Aldehyde Dehydrogenase 1A1 Possesses Stem-Like Properties and Predicts Lung Cancer Patient Outcome  Xiao Li, MD, Liyan Wan, MD, Jian Geng, MD, Chin-Lee Wu, MD, PhD, Xiaoyan Bai, MD, PhD  Journal of Thoracic Oncology  Volume 7, Issue 8, Pages 1235-1245 (August 2012) DOI: 10.1097/JTO.0b013e318257cc6d Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 ALDH1A1 expression in lung carcinomas was associated with poor prognosis. A, Expression analysis of ALDH1A1 protein in normal lung tissues and lung carcinomas using tissue microarrays (left panel: scale bar 100 μm) and the corresponding lower right quadrant magnified images (right panel: scale bar 50 μm) by immunohistochemistry. ALDH1A1 staining was localized in the cytoplasm. Aa and Ag, Weak ALDH1A1 expression in the alveolar epithelial cells of normal lung tissues. Ab and Ah, Strong ALDH1A1 expression in the basal cells and globular cells of the normal bronchus. Ac and Ai, Moderate ALDH1A1 expression in squamous-cell lung carcinomas; (Ad and Aj) strong ALDH1A1 expression in squamous-cell lung carcinomas; (Ae and Ak) moderate ALDH1A1 expression in lung adenocarcinomas; (Af and Al) strong ALDH1A1 expression in lung adenocarcinomas. B, Increased ALDH1A1 levels in human lung adenocarcinomas (Ba) and squamous-cell lung carcinomas (Bb) compared with corresponding normal lung tissues detected by immunoblotting. Expression levels of ALDH1A1 were normalized by β-actin. C, Kaplan-Meier survival analysis of overall survival in all patients according to ALDH1A1 expression. The log-rank test was used to calculate the p value (p < 0.001). ALDH1A1, aldehyde dehydrogenase 1A1. Journal of Thoracic Oncology 2012 7, 1235-1245DOI: (10.1097/JTO.0b013e318257cc6d) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 ALDH1A1 promoted aggressive phenotypes of lung cancer cells and possessed tumor stem-cell properties in vitro. A, FACS analysis of lung cancer cells using the ALDH1A1 assay. The brightly fluorescent ALDH1A1+ cells were detected in the green fluorescence channel. The cells incubated with diethylaminobenzaldehyde were used to establish the baseline fluorescence of these cells (R1) and define the ALDH1A1 (ALDH1A1+) region (R2). B, Immunoblotting analysis of the unsorted, ALDH1A1+, and ALDH1A1− cells, and the relative quantification of ALDH1A1 protein expression. C, Immunofluorescence images of the unsorted, ALDH1A1+ and ALDH1A1− cells stained for ALDH1A1 (red), Hoechst33342 (blue) and colocalization of the merged images (scale bar 50 μm). D, ALDH1A1+ lung cancer cells formed larger and more colonies as compared with the ALDH1A1− cells, by colony-formation assays. ALDH1A1+ cells possessed significantly higher colony-forming efficiency compared with ALDH1A1− populations. The unsorted, ALDH1A1+ and ALDH1A1− cells were plated in a 6-well dish. Two weeks after plating, clones were counted and results were presented as the percentage of cloning efficiency (y axis). Columns indicate mean from three independent experiments; bars represent SD; *p < 0.05. E, ALDH1A1+ cells promoted cell growth and proliferation. Data were presented as mean ± SD of the absorbance value (optical density) of cells. There was significant difference between ALDH1A1+ and ALDH1A1− cells. F, Transwell assay showed that ALDH1A1+ cells had more migratory potentialities as compared with ALDH1A1− cells. G, Cell cycles were detected with flow cytometry analysis. Increased S and G2/M phases were observed in ALDH1A1+ cells compared with ALDH1A1− cells. Each bar represented the mean ± SD. Results were representative of three independent experiments, *p < 0.05. ALDH1A1, aldehyde dehydrogenase 1A1; FACS, Fluorescence Activating Cell Sorter. Journal of Thoracic Oncology 2012 7, 1235-1245DOI: (10.1097/JTO.0b013e318257cc6d) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Light microscopic and SEM images of FACS-sorted and siRNA-transfected cells. A, Light microscopic images of unsorted (a), ALDH1A1+ (b), ALDH1A1− (c), NT siRNA (d), ALDH1A1 siRNA #1 transfected cells (e), and ALDH1A1 siRNA #2 transfected H2087 lung cancer cells (f). B, SEM images of unsorted (g), ALDH1A1+ (h), ALDH1A1− (i), NT siRNA (j), ALDH1A1 siRNA #1 transfected cells (k), and ALDH1A1 siRNA #2 transfected H2087 lung cancer cells (l). Images were taken at the magnification of ×2000. ALDH1A1, aldehyde dehydrogenase 1A1; SEM, Scanning Electron Microscope; SiRNA, small interfering RNA; FACS, Fluorescence Activating Cell Sorter; NT, nontargeting. Journal of Thoracic Oncology 2012 7, 1235-1245DOI: (10.1097/JTO.0b013e318257cc6d) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 In vitro silencing of ALDH1A1 inhibited proliferation and migration of lung cancer cells. A, Oligo siRNA-mediated knockdown of H2087 cells was confirmed by RT-PCR and Western blot. ALDH1A1 mRNA expression detected by RT-PCR and the relative quantification of ALDH1A1 mRNA expression were shown in the left panel. ALDH1A1 protein expression detected by Western blot and the relative quantification of ALDH1A1 protein expression were shown in the right panel. ALDH1A1 expression was normalized by β-actin expression. B, Immunofluorescence analysis of H2087 lung cancer cells transfected with NT siRNA and ALDH1A1 siRNA stained with Hoechst33342 (blue) and ALDH1A1 (red) (scale bar 50 μm). C, In vitro silencing of the ALDH1A1 gene suppressed cell proliferation. Data were presented as mean ± SD of the OD of cells. There was significant difference in the OD between NT siRNA transfected and ALDH1A1 siRNA transfected cells. D, Cell cycles were detected with flow cytometry analysis. Increased S phase and decreased G2/M phase were detected in the ALDH1A1 siRNA transfected cells compared to the control cells. E, Decreased migratory potentialities were observed in ALDH1A1 siRNA transfected cells compared with the NT siRNA control cells. Each bar represents the mean ± SD. Results are representative of three independent experiments. *p < 0.05. ALDH1A1, aldehyde dehydrogenase 1A1; siRNA, small interfering RNA; RT-PCR, reverse transcription-polymerase chain reaction; mRNA, messenger RNA; OD, optical density; NT, nontargeting control. Journal of Thoracic Oncology 2012 7, 1235-1245DOI: (10.1097/JTO.0b013e318257cc6d) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 ALDH1A1 promoted tumor growth in vivo. A, The tumor-formation ability of ALDH1A1+ cells was greater than that of ALDH1A1− and unsorted cells. Representative images of tumor formation after injection of unsorted, ALDH1A1+, and ALDH1A1− cells were shown. Arrows indicate subcutaneous tumors. B, A dose of 103 or 105 unsorted, ALDH1A1+, and ALDH1A1− cells were injected into the left flank of 10 balb/c nude mice respectively, as shown on the y axis. After 4 weeks, 103 ALDH1A1+ cells generated tumors in all mice, whereas the same amount of ALDH1A1− cells did not produce tumor mass in any of the 10 mice. The dose of 105 ALDH1A1+ cells yielded tumors in all mice, whereas the same amount of ALDH1A1− cells produced a small tumor mass in three mice. C, ALDH1A1+ cells generated larger tumors compared with ALDH1A1− cells. A dose of 105 ALDH1A1+ cells yielded tumors with an average of 31.0 ± 0.9 mm3, whereas the same dose of ALDH1A1− cells produced a smaller tumor mass (9.0 ± 0.7 mm3) in three of the mice. D, Histopathologic examination of the engrafted tumors formed by unsorted, ALDH1A1+ and ALDH1A1− lung cancer cells showed a highly cellular mass recapitulating the morphology of the primary lung tumors. The expression of ALDH1A1 was higher in tumors produced by ALDH1A1+ cells, compared with those produced by the unsorted and ALDH1A1− cells. ALDH1A1, aldehyde dehydrogenase 1A1. Journal of Thoracic Oncology 2012 7, 1235-1245DOI: (10.1097/JTO.0b013e318257cc6d) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions