Isolation, expansion and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer

Abstract

The cancer stem cell (CSC) theory suggests that cancer is maintained by a subpopulation of cancer cells which possesses stem cell characteristics, including self renewal, tumor initiation and pluripotent differentiation abilities. A large series of CSC markers has been reported in various cancer types. CD133 is the most frequently used marker and has been applied to isolate cancer stem cells from cancers of the liver, brain, colon and lung, etc. CD44+/CD24-/low were used as CSC markers in breast cancers. CD34 and Sca-1 were used for identification of murine lung stem cells but Sca-1 is not expressed in human tissues. Expression of embryonic genes such as BMI1 and POU5F1 were found in CSC from different tissues and both proteins are key components in maintaining ‘stemness’ of embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells. It still remains unclear whether additional markers are needed to detect lung CSC. To our knowledge, limited studies using CSC markers on a panel of non-small cell lung cancer (NSCLC) cell lines including those raised from Chinese lung cancer patients have been documented. This study aimed to examine the expression profiles of 6 CSC markers (CD24, CD34, CD44, CD133, BMI1 and POU5F1) in a panel of 10 NSCLC cell lines, 163 NSCLC tumor specimens, fetal lung, reactive and regenerating lung tissues. NSCLC cell lines showed expression of CSC markers at various frequencies that ranged from 0-99.7%, suggesting absence of a single universal marker. Six cell lines expressed CD44 while only HCC1833 expressed CD133. Expression of CD44 and CD133 were mutually exclusive. The CD133+ and CD44+ populations showed distinct properties from CD133- and CD44- cells, respectively. Both CD133+ and CD44+ expanded into tumor-spheroids in vitro in non-adherent medium but not CD133- and CD44- cells. CD44+ H1299 cells expressed ‘stemness’ markers including Oct4, Nanog and Sox2 by RT-PCR and immunofluorescence but were lost after induced-differentiation. The clonogenicity of CD44+ cells is significantly higher than CD44- cells and self-renewal ability was maintained after serial passages. CD44+ cells were cisplatin-resistant and initiated tumor formation in nude mice. The xenograft tumors showed poor differentiation that resembled the parental cell line. Freshly sorted tumor cells showed both CD44+ and CD44- populations, suggesting in vivo differentiation. Immunohistochemistry (IHC) results showed CD44 expression in epithelia of developing airways and regenerating lung. In lung cancers, CD44 expression was associated with squamous cell carcinoma (SCC) (P=0.046). When only 96 adenocarcinomas (AD) were included, CD44 expression was associated with non-smokers (P=0.024). Overall, CD44+ cells in NSCLC are capable of tumor-initiation and possess stem cell characteristics.