Volume 118, Issue 3, Pages 497-506 (March 2000) Integrin α6β4 as a suppressor and a predictive marker for peritoneal dissemination in human gastric cancer  Yoshiyuki Ishii*,‡, Atsushi Ochiai*, Tesshi Yamada*, Shingo Akimoto*, Kazuyoshi Yanagihara§, Masaki Kitajima‡, Setsuo Hirohashi*,∥  Gastroenterology  Volume 118, Issue 3, Pages 497-506 (March 2000) DOI: 10.1016/S0016-5085(00)70255-1 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 FACS analysis of integrin β4 subunit expression in 10 human gastric cancer cell lines. Cells were either immunostained with a mouse anti-human integrin β4 monoclonal antibody or left untreated, then incubated with a fluorescein isothiocyanate–labeled secondary antibody and subjected to FACS analysis. Expression levels were estimated from the mean fluorescence intensities of the samples (the numbers under histograms), and correlations between the numbers of peritoneal nodules and β4 subunit expression levels of the cell lines were evaluated by determining the Pearson (r) rank correlation coefficients: r = 0.735; P = 0.0013. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Biological features of TMK-1 β4 transfectants. (A) Western blotting of integrin β4 subunit and the numbers of peritoneal nodules in SCID mice. The extracted proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (5%) under nonreducing conditions, and β4 subunit expression levels were evaluated by comparing the densities of the bands with that of Na+,K+-ATPase. The peritoneal nodules were counted 15 days after intraperitoneal injection of the cells (means ± SD). (B) FACS analysis of integrin expression in transfectants. The transfectants were either immunostained with mouse anti-human integrin monoclonal antibodies or left untreated, then incubated with a fluorescein isothiocyanate–labeled secondary antibody and subjected to FACS analysis. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Western blotting of exogenous integrin β4 subunit (cytoplasmic domain-deleted β4 subunit), the levels of endogenous β4 and β1 subunit based on immunoprecipitation with anti-integrin α6 subunit antibody, and the numbers of peritoneal nodules in SCID mice after intraperitoneal injection of MKN28 transfectants. Exogenous β4 subunit was detected as a band with a molecular mass of 95 kilodaltons by Western blotting. The levels of endogenous β4 and β1 subunit that associated with α6 were estimated from the ratio to control (C1) by immunoprecipitation analysis. The peritoneal nodules were counted 15 days after intraperitoneal injection of cells (means ± SD); correlations between the numbers of peritoneal nodules and the level of endogenous β4 subunit that associated with α6 of the transfectants were evaluated by determining the Pearson (r) rank correlation coefficients. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Histological appearances of the peritoneal nodules of TMK-1 β4 transfectants. H&E and TUNEL staining revealed apoptotic bodies with characteristic nuclear fragments in (B and F) β4 transfectant (2A1) cells but not in (A and E) mock transfectant (TMKC-3) cells. Immunohistochemical staining with the anti–integrin β4 subunit antibody revealed strong reactivity of (D) the 2A1 transfectant with apoptotic bodies but not of (C) TMKC-3 cells. Bar = 50 μm. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 In vitro cell growth of TMK-1 integrin β4 transfectants, MKN7, and MKN28 cells under conditions of ECM ligand and EGF stimulation. Viable cells were counted 4 days after plating in triplicate dishes coated with ECM and culture in the presence or absence of EGF (20 ng/mL). Growth of each cell was evaluated based on the percentage of the number of viable cells that grew on the noncoated dishes in the absence of EGF as a control. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunohistochemical staining with the anti–integrin β4 subunit antibody of surgically resected gastric cancer tissues. (A) Normal gastric epithelium. (B) Differentiated-type cancer. (C and D) Undifferentiated-type cancer. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Recurrence and (B) survival of 84 gastric cancer patients who had no peritoneal dissemination at surgery. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Recurrence and (B) survival of 84 gastric cancer patients who had no peritoneal dissemination at surgery. Gastroenterology 2000 118, 497-506DOI: (10.1016/S0016-5085(00)70255-1) Copyright © 2000 American Gastroenterological Association Terms and Conditions