Abstract
Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.
Highlights
Lung cancer stem cells (CSCs) are a group of cells with self-renewal capability, which may play a key role in the initiation and development of lung cancer [1,2,3]
We report the cloning, identification and characterization of the symmetrical division (SD) and asymmetric division (ASD) cancer cells derived from mouse parental Lewis lung carcinoma cell line (LLC-Parental) which were named Lewis lung cancer cells (LLC)-SD and LLC-ASD respectively
The experiment of tumor growth in nude mice which is considered the gold standard for characterization of lung CSCs in vivo were carried out for LLC-SE and LLCParental
Summary
Lung cancer stem cells (CSCs) are a group of cells with self-renewal capability, which may play a key role in the initiation and development of lung cancer [1,2,3]. After the lung CSCs were identified and obtained through CD133 marker sorting, more recent research efforts have been put forward to understand how lung CSCs maintain their ability of self-renewal [1, 4,5,6]. The stemness is maintained by the symmetrical division(SD) for self-renewal and the asymmetric division(ASD) for undergoing differentiation [8, 9]. Illustration of the ability of CSCs to undergo terminal differentiation is prevented by the constitutive activation of the growth signaling pathways [10,11,12]. Since ASD is a unique feature of normal stem cells, the stemness of CSCs can be probed through the demonstration of whether the CSCs can under go ASD
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