink J, Sweep FCGJ, Span PN. Generation of multicellular tumor spheroids of breast cancer cells: how to go three-dimensional. Anal Biochem. 2013;437(1):17–19. doi: 10.1016/j.ab.2013.02.004

[94] Nagelkerke A., Bussink J., Sweep F.C.G.J., Span P.N. Generation of multicellular tumor spheroids of breast cancer cells: how to go three-dimensional // Anal Biochem. 2013. Vol. 437, N 1. P. 17–19. doi: 10.1016/j.ab.2013.02.004

[95] Froehlich K, Haeger JD, Heger J, et al. Generation of multicellular breast cancer tumor spheroids: comparison of different protocols. J Mammary Gland Biol Neoplasia. 2016;21(3-4):89–98. doi: 10.1007/s10911-016-9359-2

[96] Froehlich K., Haeger J.D., Heger J., et al. Generation of multicellular breast cancer tumor spheroids: comparison of different protocols // J Mammary Gland Biol Neoplasia. 2016. Vol. 21, N 3-4. P. 89–98. doi: 10.1007/s10911-016-9359-2

RIGHTS & PERMISSIONS

Eco-Vector