Effects of growth factor deprivation on MKN-45 spheroid cells.

Abstract

Background/aim: Serum and growth factor deprivation, a common cellular stressor in solid tumors, arises upon irradiation, chemotherapy, and antiangiogenesis. Spheroid body culture aims to enrich cancer stem cells by using low attachment conditions and some growth factors, such as basic fibroblast growth factor and epidermal growth factor to support the spheroid formation in serum-free spheroid culture. However, spheroid culture without any growth factors can imitate the tumor environment more realistically.In this study, we aimed to identify the effect of growth factor deprivation on the MKN-45 gastric cancer cell line in terms of stemness characteristics.

Materials and methods: The spheroids were obtained by culturing MKN-45 gastric cancer cells in low attachment conditions, and then spheroids were dissociated to obtain cells for further analyses. Self-renewal, multipotency, cellular transformation, invasiveness, chemoresistance, and the expression of stemness-related genes were analyzed using tumor spheroid formation assay, soft agar colony formation assay, transwell invasion assay, chemosensitivity assay, and quantitative RT-PCR assay, respectively.

Results: Fetal bovine serum and growth factor deprivation caused an increase in stemness markers of OCT4, NANOG, SOX2, MUC1, CD24 and CD90. Increasing functional aggressiveness-related properties, such as self-renewal, chemoresistance, and invasive ability, have also been observed in fetal bovine serum-growth factor-free conditions.

Conclusion: Growth factors may not be essential for spheroid culture to enrich cancer stem cells. The deprivation of both fetal bovine serum and growth factors also induces a more aggressive phenotype in MKN-45 cells; thus, it provides an opportunity for further studies targeting tumor cells.