Abstract
BackgroundsCancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC.MethodsBased on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS.ResultsOnly Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(−) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(−), CD13(−)/CD166(−) and CD13(−)/CD166(+) fractions, whereas CD13(−)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(−) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(−) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(−) and CD13(−)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(−) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone.ConclusionsWe identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a “population change” upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of “heterogeneous, unstable” cell line may prove more useful in the CSC era than conventional “homogeneous, stable” cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1297-7) contains supplementary material, which is available to authorized users.
Highlights
For a long time, tumor progression was explained on the basis of a stochastic model in which every cancer cell in a tumor could repopulate the entire tumor mass
We found that the Li-7 cell line exhibited a “population change” from CD13(+)/CD166(−) slow-glowing Cancer stem cell (CSC) to CD13(−)/CD166(+) rapidly-growing progenitor cells
The Li-7 cell line showed a population change: the FACS analysis indicated that in this cell line the proportion of CD13(+) cells decreased, while that of CD166(+) cells increased after 2 months in culture (Table 1)
Summary
Tumor progression was explained on the basis of a stochastic model in which every cancer cell in a tumor could repopulate the entire tumor mass. Slow-growing CSCs, which are at the top of this hierarchy, are resistant to conventional chemotherapy or radiotherapy and account for the progression, metastasis and recurrence of cancers [2,3]. This new CSC model has deepened our understanding of the complexity of tumor tissues [4]. A range of therapeutic options is currently available for HCC depending on the clinical stage of the disease [6]. There is considerable interest in developing more effective therapeutic strategies, especially for advanced stage HCC patients. CSCs for HCC have been visualized by their low levels of proteasome and reactive oxygen species (ROS) [19]
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