Analogy between sphere forming ability and stemness of human hepatoma cells

Abstract

Abstract. Increasing evidence suggests that cancers contain a small subset of cancer-initiating cells, so-called cancer stem cells (CSCs) that are capable of regenerating a tumor after chemoradiation therapy. Sphere forming ability is known to be one of properties of CSCs, but the significance remains unclear. The present study focused on sphere formation of human hepatoma cells in three-dimensional culture in order to evaluate the analogy between sphere forming ability and stemness of cancer cells in vitro. Under three-dimensional culture condition, HepG2, Hep3B and PLC/PRF/5 cells demonstrated the sphere formation while SK-Hep1 and Huh-7 cells did not. The population of G0/G1 phase increased in the spheres compared with the monolayer (67 vs. 38%). In spite of no significant difference in stem cell surface markers (CD44, CD90, CD133, EpCAM and ABCG2), remarkable upregulation of p27 CDK inhibitor was observed in sphere forming cells. Immunofluorescence analysis revealed the nuclear expression of p27 in the whole of the sphere, but weak expression of p21 only at the peripheral area. The spheres acquired chemoresistance to cisplatin compared with the monolayers (58.9 vs. 16.2 μM in IC50). This model was useful for assessment of the role of cell-cycle quiescence in the stemness and chemoresistance of cancer cells. IntroductionThere is an emerging concept of cancer stem cells (CSCs) that cancer cells do not consist of homogeneous population but include a small subpopulation of cells having the ability of self-renewal and differentiation into multiple phenotypes (1). Several unique properties of CSCs have been identified including drug-efflux ability (side population) (2,3), maintenance of quiescence, sphere formation, high tumorigenicity, and resistance to hypoxia and chemoradiation (4). After anticancer treatment that kills most cancer cells, such drugresistant CSCs might survive and finally generate new populations, resulting in cancer recurrence and metastasis. Detailed analysis on biological characteristics of CSCs are required to overcome the resistance of cancer.Recent studies revealed that the sphere formation might be essential for cancer-initiating ability of CSCs (5-7), but its significance and mechanism remain unclear. The sphereforming cells in human hepatoma were reported to associate with the expression of stemness markers such as CD90 (8,9), CD133 (10,11), EpCAM (12), ABCG2 (13) and CD44 (14). In this study, we focused on the sphere formation ability using 3D culture system to evaluate the in vitro analogy to the stemness phenotypes in human hepatoma cells. Materials and methodsCell lines. Human hepatoma cell lines, HepG2, Huh-7, PLC/ PRF/5 and SK-Hep1 were analyzed (15). Additionally, we used p53-deficient hepatoma cell line Hep3B as well as Hep3B transfected with wild-type p53 gene (16); named as Hep3B-p53(-) and Hep3B-p53(+), respectively. Culture media were the recommended media supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. All cell lines were cultivated...

The mitochondrial Ca2+channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation

“…In addition to identifying a critical role of MCU in tumor growth, we determined that MCU deletion in transformed fibroblasts diminished malignant capabilities in vitro. Our studies indicate that inhibition of MCU-mediated Ca 2+ uptake limits the ability of transformed fibroblasts to form clonally-derived spheres and invade, capabilities associated with initiation and progression of metastatic tumors (Uchida et al, 2010;Ishiguro et al, 2017), as previously observed (Tosatto et al, 2016). Previous studies have suggested a role for MCU as a promoter of invasion and metastasis in breast cancer and colorectal carcinoma-derived cell lines (Curry et al, 2013;Marchi et al, 2013;Tang et al, 2015;Cardenas et al, 2016;Tosatto et al, 2016;Ren et al, 2017;Yu et al, 2017;Liu et al, 2020).…”

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation

“…The ability to form clonally-derived spheres on non-adherent substrates is a stem cell-like capacity related to metastatic tumor initiation and progression ( Uchida et al, 2010 ). In a sphere-formation assay, the transformed fibroblasts, but not the immortalized cells, readily formed spheres (>30 spheres per well) ( Figure 4A ), consistent with their differential abilities to form tumors in vivo ( Figure 1B ).…”

GNS561, a New Autophagy Inhibitor Active against Cancer Stem Cells in Hepatocellular Carcinoma and Hepatic Metastasis from Colorectal Cancer

“…Moreover, there is a lack of consensus biomarkers for CSCs in HCC 24 , 29 . Consequently, the CSC subpopulation is commonly identified in HCC based on functional criteria such as their capacity to efflux Hoechst 33342 dye (side population) 30 or to form tumorspheres 31 , 32 , as only these cells have the ability to proliferate in non-adherent conditions 33 , 34 . Tumorsphere formation assay was used to isolate the CSC subpopulation of HCC cell lines.…”