Türksoy Terzioğlu Özlem, Terzioğlu Gökhan
University of Health Sciences, Hamidiye Institute of Health Sciences, Department of Molecular Biology and Genetics, İstanbul, Turkey.
University of Health Sciences, Validebağ Research Center, İstanbul, Turkey.
Turk J Biol. 2023 Jan 18;47(2):109-119. doi: 10.55730/1300-0152.2646. eCollection 2023.
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.
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.
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.
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.
背景/目的：血清和生长因子剥夺是实体瘤中常见的细胞应激源，在放疗、化疗和抗血管生成时会出现。球体培养旨在通过使用低附着条件和一些生长因子（如碱性成纤维细胞生长因子和表皮生长因子）来富集癌症干细胞，以支持无血清球体培养中的球体形成。然而，不添加任何生长因子的球体培养可以更真实地模拟肿瘤环境。在本研究中，我们旨在确定生长因子剥夺对MKN-45胃癌细胞系干性特征的影响。
通过在低附着条件下培养MKN-45胃癌细胞获得球体，然后将球体解离以获得细胞用于进一步分析。分别使用肿瘤球体形成试验、软琼脂集落形成试验、Transwell侵袭试验、化学敏感性试验和定量RT-PCR试验分析自我更新、多能性、细胞转化、侵袭性、化学抗性以及干性相关基因的表达。
胎牛血清和生长因子剥夺导致OCT4、NANOG、SOX2、MUC1、CD24和CD90等干性标志物增加。在无胎牛血清-生长因子的条件下，还观察到自我更新、化学抗性和侵袭能力等与功能侵袭性相关的特性增加。
生长因子对于富集癌症干细胞的球体培养可能不是必需的。胎牛血清和生长因子的剥夺也会在MKN-45细胞中诱导出更具侵袭性的表型；因此，它为针对肿瘤细胞的进一步研究提供了机会。
