The cancer stem cell (CSC) hypothesis proposes that a small population of cells with self-renewing tumorigenic potential exists within a tumor, and may be responsible for disease relapse after therapeutic intervention. In lung cancer, the search for CSCs and biomarkers defining them is complicated by the morphological and functional heterogeneity of the tumor tissue, and the site of carcinogenesis. Biomarkers for lung CSCs are not clearly defined. To aid in understanding the nature of human lung CSCs, we are developing in vitro culture sphere-forming conditions in an attempt to isolate and propagate cells with proposed functional properties of CSCs from early stage lung tumors resected from patients, without bias toward biomarker expression. We have used human NSCLC cell lines to optimize sphere-forming conditions which allow for propogation of cells under clonogenic, yet gentle, in vitro culture conditions. Utilizing defined “stem cell” media in soft agarose, anchorage independent conditions, we have cultured and isolated clonal spheres. These spheres were expanded in vitro, cryopreserved, and recultured in vitro for further analyses. To date, we have generated clonal sphere populations from six different NSCLC lines: one adenocarcinoma (A549), one squamous cell carcinoma (SW900), one NSCLC of neuroendocrine origin (H1299), and three large cell carcinomas (H460, H661, and H1581). Efficiency of sphere formation varied among the 6 lines, with H460, H1581, and SW900 having about 50% sphere formation under clonal conditions, approximately 10% for H1299, less than 3% for A549, and less than 1% for H661. Cell surface biomarkers were evaluated by western blot analysis of detergent solubilized membranes from paired NSCLC cell lines and their respective spheres. CD133, a proposed biomarker for lung CSCs, was detected albiet at different levels on membranes of each of the six sphere cultures. In contrast, CD133 was absent from the membranes of five of the six parent NSCLC cell lines grown under adherent conditions. The single parent cell line with expression of CD133 is H1581. To determine whether there are changes in epithelial-to-mesenchymal transition (EMT) markers between the parent cell lines and their corresponding spheres, we examined E-cadherin and fibronectin expression by western blot. No relationship between loss of the epithelial marker E-cadherin and sphere formation was observed. In fact, both A549 and its corresponding spheres expressed very high levels of E-cadherin, while most of the other parent cell lines and their corresponding spheres expressed low levels of this marker. Expression of fibronectin, which is upregulated during EMT, showed no consistent pattern between cell lines and spheres, suggesting that there is no relationship between in vitro sphere formation and EMT in these NSCLC cell lines. Expression of ALDH1, a marker whose expression in NSCLC tumors has been associated with poor prognosis, along with nuclear expression of OCT4, a transcription factor important in maintenance of normal stem cell states, and cKit, the receptor for Stem Cell Factor, are under examination.

We expect that the development and analysis of spheres generated from human NSCLC cell lines under clonal conditions will assist us in developing conditions that will promote the in vitro isolation and expansion of sphereforming cells from early stage lung cancer patients.

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