Volume 141, Issue 5, Pages 1728-1737 (November 2011) Identification of Cancer Stem Cells in Human Gastrointestinal Carcinoid and Neuroendocrine Tumors  Puja Gaur, Eric L. Sceusi, Shaija Samuel, Ling Xia, Fan Fan, Yunfei Zhou, Jia Lu, Federico Tozzi, Gabriel Lopez–Berestein, Pablo Vivas–Mejia, Asif Rashid, Jason B. Fleming, Eddie K. Abdalla, Steven A. Curley, Jean–Nicolas Vauthey, Anil K. Sood, James C. Yao, Lee M. Ellis  Gastroenterology  Volume 141, Issue 5, Pages 1728-1737 (November 2011) DOI: 10.1053/j.gastro.2011.07.037 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 ALDH+ N-CSCs are present in human surgical specimens. Single-cell suspensions from surgical specimens were analyzed for the presence of ALDH+ cells. The ALDH+ cells are indicated by a black arrow. The gates are set for analytic purposes only and were not used for sorting. DEAB, diethyl-aminobenzaldehyde. Gastroenterology 2011 141, 1728-1737DOI: (10.1053/j.gastro.2011.07.037) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 ALDH+ N-CSCs are present in the NET cell line CNDT2.5. The CNDT2.5 ALDH+ population ranged from 1.6% to 3% of viable cells, indicated by a black arrow. The boxes in these figures are the sorting gates used to isolate ALDH+ and ALDH- populations in future experiments. DEAB, diethyl-aminobenzaldehyde. Gastroenterology 2011 141, 1728-1737DOI: (10.1053/j.gastro.2011.07.037) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 NET ALDH+ cells form spheres. (A) CNDT96 ALDH+ cells formed spheres by 8 days that were viable at 21 days. The ALDH- cells died by day 8. (B) ALDH+ CNDT2.5 cells formed spheres whereas ALDH- cells did not. (C) CNDT2.5 spheres greater than 50 μm on day 21 (P < .001, mean ± standard error of the mean). Gastroenterology 2011 141, 1728-1737DOI: (10.1053/j.gastro.2011.07.037) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Src inhibition targets ALDH+ cells. (A) Src, Erk, Akt, and mTOR were activated in CNDT2.5 ALDH+ cells. (B) CNDT2.5 cells treated with PP2 (Src inhibitor) had fewer ALDH+ cells than the DMSO control (P < .0001). (C) Src inhibition markedly decreases proliferation of ALDH+ CNDT2.5 cells, compared with ALDH- cells (P < .0001). Gastroenterology 2011 141, 1728-1737DOI: (10.1053/j.gastro.2011.07.037) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 In vivo Src inhibition reduces NET tumor size. (A) Src-siRNA knock-down of CNDT2.5 Src expression. (B and C) Src-SiRNA (siSrc-DOPC) treatment decreased average CNDT2.5 tumor size in vivo (P < .001) in tumors generated by unsorted CNDT2.5 cells and tumors generated by ALDH+ CNDT2.5 cells. (D and E) Total tumor mass was greatly decreased by siSrc-DOPC treatment in tumors generated from unsorted parental cells and resulted in a 91% total volume reduction compared with the siControl-DOPC–treated group in ALDH+ derived tumors. Gastroenterology 2011 141, 1728-1737DOI: (10.1053/j.gastro.2011.07.037) Copyright © 2011 AGA Institute Terms and Conditions