Volume 116, Issue 2, Pages 363-371 (February 1999) Liver colonization by human colon cancer cells is reduced by antisense inhibition of MUC2 mucin synthesis  Lawrence R. Sternberg, James C. Byrd, Christopher K. Yunker, Steven Dudas, Vijendra K. Hoon, Robert S. Bresalier  Gastroenterology  Volume 116, Issue 2, Pages 363-371 (February 1999) DOI: 10.1016/S0016-5085(99)70133-2 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Stable integration of expression constructs into human colon cancer cell lines transfected with antisense to MUC2 (MUC2-ASen) or a sense control (MUC2-C). (A) Neo-specific PCRs show appropriately sized amplification products from MUC2-ASen genomic DNA, MUC2-C genomic DNA, and a positive control (a derivative of LS174T previously established to contain the Neo gene12). Note absence of product from parental LS LiM6 genomic DNA. (B) Southern blot hybridization showing the presence of novel, appropriately sized, MUC2-specific restriction fragments in EcoR1- and BamH1-digested genomic DNA from MUC2-ASen and MUC2-C (but not from LS LiM6 cells) (arrowheads) corresponding to the integration of a single copy of respective expression constructs into each derivative cell line. A single high-molecular-weight species corresponding to the endogenous MUC2 gene is also detected in each cell line (arrows). Horizontal lines represent size markers. Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Determination of the relative abundance of MUC2 RNA in human colon cancer cells. Competitive RT-PCR analyses contained an identical amount of cDNA target (MUC2-C cDNA, lanes 1–5; MUC2-ASen cDNA, lanes 6–10) and sequentially decreasing amounts of MUC2 MIMIC (lanes 1 and 6, 10 amol; lanes 2 and 7, 1.0 amol; lanes 3 and 8, 0.1 amol; lanes 4 and 9, 0.01 amol), cDNA target but no mimic (lanes 5 and 10), or neither (lane 11). Comparison of intensity of the 440-bp MUC2 band with that of the 340-bp mimic band was used to calculate the amount of MUC2 cDNA based on known input amount of standard. ld, 100-bp molecular-weight ladder. Bands (top to bottom) represent 600, 500, 400, and 300 bp, respectively. Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of stable introduction of MUC2 antisense construct on MUC2 protein expression in human colon cancer cells. Lysates of LS LiM6, MUC2-ASen, and MUC2-C cells were subjected to Western analysis using MUC2-specific monoclonal antibody CCP58. Equal amounts of homogenate protein (30 μg) were added to each lane, and proteins were separated by electrophoresis on 7.5% SDS-polyacrylamide gels. MUC2 has a reported Mr of >600 kd34 and therefore migrates much more slowly than the slowest migrating standard. Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Reduction of high-molecular-weight glycoprotein (HMG) in human colon cancer cells after MUC2 antisense transfection. Cells were metabolically labeled with [3H]GlcN, and labeled glycoproteins secreted into the medium (▨) or in the cytosol fraction (□) were quantitated by size-exclusion chromatography on Superose 6 columns. Radioactivity in the void volume was normalized to cellular protein (mean ± SEM; n = 3; P = 0.0084). Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Mucin-associated carbohydrates secreted by MUC2-ASen and MUC2-C cells. Cells were grown for 72 hours in serum-free medium, and medium from control (open symbols) or antisense-transfected (closed symbols) cells was subjected to size-exclusion chromatography on Superose 6 columns. Column fractions were adsorbed to polystyrene microtiter plates and assayed for mucin-associated carbohydrates by enzyme-linked immunosorbent assay. (A) Sialyl-LewisX recognized by monoclonal antibody CSLEX-1. (B) Sialyl-Tn recognized by monoclonal antibody JT10e. (C) Tn recognized by Vicia villosa agglutinin B4 (VVA). (D) Superose 6 profile of glucosamine-labeled glycoproteins secreted by MUC2-C (open symbols) and MUC2-ASen (closed symbols) cells. Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Binding of human colon cancer cells to E-selectin. Colon cancer cells were incubated in microtiter wells containing a soluble E-selectin chimeric protein consisting of E-selectin and a fragment of human IgG1 adsorbed to the plate. ○, Binding of control cell line MUC2-C; □, binding of MUC2 antisense-transfected cell line MUC2-ASen. Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Liver colonization by human colon cancer cells. Representative livers from athymic nude mice injected intrasplenically with 106 human colon cancer cells and killed 4 weeks later. (A) Livers from animals injected with MUC2-C control cells. (B) Livers from animals injected with MUC2-ASen cells. Control livers all contained >500 tumor nodules. Liver colonization was significantly inhibited in animals injected with antisense-transfected cells (0–34 nodules). Gastroenterology 1999 116, 363-371DOI: (10.1016/S0016-5085(99)70133-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions