Volume 141, Issue 6, Pages 2218-2227.e5 (December 2011) c-Met Is a Marker of Pancreatic Cancer Stem Cells and Therapeutic Target  Chenwei Li, Jing–Jiang Wu, Mark Hynes, Joseph Dosch, Bedabrata Sarkar, Theodore H. Welling, Marina Pasca di Magliano, Diane M. Simeone  Gastroenterology  Volume 141, Issue 6, Pages 2218-2227.e5 (December 2011) DOI: 10.1053/j.gastro.2011.08.009 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Isolation of tumorigenic pancreatic cancer cells and tumor formation in NOD/SCID mice. Plots depicting representative staining patterns of (A) c-Met and (B) c-MethighCD44+ cells in primary human pancreatic tumors. (C) Tumor formation in a mouse at the injection site of 500 c-MethighCD44+ cells, with no tumor formation at the injection site of 500 c-Met−CD44− cells. (D) Tumors derived from c-MethighCD44+ cells appeared histologically similar to the patient's primary tumor. (E, left) Staining pattern from a patient tumor that had been passaged once in NOD/SCID mice. c-MethighCD44+ tumorigenic cells from the tumor were then doubly sorted (middle) and injected into NOD/SCID mice. (Right) The staining pattern of the resultant tumor that arose from the c-MethighCD44+ cells. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 c-Met inhibition prevents pancreatic tumorsphere formation. (A) c-Methigh cells formed tumorspheres, whereas c-Met− cells did not. (B and C) XL184 (10 μmol/L) inhibited tumorsphere formation. (D) Effect of treatment with XL184 (3 or 10 μmol/L) for 72 hours on expression of phospho-c-Met, phospho-ERK, and total c-Met in tumorspheres. (E) The effect of XL184 (10 μmol/L) treatment on the induction of apoptosis in both CSCs and non-CSCs, measured by active caspase-3 staining. (F) Western blotting using an anti–c-Met antibody to verify knockdown of c-Met expression in tumorspheres using 2 different c-Met shRNAs (top panel). Tumorsphere formation is inhibited in c-MethighCD44+ cells infected with lentiviral constructs expressing c-Met shRNA1 and shRNA2 (bottom panel). *P < .05 vs control. Each experiment was performed using cells from 2 different patient tumors, with each experiment repeated 3 times and in triplicate. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Effect of c-Met inhibition with or without gemcitabine on subcutaneous tumor growth in NOD/SCID mice. (A) Depicted are representative tumors from each treatment group. (B) Pooled results of the average tumor size ± SEM from each treatment group over time. (C) Flow cytometry to measure pancreatic cancer cells expressing c-MethighCD44+ 4 weeks after initiation of treatments. (D) Flow cytometry to measure CD44+CD24+ESA+ 4 weeks after the initiation of treatments. The percentage of the CD44+CD24+ESA+ cancer cells/total cancer cells is indicated in the upper right box of the lower panel of fluorescence-activated cell sorting plots. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Effect of XL184 and gemcitabine on orthotopic tumor growth. Pancreatic cancer cells infected with a luciferase-expressing lentivirus were injected into the pancreatic tail of NOD/SCID mice (n = 6 per group). (A) Representative bioluminescent images of animals in each group are shown at 4 weeks after treatment depicting the extent of tumor burden. (B) Pooled results of the average tumor size ± SEM from each treatment group over time. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 XL184 blocks metastasis in an intracardiac injection model. (A) Images taken 6 weeks after intracardiac injection of cancer cells (representative animals in each group depicted). (B) Tumor burden measurement 6 weeks after intracardiac injection. (C) Representative histologic images of liver, lung, and pancreatic tissue from a control (top panel) or XL184-treated animal (bottom panel) 6 weeks after intracardiac injection of pancreatic cancer cells. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Single cell assays further confirmed the ability of XL184 to inhibit tumorsphere formation. (A) Representative pictures of tumorspheres derived from single cells in the different treatment groups. (B) XL184 (10 μmol/L) inhibited tumorsphere formation. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 (A) Cell cycle analysis of CSCs and non-CSCs left untreated or treated with 10 μmol/L XL184 for 24 hours. (B) Phospho-H3 staining showing that the accumulation of cells at G2/M is due to G2 cell cycle arrest. (C) Quantitation of the percentage of CSC and non-CSC cells in G2 arrest before and after XL184 treatment. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Tumorspheres were left untreated or treated with XL184 (10 μmol/L) for 24 hours. Western blotting was performed to measure levels of phosphorylation of RET, KIT, and VEGFR2. Total RET, KIT, and VEGFR2 were measured and served as loading controls. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Growth of tumors initiated by implantation of viable tumorsphere cells infected first for 48 hours with lentiviral constructs expressing control shRNA or c-Met shRNA1 or shRNA2. The experiment was performed using a single tumor and repeated 3 times, with 5 animals in each treatment group. Gastroenterology 2011 141, 2218-2227.e5DOI: (10.1053/j.gastro.2011.08.009) Copyright © 2011 AGA Institute Terms and Conditions