Volume 143, Issue 1, Pages 234-245.e7 (July 2012) Identification of Pancreatic Cancer Stem Cells and Selective Toxicity of Chemotherapeutic Agents  Rama Adikrisna, Shinji Tanaka, Shunsuke Muramatsu, Arihiro Aihara, Daisuke Ban, Takanori Ochiai, Takumi Irie, Atsushi Kudo, Noriaki Nakamura, Shoji Yamaoka, Shigeki Arii  Gastroenterology  Volume 143, Issue 1, Pages 234-245.e7 (July 2012) DOI: 10.1053/j.gastro.2012.03.054 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Real-time imaging of human pancreatic CSCs. (A) Asymmetric cell division of Gdeghigh cells observed by time-lapse microscopy (Supplementary Video 1). Bar, 20 μm. (B) Symmetric cell division of Gdeglow cells observed by time-lapse microscopy (Supplementary Video 2). Nonfluorescent Gdeglow cells divided rapidly as compared with fluorescent Gdeghigh cells. Bar, 20 μm. (C) Real-time imaging of sphere formation (Supplementary Video 3) revealed that the Gdeghigh cells attached to each other (green arrow) and formed complete spheres with the exclusion of the generated Gdeglow cells (white arrow). Bar, 50 μm. (D) Complete sphere formation was observed by the sorted Gdeghigh cells (left), whereas the Gdeglow cells could not form such spheres (right). Bar, 50 μm. (E) Increased tumorigenicity of Gdeghigh cells when injected subcutaneously into NOD/SCID mice. (F) Gdeghigh and Gdeglow cells, sorted by FACS, were injected subcutaneously into both flanks of NOD/SCID mice, and the size of the tumors was measured at regular intervals. Interestingly, the Gdeghigh cells grew slowly in vitro, but grew more rapidly as a solid tumor. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Pancreatic CSCs are resistant to conventional chemotherapy. (A) Real-time resistance of Gdeghigh cells to gemcitabine observed by time-lapse microscopy (Supplementary Video 4). After the administration of 0.1 μg/mL gemcitabine, cell death was observed only in the Gdeglow cells and not the Gdeghigh cells. (B) FACS cell number assay for Gdeghigh and Gdeglow populations with or without the administration of 0.1 μg/mL gemcitabine (GEM). The proliferation of Gdeglow cells was inhibited by gemcitabine, but no significant difference was detected in the Gdeghigh cells. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Synthetic lethal screen targeting pancreatic CSCs. (A) Schematic of the primary screening. After FACS sorting, 3 × 103 cells from the Gdeghigh and Gdeglow populations were plated separately onto 96-well microtiter plates. After incubation with the chemical compounds for 48 hours, the living cells were stained with SYTO 59 red fluorescent marker at 500 nmol/L, and the green or red fluorescence intensity of each well was quantified using an InCell Analyzer 2000 (GE Healthcare). (B) Green or red fluorescence intensity showed that quercetin and isoquercetin were selectively toxic to Gdeghigh but not Gdeglow cells, whereas Gdeglow but not Gdeghigh cells were sensitive to gemcitabine and cisplatin. (C) The CSC-specific effects of quercetin observation by time-lapse microscopy (Supplementary Video 5). Treatment with 25 μmol/L quercetin induced cell death in the Gdeghigh pancreatic cancer cells, whereas the Gdeglow cells were still proliferating. (D) FACS cell number assay for Gdeghigh and Gdeglow populations with or without the administration of 25 μmol/L quercetin (QRN). The proliferation of Gdeghigh cells was sufficiently inhibited by quercetin, but no significant effect of quercetin was detected in the Gdeglow cells. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Immunocytochemical analysis of β-catenin in human pancreatic non-CSCs and CSCs. (A) Gdeglow. β-catenin was located in the cell membrane, but was not translocated into the nucleus. (B) Gdeghigh. The nuclear accumulation of β-catenin was detected. (C) After the Gdeghigh cells were treated with 25 μmol/L quercetin (QRN) for 12 hours, the nuclear translocation of β-catenin was no longer detected. DAPI, 4′,6-diamidino-2-phenylindole; ZsGreen, Zoanthus Green. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 In vitro combination therapy with gemcitabine and quercetin. (A) Time-lapse microscopic observation (Supplementary Video 6). The combined administration of 0.1 μg/mL gemcitabine plus 25 μmol/L quercetin induced cell death in Gdeghigh cells as well as Gdeglow cells. (B) FACS cell number assay for Gdeghigh and Gdeglow populations with or without the combined administration of 0.1 μg/mL gemcitabine plus 25 μmol/L quercetin (GEM+QRN). The combination therapy significantly reduced the cell number in both the Gdeghigh and Gdeglow populations. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 In vivo combination therapy with gemcitabine and quercetin. Established subcutaneous tumors derived from (A) 1 × 106 sorted Gdeghigh cells or (B) sorted Gdeglow cells were treated with gemcitabine alone (GEM; 100 mg/kg), quercetin alone (QRN; 12.5 mg/kg), a combination of gemcitabine and quercetin (GEM+QRN), or control buffer intraperitoneally every 3 days for 5 times. The upper panels show tumor volumes measured and plotted every other day. The lower panels show the representative images of subcutaneous tumors in NOD/SCID mice after treatment (day 14). Monotherapy using gemcitabine showed only limited antitumor effects in the Gdeghigh group (A), but sufficient effects in the Gdeglow group (B). (A) The Gdeghigh-derived aggressive tumors were significantly sensitive to only combined therapy with quercetin and gemcitabine. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 The CSC imaging system was applied to other human pancreatic cancer cells derived from a Japanese patient (KLM1). (A) Immunocytochemistry for β-catenin in KLM1 human pancreatic cancer cells. Upper panel: Gdeghigh KLM1 cells showing the accumulation of β-catenin in the nucleus. Lower panel: Gdeglow KLM1 cells showing that β-catenin was located in the membrane without nuclear translocation. (B) FACS cell number assay for Gdeghigh and Gdeglow KLM cells with or without the administration of 0.1 μg/mL gemcitabine (GEM). The proliferation of Gdeglow KLM1 cells was inhibited by gemcitabine, but no significant difference was detected in the Gdeghigh KLM1 cells. (C) FACS cell number assay for Gdeghigh and Gdeglow populations of KLM1 with or without the administration of 25 μmol/L quercetin (QRN). The proliferation of Gdeghigh KLM1 cells was inhibited sufficiently by quercetin, but no significant effect from quercetin was detected in the Gdeglow KLM1 cells. (D) FACS cell number assay for Gdeghigh and Gdeglow populations of KLM1 with or without the combined administration of 0.1 μg/mL gemcitabine plus 25 μmol/L quercetin (GEM+QRN). The in vitro combination therapy significantly reduced the cell number in Gdeghigh as well as Gdeglow populations. (E) Established subcutaneous tumors derived from 1 × 106 sorted Gdeghigh KLM1 cells were treated with gemcitabine alone (GEM; 100 mg/kg), quercetin alone (QRN; 12.5 mg/kg), a combination of gemcitabine plus quercetin (GEM+QRN), or a control buffer intraperitoneally every 3 days for 5 times. The upper panels show tumor volumes measured and plotted every other day. The lower panels show representative images of the subcutaneous tumors in NOD/SCID mice after treatment (day 14). In vivo, the pancreatic CSCs from the KLM1 cells were quite resistant to gemcitabine, but were significantly sensitive to only the combination of gemcitabine plus quercetin. DAPI, 4′,6-diamidino-2-phenylindole; ZsGreen, Zoanthus Green. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 PANC1 cells, engineered to stably express ZsGreen-Gdeg using retroviral vector, showed an approximately 0.5% proportion of cells displaying fluorescence (Gdeghigh) at low magnification (left) and high magnification (middle). Stable transfection was confirmed by treatment with MG132 for 12 hours, resulting in 100% of the cells appearing fluorescent (right). Bar, 20 μm. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Observations using time-lapse microscopy on sorted cells within 48 hours. (A) There were 14 asymmetric divisions of the Gdeghigh cells observed among the 16 divisions that occurred overall (87.5%). (B) The Gdeglow cells performed only symmetric divisions. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 The histologic analysis of tumor sections treated with quercetin and gemcitabine, as compared with controls (day 14). (A) Tumor sections stained with H&E (×200) showed dense viable tumor cells in the control tumors (upper), as well as increased tumor stroma and necrotic tissues more than viable tumor cells in the tumors treated with quercetin and gemcitabine (lower). (B) Evaluation of the viable cell numbers per field in tumor sections treated with quercetin and gemcitabine, as compared with the control tumor sections (P = .000002). The viable cells were counted in 5 different random fields (×200). Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Analysis of Gdeghigh and Gdeglow pancreatic cancer cells based on various stem cell markers. (A) An analysis of PANC1 cells revealed an up-regulation of ESA and CXCR4 markers in the Gdeghigh cells, but the other stem cell markers were not increased. (B) An analysis of KLM1 cells showed that CD90 was up-regulated in the Gdeghigh cells, but other markers were not. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Simultaneous administration of quercetin with the implantation of 106 Gdeghigh cells in NOD/SCID mice. (A) Subcutaneous tumors in NOD/SCID mice treated with 12.5 mg/kg quercetin (right) and control buffer (left) intraperitoneally every 3 days for 5 times (day 14). (B) The tumor volumes were evaluated in each group (n = 4). The simultaneous administration of quercetin could block Gdeghigh xenograft formation. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 Quantification of the immunocytochemical analysis of β-catenin in human pancreatic CSCs. (A) Fluorescent microscopic images at low magnification (×100). Upper: Gdeghigh cells (control). Lower: Gdeghigh cells after treatment with quercetin. (B) The frequency of nuclear β-catenin was decreased significantly after quercetin treatment (P = .000006). The nuclear accumulation of β-catenin was evaluated by counting in 5 different random fields. Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 Knockdown of β-catenin in human pancreatic cancer cells. (A) Immunocytochemical analysis confirmed the β-catenin inhibition in the Gdeghigh cells after transfection with β-catenin siRNA (below) as compared with transfection with control small interfering RNA (siRNA) (above). (B) Flow cytometry analysis showed that the Gdeghigh rates were not affected by β-catenin siRNA as compared with quercetin (*P < .0002). Gastroenterology 2012 143, 234-245.e7DOI: (10.1053/j.gastro.2012.03.054) Copyright © 2012 AGA Institute Terms and Conditions