Evaluation of Impacts of Cellular Metabolism on the Migration of Ovarian Cancer Cells by Two in Vitro Assays: A Method Comparison Study
Abstract
Background: Alteration of metabolic pathways in cancer cells can intensely modulate their migration as an important step in invasion and metastasis. Ketogenic diet showed some contradictory results in cancer patients. In this study the impact of metabolic reprogramming of A2780CP as a model of ovarian cancer stem-like cells on cell migration by two in vitro methods: wound healing and soft agar colony-forming assays. Materials and Methods: short term and long term metabolic reprogramming were done by restriction of glucose to 250mg/L with or without enrichment with beta-hydroxybutyrate (5 milimolar) for 48 hours and 30 days, respectively. Wound healing assay was done and the wound ratio was calculated for 24 and 48 hours. Soft agar colony formation assay was also done in treated and control cells. For method comparison, ten biological replicates were analyzed in triplicate. Results: Migration of A2780CP ovarian cancer stem-like cells were significantly alleviated by long term glucose restriction but no significant changes were observed in short term study. Beta-hydroxybutyrate enrichment did not produce significant impacts on glucose restriction in short or long term studies. Conclusion: The results of colony formation in soft agar and wound or scratch healing assay were in good correlation and convergence which could be used interchangeably in the investigation of metabolic reprogramming in cancer cells. [GMJ.2020;9:e1831]References
Pijuan J, Barcelo C, Moreno DF, Maiques O, Siso P, Marti RM et al. In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. Front Cell Dev Biol. 2019;7:107.
https://doi.org/10.3389/fcell.2019.00107
PMid:31259172 PMCid:PMC6587234
Cappiello F, Casciaro B, Mangoni ML. A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration. J Vis Exp. 2018(133).
https://doi.org/10.3791/56825
PMid:29608162 PMCid:PMC5931780
Grada A, Otero-Vinas M, Prieto-Castrillo F, Obagi Z, Falanga V. Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay. J Invest Dermatol. 2017;137(2):e11-e6.
https://doi.org/10.1016/j.jid.2016.11.020
PMid:28110712
Martinotti S, Ranzato E. Scratch Wound Healing Assay. Methods Mol Biol. 2019.
https://doi.org/10.1007/7651_2019_259
PMid:31414347
Borowicz S, Van Scoyk M, Avasarala S, Karuppusamy Rathinam MK, Tauler J, Bikkavilli RK et al. The soft agar colony formation assay. J Vis Exp. 2014(92):e51998.
https://doi.org/10.3791/51998
PMCid:PMC4353381
Horibata S, Vo TV, Subramanian V, Thompson PR, Coonrod SA. Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J Vis Exp. 2015(99):e52727.
https://doi.org/10.3791/52727
PMid:26067809 PMCid:PMC4542786
Finley LWS. Metabolic signal curbs cancer-cell migration. Nature. 2019;571(7763):39-40.
https://doi.org/10.1038/d41586-019-01934-9
PMid:31263261
Magee BA, Potezny N, Rofe AM, Conyers RA. The inhibition of malignant cell growth by ketone bodies. Australian Journal of Experimental Biology and Medical Science. 1979;57(5):529-39.
https://doi.org/10.1038/icb.1979.54
PMid:548019
Seyfried TN, Flores RE, Poff AM, D'Agostino DP. Cancer as a metabolic disease: implications for novel therapeutics. Carcinogenesis. 2013;35(3):515-27.
https://doi.org/10.1093/carcin/bgt480
PMid:24343361 PMCid:PMC3941741
Woolf EC, Curley KL, Liu Q, Turner GH, Charlton JA, Preul MC et al. The ketogenic diet alters the hypoxic response and affects expression of proteins associated with angiogenesis, invasive potential and vascular permeability in a mouse glioma model. PLoS One. 2015;10(6):e0130357.
https://doi.org/10.1371/journal.pone.0130357
PMid:26083629 PMCid:PMC4470583
Tseng P-L, Chen C-W, Hu K-H, Cheng H-C, Lin Y-H, Tsai W-H et al. The decrease of glycolytic enzyme hexokinase 1 accelerates tumor malignancy via deregulating energy metabolism but sensitizes cancer cells to 2-deoxyglucose inhibition. Oncotarget. 2018;9(27):18949-69.
https://doi.org/10.18632/oncotarget.24855
PMid:29721175 PMCid:PMC5922369
O'Flanagan CH, Smith LA, McDonell SB, Hursting SD. When less may be more: calorie restriction and response to cancer therapy. BMC medicine. 2017;15(1):106.
https://doi.org/10.1186/s12916-017-0873-x
PMid:28539118 PMCid:PMC5442682
Chung H-Y, Park YK. Rationale, feasibility and acceptability of ketogenic diet for cancer treatment. Journal of cancer prevention. 2017;22(3):127.
https://doi.org/10.15430/JCP.2017.22.3.127
PMid:29018777 PMCid:PMC5624453
Karakuş F, Eyol E, Yılmaz K, Ünüvar S. Inhibition of cell proliferation, migration and colony formation of LS174T Cells by carbonic anhydrase inhibitor. Afr Health Sci. 2018;18(4):1303-10.
https://doi.org/10.4314/ahs.v18i4.51
PMid:30766596 PMCid:PMC6354875
Gupta R, Yang Q, Dogra SK, Wajapeyee N. Serine hydroxymethyl transferase 1 stimulates pro-oncogenic cytokine expression through sialic acid to promote ovarian cancer tumor growth and progression. Oncogene. 2017;36(28):4014-24.
https://doi.org/10.1038/onc.2017.37
PMid:28288142 PMCid:PMC5509519
Saha S, Ghosh M, Dutta SK. The dual-hit metabolic modulator LDCA synergistically potentiates doxorubicin to selectively combat cancer-associated hallmarks. RSC advances. 2017;7(84):53322-33.
https://doi.org/10.1039/C7RA08625C

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