| Background & ObjectiveAs the concept of stem cell was introduced into oncology research, the theory of cancer stem cell has been put forth which stipulates that it is a small population of cells called cancer stem cells that initiates tumor formation and maintains tumor growth. These cells are rare within the bulk of the tumor mass, and possess stem cell like properties including self-renewal, differentiation, and resistance to therapies, and high tumorigenicity in vivo. Isolation of cancer stem cells is of great significance in oncology research. In the past several years, the existence of liver cancer stem cells (LCSCs) has been substantially proven by the identification and isolation of a subpopulation of liver cancer cells using side population technique or fluorescence-activated cell sorting or magnetic-activated cell sorting based on surface markers such as CD 133, CD90, OV6 and EpCAM. However, there still have been controversies over the specific maker of LCSCs and direct evidence that liver cancer stem/progenitor cells could be cultured and propagated in vitro is still lacking. In the current study, we propose to establish a method which could effectively enrich and propagate subpopulations with LCSCs characteristics in vitro.MethodsUsing the stem cell culture system, undifferentiated cellular spheres were enriched and amplified from liver cancer cell line PLC/PRF/5, Huh7, HepG2, MHCC97-H. Series characteristics of PLC/PRF/5 spheres were identified as followed.1. Sphere formation assay.2. Colony formation assay.3. Chemotherapy sensitivity assay.4. Assessments of sphere differentiation.5. In vivo tumorigenicity experiments.6. Western blotting and immunofluorescent staining analysis of stem cell genes.7. Real-time PCR microarray analysis of stem cell genes. Results1. One PLC/PRF/5 cell forming a nonadherent three dimensional sphere.2. Cells from spheres possessed the capability for maintenance of their high clonogenicity.3. Compared with PLC/PRF/5 cells, the survival rates of spheroid cells under 12 hour-treatment of 3μg/ml,5μg/ml and 9μg/ml cisplatin were greater (P＜0.01) (1.4-fold, 1.9-fold,1.8-fold respectively), whereas under 24 hour-treatment, values also increased (P＜0.01) (1.5-fold,2.1-fold and 2.3-fold, respectively).4. A subpopulation of spheres under differentiation conditions appealed longitudinal stretched cells, lost or greatly reduced for hepatic cell markers albumin and Hep Parl, and increased expression for skeletal and cardiac cell maker MEF2C, indicating that spheres maybe have the capacity to differentiate into lineages with skeletal and cardiac phenotype.5. At least 500 cells from spheres were able to form a tumor when subcutaneous injected into NOD/SCID mice, while 200,000 normally cultured PLC/PRF/5 liver cancer cells were needed to form a tumor, which was 400 times of spherical cells.6. The makers of liver cancer stem cell previously reported, including CD133, EpCAM and OV6 were up-regulated as compared to culture of cells in monolayer dishes.7. DTX1 and Ep300, the downstream proteins of CSL-independent Notch signaling pathway, were up-regulated in PLC/PRF/5 spheres, suggesting that the CSL-independent Notch signaling pathway may play a significant role in liver cancer stem cells.ConclusionThe method of celluar spheres culturing could efficiently enrich liver cancer stem-like cells, and long-term cultures of these cells would represent a step of crucial importance, which could also be used as a convenient tool to study biological properties and mechanisms of drug resistance of liver cancer stem cells ex-vivo, or establish an appropriate model to study roles of cancer stem cells in the development, progression and treatment of liver cancer. |
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