There are two classical models of carcinogenesis [29, 30]. One is the stochastic model, which is based on the belief that most of tumor cells are capable of extensive proliferation and contribute substantially to tumor maintenance; carcinogenesis could results from the random mutations and the subsequent clonal selections. The other is the hierarchical model, which is based on the belief that there are hierarchical differences among tumor cells, and only a small number of specific cells capable of extensive proliferation can contribute to carcinogenesis. The discovery of CSCs in solid tumors strongly supports the hierarchical model. The CSC hypothesis considers that CSCs subsets are located in the top of the hierarchical structure of tumor cells and directly affect the organization and construction of lower hierarchical cells. Therefore, the identification of tumorigenic liver CSCs could provide new insight into the HCC tumorigenic process and possibly bear great therapeutic implications.
Usually, the isolation and identification of liver CSCs involve two types of methods: one is based on the sorting of side population (SP) cells that can exclude the hoechst 33342 dye . However, Hoechst 33342 is cytotoxic; consequently, SP cells are protected by their membrane transport properties, whereas unprotected non-SP cells suffer toxicity and are unable to grow. Thus the differing tumor-initiation abilities of SP and non-SP cells are most likely due to an artifact of Hoechst 33342 toxicity, rather than due to intrinsic stem-cell properties . The other type includes the fluorescence activated cell sorting (FACS) and the magnetic activated cell sorting (MACS), which are based on cell surface markers. The proposed markers for liver CSCs include CD133, CD90, CD44, CD13, EpCAM and OV6, on the basis of the hypothesis that CSCs are originated from somatic stem cells and accordingly express the same surface markers [33, 34]. Although they have been reported to be used to enrich CSC fraction, their sensitivity and specificity for identifying liver CSCs are being challenged. For example, Kimura et al reported CD133+ fraction in Hep3B and Huh7 were 16.8% and 2.7%, respectively , whereas some other groups reported more than 90% in Hep3B and 60% in Huh7 [5, 6]. In Huh7 the CD13-positive cells typically existed in a CD133strong fraction, but in PLC/PRF/5 the CD13-positive cells were CD133-negative . Some researchers indicated that different culture conditions and differentiated degree of the cells, especially the latter, were important factors. The roles of these phenotypes in defining functionally distinct populations of cells from progenitor to differentiated hepatocytes need to be systemically studied.
Recently, sphere culture has been increasingly used as a method for enriching stem cells which relies on their property of anchorage independent growth. Researchers have reported the application of sphere culture to isolate, enrich, maintain or expand potential CSC subpopulations from various types of cancers [18–25]. The sphere-forming cells from primary tumors, such as breast cancer and ovarian cancer, showed stem-like properties and expressed their CSC markers [19, 23]. It is generally agreed that, like all stem cells, the tumor sphere-forming cells are capable of proliferation, self-renewal and possess higher tumorigenicity. Using neural crest stem cell conditions and sphere formation system, Hansford et al  for the first time successfully expanded tumor cells both from low-risk neuroblastomas and from the bone marrow metastases of high-risk tumors. The latter formed metastatic tumors in a murine xenograft model with as few as 10 cells and could also be serially passaged . To our knowledge, there have been few reports on the isolation and long-term propagation of liver CSCs by the method of sphere culture.
In the present study, using stem cell conditioned culture system, we tested three human hepatoma cell lines, PLC/PRF/5, MHCC97H and HepG2. Cells are plated at a low density (< 5000 cells/well in 6-well plate) to avoid spontaneous cell aggregation. The three cell lines could form clonal nonadherent 3-D spheres, and without exceptions, could also be serially passaged. We evaluated the PLC/PRF/5 sphere-forming cells for their stemness characteristics. It has been showed that they were capable of self-renewal, proliferation, drug resistance, and overexpressing liver CSC related proteins. Xenotransplantation is the gold standard for evaluating tumorigenicity of tumor cells. We tested the third, sixth and ninth generations of the PLC/PRF/5 sphere-forming cells for their tumor initiating capability. It was demonstrated that as few as 500 cells from the PLC/PRF/5 spheres were able to form a tumor when subcutaneously injected into NOD/SCID mice, while 2 × 105 parental cells were needed. This was 400 times higher than that of sphere-forming cells. Moreover, the tumor initiating capability was not decreased as the spheres were passaged. Similar CSC properties of self-renewal, strongly proliferation, drug resistance and tumorigenicity are also found in the MHCC97H and HepG2 spheres. Indeed, the HepG2 parental cells at 106 cells/mouse could not form visible xenografts in nude mice, but its sphere-forming cells at the same amount of cells could form xenograft tumors, suggesting the tumorigenic efficacies of sphere-forming cells were enhanced compared with the parental cancer cells.
To further explore the CSC properties of sphere-forming cells, we examined the sensitivity of sphere-forming cells to chemotherapeutics and the expression of candidate CSC markers. The PLC/PRF/5 sphere-forming cells exhibited general resistance to cisplatin, 5-Fu, gemcitabine, mitomycin and sorafenib, and showed higher survival percentages compared with its parental cells. Synchronously, we found that CD44 expression was obviously enriched in HepG2 and MHCC97H sphere-forming cells compared with their parental cells. CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins, normally takes part in cell-cell and cell-matrix adhesion interactions, which is involved in cancer cell migration, proliferation and metastasis. Accordingly, CD44 expression enrichment in sphere-forming cells may account for their increased survival ability and tumorigenicity. Therefore, we propose that the nonadherent tumor spheres cultured in serum-free condition possess liver CSC properties. This long-term culture system may also provide the means of further purifying and functionally characterizing the biological properties of the liver CSC fraction, with the goal of developing new therapeutic strategies directed specifically against liver CSCs.
Accumulating evidence has been established that the Notch signaling pathway plays vital and universal roles not only in cell differentiation, embryonic development and tissue self-renewal, but also in pathogenesis of some types of human cancers and genetic disorders. Recent advancements have further revealed that the Notch signals produce a marked effect either in stem cells or CSCs. The activated Notch signals can inhibit hematopoietic stem cell differentiation and maintain their pluripotency [36, 37], and maintaining the stem cell population in several solid tissue types, including several neuroectodermal tissues . Only when the Notch signals are activated the cancer stem cell activity could be enhanced to promote intestinal tumor formation . Generally, the Notch signaling pathway is mediated in two different pathways. One is through CSL-DNA binding proteins; the other is the CSL-independent pathway. DTX1 (Deltex-1) is an important transcriptional regulator that is downstream of the Notch receptor in the CSL-independent Notch signaling pathway . Ep300, also known as p300, is a transcriptional co-activator protein. It functions as a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation. It has been reported that Ep300 can work as a transcriptional co-activator of DTX1. Yamamoto et al  reported that DTX1 inhibited the transcriptional activation of the neural-specific helix-loop-helix type transcription factor MASH1 by binding to Ep300. This mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. In our study, the result of stem cell microarrays showed that DTX1 and Ep300 were highly expressed in liver cancer stem-like cells. This was further confirmed by Western blotting. Although the molecular mechanism and function of the CSL-independent Notch signaling pathway have not been elucidated, and little has been known about its involvement in HCC, we suppose that the CSL-independent Notch signaling pathway play an important role in the differentiation and propagation of liver CSCs.