Enhanced expression of stem cell markers and drug resistance in sphere-forming non-small cell lung cancer cells

Abstract

There is growing evidence suggesting that cancer stem cells (CSCs) are playing critical roles in tumor progression, metastasis and drug resistance. However, the role of CSCs in non-small cell lung cancer (NSCLC) remains elusive. In this study, we enriched for stem-like cells from tumor spheres derived from NSCLC cell line A549 cultured in serum-free medium. Our results showed that sphere-derived cells expressed various stem cell markers such as CD44, CD133, Sox2 and Oct4. Compared with the corresponding cells in monolayer cultures, sphere-derived cells showed marked morphologic changes and increased expression of the stem cell markers CD133. Furthermore, we found that sphere-derived cells exhibited increased proliferation, cell-cycle progression as well as drug-resistant properties as compared to A549 adherent cells. Consistently, expression of several drug resistance proteins, including lung resistance-related protein (LRP), glutathion-S-transferase-π (GST-π) and multidrug resistance proteins-1 (MRP1) were all significantly enhanced in sphere-derived cells. These results indicate the enrichment of CSCs in sphere cultures and support their role in regulating drug resistance in NSCLC.

Keywords: CD133; Cancer stem cell; GST-π; LRP; MRP1; multidrug resistance.

Figure 1

Isolation and identification of LCSCs. A. Three types of colonies formed by A549 cells. (400×). B. Immunostaining of stem cell markers in secondary spheres. Nuclei were stained using DAPI (blue). LCM, scale bar = 25 10, 50, 75, 10, 30 μm (left to right).

Figure 2

A549 cancer spheres have high express CD133 and more self renewal capability. A. The morphology of tumor spheres. a. Primary tumor spheres formed in the CSM medium. b. Secondary spheres derived from single parental cells of primary tumor spheres under microscopy. a and b. 200×. B. Flow cytometry analysis of CD133 in A549 cancer spheres (left) and A549 cells lines (right). Cells were labeled with fluorescent anti-CD133 antibody, which are shown as black areas. The white area on each box represents the corresponding negative control labeling, and the line denotes a positive gate. Numbers are the percentage of positive cells. Data are representative of three independent experiments. C. Detection of CD133 positive rate among four groups cells. The results are expressed as the mean ± SD of three experiments (n = 3). #P<0.05, compared to A549 cells. D. Growth curves 1. A549 cancer spheres cultured in DMEM with 10% FBS; 2. A549 cancer spheres cultured in serum-free Cancer Stem Cell medium; 3. A549 cells line cultured in DMEM with 10% FBS. *P < 0.05, compared to Tumor stem cells in serum-free stem cell culture medium. #P < 0.05, ##P < 0.01, compared to A549 cells line cultured in DMEM with 10% FBS.

Figure 3

Measurement of the sensitivity of A549 cancer spheres and A549 cells line to anticancer drugs (DDP and GEM). A. The lethal doses (IC50) of DDP (left) and GEM (right) in A549 cancer spheres and A549 cells line, The data shown represent the mean ± SD of three independent experiments(n = 3). #P<0.05, compared to A549 cells line. B. Graphs shows flow cytometry of the apoptotic induced by DDP and GEM. The cells were stained with annexin V-FITC and Propidium Iodideed. a. A549 cells line; b. A549 cancer spheres; c. A549 cells line/DDP; d. A549 cancer spheres/DDP; e. A549 cells line/GEM; f. A549 cancer spheres/GEM. C. Histograms represent the percents of Apoptotic of DDP and GEM in A549 cancer spheres and A549 cells line. The data shown represent the mean ± SD of three independent experiments (n = 3), #P < 0.05, compared with A549 spheres/DDP; ★P < 0.05, compared with A549 spheres/GME.

Figure 4

Effect of the IC50 concentration of Cisplatin (DDP) and Gemcitabine (GEM) on the cell cycle profile of A549 cells line and A549 spheres. A. A549 cells line; B. A549 cells line/DDP; C. A549 cells line/GEM; D. A549 cancer spheres; E. A549 cancer spheres/DDP; F. A549 cancer spheres/GEM. This experiment was repeated three separate times, and similar results were obtained. The representative flow cytometry pattern is shown.

Figure 5

Enhanced expression of GST-π, MRP1 and LRP in A549 spheres. A. Western-blot of GST-π, MRP1 and LRP in HEB, A549 cells and A549 spheres. B. Immunofluorescence of GST-π expression in HEB, A549 cells and A549 spheres using confocal microscopy. Scale bars: 25 µm.