Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis

GASTROENTEROLOGY 1996;110:1354–1367

Enhanced Sialylation of Mucin-Associated Carbohydrate Structures in Human Colon Cancer Metastasis ROBERT S. BRESALIER,*,‡ SAMUEL B. HO,§ HARALD L. SCHOEPPNER,* YOUNG S. KIM,x MARVIN H. SLEISENGER,x PNINA BRODT,Ø and JAMES C. BYRD* *Department of Medicine, Henry Ford Health Sciences Center, Detroit, Michigan; ‡Department of Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan; xDepartment of Veterans Affairs Medical Center and University of California, San Francisco, California; § Department of Veterans Affairs Medical Center, University of Minnesota, Minneapolis, Minnesota; and ØDepartment of Surgery, McGill University, Montreal, Quebec, Canada

Background & Aims: Patients with mucinous colon cancers often have a poor prognosis. The aim of this study was to determine whether metastatic potential depends on specific alterations in mucin-associated carbohydrate structures. Methods: A quantitative scoring system was used to examine the expression of mucinassociated carbohydrates in paired human primary colon cancers and metastases and in cecal tumors and liver metastases from an animal model of metastasis. Adhesion of metastatic cells to basement membrane and endothelial ligands was examined. Results: Metastases expressed a decrease in mucin core structures Tn and T, a reciprocal increase in sialyl T and sialyl Tn, and an increase in peripheral sialyl Lex compared with the primary tumors from which they arose. Altered expression of sialylated mucin structures resulted from selective metastasis of cells that produce sialomucins. Antibodies to sialylated epitopes or desialylation inhibited adhesion of metastatic cells to basement membranes. Neutralizing antibody to endothelial-associated E-selectin (a ligand for sialyl Lex) inhibited adhesion of metastatic cells to cytokine-activated hepatic endothelial cells, and inhibition of sialomucin with antisense to the MUC2 gene inhibited adhesion to E-selectin. Conclusions: Increased sialylation of mucin-associated carbohydrates is characteristic of colon cancer cells that are most likely to metastasize. Sialylated carbohydrate structures on mucin play a role in adhesive interactions involving both basement membrane and endothelial-associated ligands.

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olorectal cancer–related mortality is most strongly related to the extent of tumor cell invasion and metastasis at diagnosis.1 Patients with mucinous colon cancers have a poor prognosis,2,3 and results using human colon cancer cells in in vivo models suggest that mucins play a role in metastasis.4,5 However, it is not yet known how colon cancer mucin affects the biological behavior of these cells or which of the many steps in metastasis

depend on specific alterations in secreted or cell-surface glycoproteins. Alterations in cell-surface and secreted glycoproteins are common during carcinogenesis and have a major influence on the homotypic and heterotypic cell-cell and cell-substratum interactions that mediate tumorigenesis and metastasis.6,7 Sialylated oligosaccharide structures are prominent in mucin-type glycoproteins from human colorectal cancers and, in many cases, are responsible for tumor-associated antigenicity.4,8 – 12 Altered sialylation seems to be important in the transition from adenoma to carcinoma13,14 and may have implications for determining prognosis in patients with colorectal cancer.15 –17 Therefore, we studied the expression of mucin-related carbohydrate structures in human primary colon cancers and their associated metastases and in primary tumors and metastases (cecum to liver) from a nude mouse model of human colon cancer metastasis.18,19 To further define the role of mucin and mucin-associated sialoglycoproteins in individual stages of metastasis, we examined the effect of carbohydrate-specific antibodies on adhesion of metastatic colon cancer cells to basement membranes and the effect of inhibition of sialomucin production on adhesion to endothelial-associated selectins. The results indicate that primary and metastatic colon cancers differ in their expression of mucin-associated carbohydrate structures and that this is caused by altered sialylation, which can enhance or mask epitope expression in metastases. This arises through the selective metastasis of colon cancer cells that produce sialomucins. These alterations lead to cell-cell and cell-substratum interactions that enhance metastasis. Abbreviations used in this paper: ACA, Amaranthus caudutus agglutinin; MAA, Maakia amurensis agglutinin; MAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazole-2yl)-2,5 diphenyl-2,4 tetrazolium bromide; PNA, peanut agglutinin; SNA, Sambucus nigra agglutinin; VVA, Vicia villosa agglutinin. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

May 1996

Materials and Methods Surgical Tissue Specimens Computerized lists of patients with surgically resected colorectal carcinomas were obtained from the Pathology Service of the Department of Veterans Affairs Medical Center (San Francisco, CA) and the Pathology Service of the Henry Ford Health Sciences Center (Detroit, MI). Formalin-fixed, paraffinembedded specimens were obtained from patients whose resected tumors were staged as modified Dukes’ classification D and from whom paired lymph node or distant metastases were available. No attempt was made to select for any other clinical or pathological features. Fifty-eight specimens were obtained from 23 patients. Metastases included those from liver (10), lymph nodes (10), lung (3), ovary (2), prostate (1), kidney (1), bone (1), brain (1), and intra-abdominal recurrences (6).

Xenografts of Primary Human Colon Cancers and Their Metastases in Athymic Mice An animal model of human colon cancer metastasis was used to examine the expression of mucin-associated sialoglycoprotein structures during progressive in vivo selection of colon cancer cells with enhanced spontaneous metastatic ability.18,19 In this model, human colon cancer cells (LS174T) were injected into the cecal wall of 3–4-week-old Balb/c NCRNU athymic mice. After 6 weeks, animals were killed, and the cecum and liver were examined for the presence of primary cecal tumors and metastases (cycle 1). Liver metastases were cultured and grown to high density, and a new cell line was established. These cells were then injected into the cecum of additional mice (cycle 2). This procedure was repeated serially to obtain tumor cells of increasing metastatic ability (six cycles). Cell lines obtained in this manner have been verified to have increasing metastatic potential and have been well characterized.4,18,20 Tissue from primary cecal tumors and liver metastases obtained at each cycle were fixed in formalin and embedded in paraffin, and serial 5-mm sections were prepared for immunohistochemical analysis.

Cell Lines Parental cell line LS174T was derived from a welldifferentiated human colonic adenocarcinoma and has been characterized extensively.4,5,18,20 LS LiM6 is a derivative of LS174T with high liver-metastasizing ability during cecal growth and liver-colonizing capacity after splenic-portal injection into athymic nude mice.4,20 All cell lines were grown in Dulbecco’s modified Eagle medium, supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin 100 mg/mL) in a 7% CO2 environment. Early-passage LSLiM6 cells (passaged fewer than 15 times) were used for all studies.

Immunohistochemistry Immunohistochemical staining. Immunohistochemistry was performed using a modified streptavidin-peroxidase

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Table 1. Lectins and MAbs Used to Label Mucin-Associated Carbohydrate Structures Structure

MAb and/or lectin

Antigen

GalNAcaThr/Ser Siaa6GalNAcaThr/Ser Galb3GalNAcaThr/Ser Siaa3Galb3GalNAcaThr/Ser Galb4(Fuca3)GlcNac-R Siaa3Galb4(Fuca3)/GlcNAc-R Siaa6Gal-R Siaa3Gal-R

VVA, 91S8 TKH2, JT10e, SNA PNA, AH 9-16 ACA BY 87 MAA, SNH3 SNA MAA

Tn Sialyl Tn T T or sialyl T Lex Sialyl Lex

GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Sia, sialic acid.

technique as described previously.9 All steps were conducted at room temperature. Briefly, 5-mm tissue sections were deparaffinized, rehydrated, incubated with fresh 3% hydrogen peroxide in methanol for 10 minutes, and then washed with phosphate-buffered saline (PBS) buffer. A panel of 11 monoclonal antibodies and lectins was used to examine the expression of mucin-associated carbohydrate structures in serial tissue sections. Antibodies, lectins, and the structures that they identify are listed in Table 1. All lectins were obtained from Vector Laboratories (Burlingame, CA) and used at concentrations of 2 mg to 5 mg/mL. Monoclonal antibodies (MAbs) TKH2 and SNH3 were kindly provided by Dr. S. Hakomori (Biomembrane Institute, Seattle, WA) and were used at a working concentration of 2.5 mg/mL. MAb AH 9-16 was obtained from Calbiomed, Ltd. (Edmonton, Alberta, Canada) and was used at 2 mg/mL. MAbs JT10e and 91S8 were developed in the laboratory of Dr. Y. S. Kim and were used at 5 mg/mL. MAb BY87 was obtained as a tissue culture supernatant from Novacastra Laboratories (Newcastle Upon Tyne, England) and was used at a 1:20 dilution. Tissues were incubated with either 5% normal rabbit serum in PBS (antibodies) or 1% bovine serum albumin (lectins) for 20 minutes. After blotting, specimens were incubated with primary antibody (90 minutes) or biotinylated lectin (60 minutes) and washed three times with buffer. Antibody-stained slides were exposed to biotinylated rabbit antimouse immunoglobulin (Ig) G / IgA / IgM (1:75 dilution in PBS) for 20 minutes and washed three times with PBS. All slides were exposed to 10 mg/mL streptavidin-peroxidase conjugate (Zymed, San Francisco, CA) for an additional 30 minutes. Finally, slides were washed three times with buffer and reacted with diaminobenzidine in 0.03% hydrogen peroxide for 10 minutes, were rinsed with tap water, and were counterstained with methyl green or Mayer’s hematoxylin. Negative controls consisted of substituting normal mouse serum for monoclonal antibodies or PBS for lectins. Additionally, lectin specificity was confirmed by preincubating equal volumes of lectins with 0.2–0.5 mol/L hapten sugars (e.g., lactose for peanut agglutinin [PNA], galactose-b-3-N-acetylgalactosamine-a-o-benzyl for Amaranthus caudatus agglutinin [ACA], N-acetylgalactosamine-a-o-benzyl for Vicia villosa agglutinin

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with a staining score of 3 would yield an average score for the entire specimen of (0 1 0) / (1 1 0.20) / (2 1 0.60) / (3 1 0.20) Å 2.0. Sections were scored by two independent observers (interobserver reliability, r Å 0.95).

Adhesion of Colon Cancer Cells to Basement Membranes

Figure 1. Immunohistochemical tumor staining intensity score. All 101 fields were scored individually for staining intensity (Is , see equation on bottom), the percentage of fields at each intensity was determined, and the scores were added to yield an average score for the entire specimen that accounts for intratumoral heterogeneity.

[VVA], Siaa6lactose for Sambucus nigra agglutinin [SNA]; see Table 1 for sugar specificities) before use. All control experiments resulted in abolition of specific staining. A representative sample (10 primary tumors and 10 metastases) was also examined for staining before and after sialidase treatment. Specimens were treated with neuraminidase type X from Clostridium perfringens (Sigma Chemical Co., St. Louis, MO) at a concentration of 0.2 U/mL in Hank’s buffered saline solution, pH 6.0, at 37⬚C for 1 hour before staining with antibody or lectin as described above. Immunohistochemical staining score. Staining of tissue specimens was scored using a semiquantitative scoring system that takes into account tumor heterogeneity and that is subject to statistical analysis (Figure 1). Using the 101 objective, staining intensity and distribution in each field was scored as absent (0), weak (1), moderate (2), or strong (3). All 101 fields in a given specimen were individually scored, the percentage of fields at each intensity was determined, and scores were added to yield an average staining intensity score (Is ) for the entire specimen. Is Å [(0 1 Fo ) / (1 1 F/ ) / (2 1 F// ) / (3 1 F/// )], where Fo is the percent 101 fields scored as 0, F/ is the percent scored as 1, F// is the percent scored as 2, and F/// is the percent of fields scored as 3. For example, a tissue specimen containing no fields with absent staining (0), 20% with a staining score of 1, 60% with a staining score of 2, and 20%

Adhesion of colon cancer cells of different metastatic potential to basement membrane matrix and the effect of specific carbohydrate inhibition were assayed in microtiter plates as described previously.5 Briefly, microtiter plates were coated with 4 mg/well matrigel (Collaborative Biomedical Products, Bedford, MA) at 37⬚C under sterile conditions for 18 hours. The coated wells were washed three times with calcium- and magnesium-free PBS, and tumor cells were added in serumfree medium at a concentration of 4 1 104 cells/well. In some cases, cells were preincubated with various concentrations of monoclonal antibody or lectin for 30 minutes. Controls consisted of substituting mouse IgG1 (Sigma Chemical Co.) for specific antibody before their addition to microtiter wells and using MAbs directed against nonsialylated epitopes. In other experiments, cells were treated with neuraminidase type X from C. perfringens (0.1 U/mL in PBS at 37⬚C for 30 minutes) before use. Cells were washed three times, resuspended in serum-free medium, and added to the microtiter wells. After a 2-hour incubation, wells were gently washed with PBS, and attached cells were detected by the 3-(4,5-dimethylthiazole2yl)-2,5 diphenyl-2,4 tetrazolium bromide (MTT; Sigma Chemical Co.) colorimetric assay reading absorbance at 550 nm.21 All experiments were performed in triplicate.

Adhesion of Colon Cancer Cells to Endothelial Selectins Adhesion of cells to a soluble E-selectin chimeric protein22 consisting of the extracellular region of E-selectin and a fragment of human IgG1 (ELAM-Rg) was measured in microtiter plates. ELAM-Rg and control chimeric protein were gifts of Dr. L. Laskey (Genentech Inc., San Francisco, CA). Affinity-purified goat anti-human IgG (10 mL/mL in 50 nmol/ L Tris, pH 9.5) was adsorbed to plates for 2 hours at room temperature, and the remaining protein-binding sites were blocked by overnight incubation with 0.1% bovine serum albumin in PBS. The coated plates were then incubated with 0.1 mg/mL ELAM-Rg in PBS and 0.1% bovine serum albumin for 1 hour at 22⬚C and washed three times with PBS. Colon cancer cells (105) were added to each well, and the plates were incubated for 20 minutes at 22⬚C with rotation at 60 rpm. After incubation, the plates were gently washed with PBS, and the attached cells were quantitated with the MTT assay as above. Nonspecific binding was determined using a control chimeric protein (CD4-FC) and subtracted to determined specific adhesion. The effect of specific mucin inhibition on selectin binding was tested after pretreatment of cells with antisense phosphorothioate oligodeoxynucleotides complementary to an 18-nucleo-

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Figure 2. Tumor staining intensity scores for primary colorectal cancers (䊐) and their associated metastases ( ) using a panel of MAbs and lectins that recognize mucin-associated carbohydrate structures. Bars indicate mean staining scores using ratio estimation analysis, which accounts for differing numbers of metastatic tumors in each patient. Carbohydrate structures recognized by each MAb or lectin are shown in Table 1. (A ) Tn antigen, (B ) sialyl Tn (SNA recognizes Siaa6Gal-R), (C ) T antigen, and (D ) sialyl Lex. *P õ 0.05; **P õ 0.005.

tide consensus sequence of the MUC2 gene tandem repeat region23 (5ⴕ-GTGGTGGTGGTGATGGGT). Cells cultured to 50% confluency in 24-well plates were incubated for 24 hours in 500 mL/well OptiMEM (GIBCO, Grand Island, NY) plus 10 mg/mL lipofectin and 5 mmol/L antisense oligodeoxynucleotide. Medium was removed, cells were washed, and then cells were reincubated for an additional 24 hours in medium containing oligodeoxynucleotide without lipofectin. Cells were then harvested, and adhesion to E-selectin was assayed as above. Controls consisted of cells treated identically but without the addition of antisense oligodeoxynucleotides and cells treated with a control oligodeoxynucleotide consisting of a scrambled 18-nucleotide sequence. Mucin inhibition was confirmed by incubating cells with 10 mCi/mL tritiated glucosamine on day 2,4 followed by superose 6-gel filtration chromotography of the media. Antisense inhibition of mucin-associated carbohydrate antigens on the cell surface, in cell homogenates, and in secreted material was confirmed by fixed-cell enzyme-linked immunosorbent assay, slot blot and Western analysis of cell homogenates, and enzyme-linked immunosorbent assay24 with biotinylated or peroxidase-labeled lectins, respectively.

Hepatic endothelial cells were obtained from athymic mice by perfusion of mouse livers with pronase and collagenase, followed by separation of parenchymal and nonparenchymal cells on metrizamide density gradients.25 Cells were cultured 5 – 7 days in 24-well plates precoated with rat tail (type I) collagen. Characterization of cells based on morphology, immunocytochemistry with antibody to von Willebrand factor, uptake of acetylated low-density lipoprotein labeled with 1,1ⴕ-dioctadecyl-1-1-3,3ⴕ,3ⴕ-tetramethylindo-carbocyanine perchlorate, and phagocytosis of latex particles26 showed that ú85% of cells in primary cultures were endothelial at the time adhesion assays were performed. Adhesion assays were performed as described previously.27 Tumor cells (2.5 1 105 activated 6 hours with 50 mg/mL tumor necrosis factor a) were radiolabeled with Na51Cr and incubated at room temperature for 45 minutes, with endothelial cells and unbound cells removed by gentle washing and with specific adhesion quantitated by counting of radioactivity. In some experiments, MAbs to E-selectin28 (a gift from Dr. B. Wolitzky, Hoffman – LaRoche, Inc., Nutley, NJ) were added to endothelial cell cultures 15 minutes be-

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Figure 3. Photomicrographs of paired primary colorectal cancers and their metastases stained with a panel of MAbs and lectins. MAb 91S8 (Tn): (A ) primary tumor and (B ) liver metastasis (methyl green counterstain). MAb TKH2 (sialyl Tn): (C ) primary tumor, (D ) lung metastasis, and (E ) liver metastasis. MAb JT10e (sialyl Tn): (F ) primary tumor and (G ) liver metastasis. SNA (Siaa6Gal-R): (H ) primary tumor and (I ) lymph node metastasis (hematoxylin counterstain).

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Figure 3. (cont’d). MAb SNH3 (sialyl Lewisx): (J ) primary tumor, (K ) abdominal recurrence, and (L ) lung metastasis. MAA (siaa3Gal-R): (M ) primary tumor and (N ) abdominal recurrence (original magnification: A, C, D, and F, 501; B and E, 251).

fore addition of tumor cells. All experiments were performed at least in triplicate.

Statistical Analysis Ratio estimation analysis29 was used to compare the scoring outcome of primary tumors with metastatic tumors for each patient. The ratio estimation accounts for differing numbers of metastatic tumors in each patient by weighting the standard error accordingly and also accounts for the paired nature of the data. Ratio estimation methods were also used to compare staining intensity scores before and after treatment with neuraminidase. Using the standard error from the ratio estimation techniques, a one-sample Student’s t test was used to test changes within a given tissue. Differences in adhesion to basement membrane, selectins, and endothelial cells were compared using the Student’s t test.

Results Expression of Mucin-Associated Carbohydrate Structures in Human Adenocarcinomas and Their Metastases Twenty-three primary adenocarcinomas of the colon and 35 associated metastases from the same patients were assessed for their expression of mucin-associated carbohydrate structures using a panel of 10 MAbs and lectins and a scoring system that accounts for intratumoral heterogeneity (Figures 2 and 3). Ratio estimation analysis was used to take into account the differing num-

ber of metastatic tumors per patient (standard errors were calculated taking into account the number of metastases for each patient). Expression of the mucin core structure Tn (GalNAc aThr/Ser) was significantly reduced in metastases compared with the primary tumor from which they arose as determined by staining with MAb 91S8 (score, 0.33 { 0.08 metastases vs. 1.09 { 0.31 primaries; P õ 0.005) or the lectin VVA (score, 0.41 { 0.12 metastases vs. 0.75 { 0.24 primaries, P Å 0.02). The expression of T antigen (Galb3 GalNAc aThr/Ser) determined by staining with MAb AH9-16 (score, 0.06 { 0.03 metastases vs. 0.52 { 0.18 primaries) or the lectin PNA (score, 0.42 { 0.13 metastases vs. 1.15 { 0.26 primaries) was also significantly lower in metastases compared with their primary tumors (P õ 0.005 metastases vs. primaries for each reagent). Conversely, expression of the sialylated mucin core structure sialyl Tn (Siaa6 GalNAc aThr/Ser) was significantly increased in metastases compared with primary tumors using MAbs TKH2 (score, 1.84 { 0.24 metastases vs. 0.95 { 0.16 primaries; P õ 0.001) and JT10e (score, 1.54 { 0.40 metastases vs. 0.83 { 0.18 primaries; P Å 0.03), as well as the lectin SNA, which recognizes a2,6-linked sialylated structures (score, 1.95 { 0.21 metastases vs. 1.24 { 0.25 primaries; P õ 0.001). Sialyl LewisX (sialyl Lex; Sia a3 Galb4 [Fuc a3]GlcNAc-R), a structure found on extended mucin carbohydrate chains, and sia a3-Gal–containing structures in general

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were also increased in metastases compared with primary tumors by staining with MAb SNH3 (score, 2.22 { 0.10 metastases vs. 1.31 { 0.22 primaries; P õ 0.001) and the lectin MAA (score, 2.01 { 0.34 metastases vs. 1.05 { 0.29 primaries; P Å 0.001), respectively. On the other hand, nonsialylated Lewis X expression (MAb BY87) was lower in metastases compared with primary tumors (1.20 { 0.14 metastases vs. 2.42 { 0.23 primaries; P õ 0.001). One explanation for the lower expression of the mucin core antigens Tn and T in metastases compared with the primary tumors from which they arose is that these antigens are cryptic, being masked by enhanced sialylation in metastases. Therefore, a representative sample of 10 primary tumors and 10 metastases was treated with neuraminidase in situ to remove sialic acid before staining with the lectins VVA and PNA. Neuraminidase treatment significantly increased staining of all tissues by these lectins. However, the mean change for metastases was significantly greater than that of the primary tumors (P õ 0.05) with desialylation completely abolishing the difference in staining between these groups (Figure 4A and B). To further confirm that enhanced sialylation is responsible for decreased binding of PNA in metastases, tissues were stained with the lectin ACA.30 Unlike PNA, where any substituent sialylation on the hydroxyl groups of galactose interferes with binding to T antigen, sialylation at the Cⴕ3-hydroxyl of galactose does not interfere with binding by ACA. In contrast to PNA, ACA bound strongly to the mucins in metastases (Figure 4C and D), confirming the presence of sialylated forms of the T antigen. Mucin-Associated Sialoglycoproteins in an Experimental Model of Human Colon Cancer Metastases The enhanced expression of sialylated mucin-associated carbohydrate structures in distant metastases could result from the selective metastasis of subpopulations of colon cancer cells producing these antigens or as a result of the influence of the microenvironment in which they grow.31 – 33 To address this question, we examined the expression of the mucin core structure sialyl Tn and the peripheral carbohydrate structure sialyl Lex during the progressive selection of metastatic cell populations in an animal model of colon cancer metastasis.18,19 Human colon cancer cell line LS174T was derived from a well-differentiated adenocarcinoma and produced xenografts of low metastatic ability when injected into the cecal wall of athymic nude mice.18 Pri-

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mary cecal carcinomas showed weak heterogeneous staining for both sialyl Tn (MAb TKH2) and sialyl Lex (MAb SNH3), whereas liver metastases (cycle 1) showed moderate and more homogeneous staining. Cells derived from liver metastases were cultured and reinjected into the cecum of additional animals, and the process was repeated through multiple cycles. Cells derived from the fourth to sixth cycles have high metastatic ability from cecum to liver in nude mice.4,18,20 Immunohistochemical analysis of xenografts from these latter cycles showed strong homogeneous staining for sialyl Tn and sialyl Lex in both metastases and primary tumors (Figure 5), suggesting that enhanced expression of sialylated antigens in metastases results from selection of metastatic subpopulations of colon cancer cells with these properties and is not simply a result of the environment in which they grow. Adhesion of Colon Cancer Cells to Basement Membranes Cell surface and secreted glycoproteins may be involved in several stages of metastasis, including adherence of metastatic cells to endothelia and the extracellular matrix.5 – 7,20,34 We have shown previously that the ability of human colon cancer cells to adhere to basement membrane components is proportional to their ability to produce mucin and that inhibition of mucin glycosylation inhibits basement membrane binding.5 To further define the potential role of mucin and mucin-associated sialoglycoproteins in individual stages of metastasis, we examined the effect of carbohydrate-specific antibodies or lectins on adhesion of metastatic colon cancer cells to the basement membrane and the effect of mucin production on adhesion to endothelial-associated selectins. Preincubation of metastatic colon cancer cell line LSLiM6 with MAb TKH2 (sialyl Tn), the lectin SNA (siaa6Gal-R), and MAb SNH3 (sialyl Lex) inhibited adhesion to basement membrane matrigel in a dose-dependent manner (Figure 6). Low metastatic cell line LS714T does not bind as well to matrigel,5 and its binding was not significantly affected by sialyl-specific antibodies or lectins. No inhibition occurred when mouse IgGl was substituted for specific antibodies or when cells were incubated with MAbs 91S8 or PS10,35 directed against nonsialylated mucin-associated carbohydrate epitopes. Adhesion of LSLiM6 was also inhibited by prior desialylation with neuraminidase (Figure 7) (P õ 0.005). Adhesion of Colon Cancer Cells to Endothelial Selectins Selectins are a group of sialic acid–binding mammalian lectins present on blood-borne cells, such as neu-

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Figure 4. Decreased expression of mucin core antigens Tn and T in metastasis is caused by masking by sialylation. (A ) Mean tumor staining scores for primary tumors (䊐) and metastases ( ) stained with VVA (Tn) and PNA (T) before and after treatment with neuraminidase. The mean change for metastases was greater than that for primary tumors (*P õ 0.05). (B ) Photomicrographs of liver metastases stained with VVA and PNA before and after desialylation with neuraminidase (Neu’ase) (methyl green counterstain). (C ) Mean tumor staining scores for primary tumors (䊐) and metastases ( ) stained with ACA. This lectin recognizes both T antigen and sialylated forms of T antigen. (D ) Photomicrograph of liver metastasis stained with ACA (same specimen as PNA in B ) (original magnification: B and D, 501).

trophils and lymphocytes, and on cells of the vascular endothelium.7,22,36 Selectins present on a variety of vascular endothelial cells have a high affinity for sialylated fucosylated lactosamine structures such as sialyl Lex.22,35 Because these structures are present on the extended carbohydrate chains of mucins, we examined the effect of inhibition of the expression of the MUC2 gene on the ability of metastatic colon cancer cells to bind to an E-selectin–Fc chimeric protein.22 MUC2 codes for the apoprotein core of the colon’s major secreted mucin, which is highly sialylated.37,38 Treatment of metastatic colon cancer cell line LSLiM6 with antisense to MUC2 inhibited production of high-molecular-weight glycosylated material by these cells by 50% (Figure 8A). Inhibition of mucin synthesis was further confirmed by a 40%– 50% inhibition of mucin-associated carbohydrate antigens recognized by VVA, PNA, JT1Oe, anti-Lex, and MAA in secreted media (P õ 0.05 for each antigen), by decreased expression on the cell surface (fixed-cell enzyme-linked immunosorbent assay; P õ 0.02 to P õ

0.001), and on slot blots and Western analysis of cell homogenates (data not shown). This was accompanied by a 50% reduction in adhesion to E-selectin (Figure 8B) (P õ 0.005) Treatment of cells with a scrambled control oligodeoxynucleotide had no effect on either mucin inhibition or selectin adhesion. To confirm that adhesion of these colon cancer cells to target endothelia involves selectinmediated adhesion, we examined the ability of antibody to E-selectin to inhibit adhesion of LSLiM6 to cytokinestimulated hepatic endothelial cells isolated from nude mice (a potential target in our experimental model). Pretreatment of hepatic endothelial cells with neutralizing antibody to murine E-selectin (MAb9A9) inhibited binding of cytokine-stimulated LSLiM6 to baseline (unstimulated) levels (P õ 0.0001) (Figure 9). No effect was observed with control antibody 14D4.

Discussion Mucins are high-molecular-weight 0-linked glycoproteins that are prominently expressed in the colon.

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Figure 5. Immunohistochemical staining for (A–D ) sialyl Tn and (E–G ) sialyl Lex of experimental primary cecal tumors (A, C, E, and G ) formed after inoculation of human colon cancer cells into nude mice and their spontaneous liver metastases (B, D, and F ). Cycle 1 (A, B, E, and F ) represents specimens from original group of animals injected with unselected colon cancer cells of limited metastatic ability. (C, D, and G ) Cycles 4 and 5. Liver metastases were cultured and reinjected into the cecum of additional animals, and the process was repeated serially for multiple cycles to select tumor cells of high metastatic ability (methyl green counterstain; original magnification 251).

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The carbohydrate moieties of colonic mucins are heterogeneous and consist of both core and peripheral antigens. Alterations in the expression of mucin-associated antigens occur during carcinogenesis, and a number of cancer-associated antigens are present on mucin glycoproteins.4,8 – 15 Core region carbohydrate antigens include Tn (GalNAcaThr/Ser), sialyl Tn (NeuAca6GalNAc), and T (Galb3GalNAc). Peripheral antigens include sialyl Lea (Siaa3Galb3[Fuca4]GlcNAc) and sialyl Lex (siaa3Galb4[Fuca3]GlcNAc). Alterations in the expression of cell surface and secreted sialoglycoproteins have been associated with enhanced carcinogenesis and the metastatic potential of tumor cells.6,7,29,32,39,40 The current study strongly indicates that sialylated mucin-associated carbohydrate structures are characteristic of colon cancer metastases and that this results from the selective metastasis of colon cancer cells that express these antigens. Human colon cancer metastases showed increased expression of both core (e.g., sialyl Tn) and peripheral (e.g., sialyl Lex) sialylated antigens and a reciprocal decrease in the mucin core antigens Tn and T compared with the primary tumors from which they arose. This was determined using a semiquantitative immunohistochemical scoring system that has the advantage of accounting for intratumoral heterogeneity and providing an average numeric score for the entire specimen that is subject to statistical analysis. Specimens of primary tumors and metastases from the same patients were compared using a pairwise analysis that accounts for differing numbers of metastases per patient. The use of multiple antibodies and lectins that recognize similar structures strengthen the data (summarized in Figure 10). The decreased expression of Tn and T antigens in metastases seems to result from increased sialylation of mucin core carbohy-

Figure 6. Effect of preincubation with carbohydrate-specific MAbs (B ) TKH2 (sialyl Tn) and (C ) SNH3 (sialyl Lex) or lectins (A ) SNA (Siaa6 Gal-R) on adhesion of colon cancer cell lines LS174T (low metastatic ability) and LSLiM6 (high metastatic ability) to basement membrane matrigel. Adhesion was assessed by the MTT colorimetric assay. No inhibition occurred when mouse IgG1 was substituted for specific antibodies. *P õ 0.05; **P õ 0.005.

Figure 7. Effect of desialylation with neuraminidase ( ) on adhesion of colon cancer cells to basement membrane matrigel. 䊐, Control. **P õ 0.005.

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drates because desialylation with neuraminidase significantly enhanced expression of these antigens, and this change was significantly greater for metastases than for primary tumors. In addition, although the lectin PNA bound significantly less to mucin in metastases compared with primary tumors, the lectin ACA bound strongly in metastases. Sialylation on the hydroxyl groups of galactose interferes with binding of PNA to T antigen, whereas sialylation at the Cⴕ3-hydroxyl of galactose does not interfere with ACA binding.29 These data are supported by the work of Hoff et al.39 who showed that detergent extracts of colon cancer metastases inhibited binding of MAb FH6 (sialyl-dimeric Lex) to HT-29 colon cancer cell monolayers to a greater extent than extracts from primary cancers. Western analysis of electrophoreti-

Figure 8. Effect of mucin inhibition by antisense to the MUC2 gene on adhesion of metastatic colon cancer cell line LSLiM6 to an Eselectin-Fc chimeric protein. (A ) Superose 6-gel filtration chromatography of glucosamine-labeled high-molecular-weight glycoconjugates produced by LSLiM6. Cells were treated with 5 mmol/L antisense complimentary to an 18-nucleotide consensus sequence of the MUC2 tandem repeat region (䊏). Control cells (䊐) were treated with a scrambled nucleotide sequence. (B ) Effect of mucin inhibition on adhesion to E-selectin. **P õ 0.005.

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cally separated tissue extracts indicated that FH6 bound to very high-molecular-weight glycoproteins, presumably mucins. Furthermore, human colon cancer cells selected for increased expression of sialyl-dimeric Lex are more invasive in an in vitro model using matrigel-coated filters.40 Most cancers, including those of the colon, consist of heterogeneous populations of cells, only some of which can metastasize.6,18,31,32 Immunohistochemical analysis of primary cecal tumor xenografts and spontaneous liver metastases derived from serial cycles in our animal model of human colon cancer metastasis indicates that enhanced expression of sialylated mucin core and peripheral structures in metastases results from such selective metastasis and is not simply the result of the organ environment in which they grow. Cecal tumors formed after injection of unselected colon cancer cells of limited metastatic ability showed low and heterogeneous expression of sialyl Tn and sialyl Lex. Spontaneous liver metastases showed stronger and more uniform expression of these antigens. When cell lines selected from liver metastases were reimplanted into the cecum, they retained their strong expression of the mucin-associated structures. Metastasis is a multistage process involving a variety of cell-cell and cell-substratum interactions.5 – 7 We have shown previously a relationship between mucin production by human colon cancer cells and metastasis,4,5 but

Figure 9. Effect of pretreatment of cytokine-activated hepatic endothelial cells with anti–E-selectin neutralizing antibody 9A9 on adhesion of metastatic colon cancer cell line LSLiM6 (**P õ 0.0001 compared with activated endothelial cells). Tumor cells were labeled with Na51Cr and specific adhesion quantitated by counting of radioactivity. Control nonneutralizing antibody 14D4 had no effect on adhesion. TNF, tumor necrosis factor.

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Figure 10. Summary of the expression of mucin-associated carbohydrate structures in primary colorectal cancers and their metastases. Areas within the ovals encompass the mean tumor staining scores { SEM. Solid line represents equal staining for primary tumors and metastases. Sia LeX, sialyl Lex; Sia T, sialyl T; Sia Tn, sialyl Tn.

it is not yet known which steps in metastasis depend on specific alterations in mucin structures. The present study suggests that sialylated carbohydrate structures on mucin play a role in adhesive interactions involving both basement membrane and endothelial-associated ligands. Antibodies and lectins recognizing both sialyl Tn and sialyl Lex specifically inhibited adhesion of metastatic colon cancer cells to basement membrane matrigel in a dosedependent manner. This adhesion was also inhibited by prior desialylation of the tumor cells. Although it remains to be determined precisely how adhesion of colon cancer cells to basement membrane components stimulates metastasis, we have shown previously that sequences on the a1 chain of laminin promote liver colonization by human colon cancer cells.20 Antibodies to mucinassociated sialylated antigens only partially inhibited adhesion of colon cancer cells to the extracellular matrix. The extracellular matrix is composed of multiple components, including types IV and V collagen, laminin, and heparin sulfate proteoglycan. Multiple receptors and ligands involving diverse molecules, such as cadherins, integrins, and the immunoglobulin supergene family of intercellular molecules, may play a role in the dynamic interactions that mediate cell adhesion and locomotion.7 The receptors involved in the residual adhesion of LSLiM6 to the extracellular matrix remain to be determined, but we have shown previously that this cell line expresses both the 32- and 67-kilodalton laminin-binding protein20 and an endogenous b-galactoside–binding lectin galectin-3,34 which has been associated with meta-

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static potential. Galectin-3 on colon cancer cells may interact with glycoproteins of the extracellular matrix. In addition, the latter molecule may be of particular interest because it has been hypothesized that colon cancer metastasis may involve interactions with galactosebinding lectins in the liver.41 Although colon cancer mucin is highly sialylated, we have shown recently the ability of galectin-3 to bind to residual nonsialylated structures on colon cancer–associated mucin.42 Selectins present on vascular endothelial cells have a high affinity for sialylated fucosylated lactosamine structures such as sialyl Lex.22,35,40 Several studies indicate that expression of E-selectin ligands in primary colon cancers correlates with poor survival16,17 and that human colon cancer cells adhere in vitro to tumor necrosis factor a– stimulated human umbilical endothelial cells.22,43 Our data support the importance of mucin-associated sialylated carbohydrate structures in binding to endothelial selectins because inhibition of sialomucin production with antisense oligodeoxynucleotides complementary to a region of the MUC2 gene significantly inhibited binding of metastatic colon cancer cells to E-selectin. Furthermore, neutralizing antibody to E-selectin specifically inhibited adhesion of cytokine-stimulated metastatic colon cancer cells to endothelial cells from the liver, the major target of distant metastasis. Metastatic colon cancer cell line LSLiM6 had a low basal adhesion to nonactivated endothelial cells. The receptor(s) involved in this basal adhesion and their potential role in metastasis have not been determined, but E-selectin–mediated adhesion to stimulated endothelial cells involving mucin-associated sialoglycoproteins seems to play an important role in tumor cell-endothelial interactions. A growing body of evidence suggests a role for mucinassociated carbohydrate structures in the progression and biological behavior of colorectal cancers. The present study extends these findings and strongly indicates that mucin-associated sialoglycoproteins play a role in colorectal cancer metastasis.

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42. Bresalier RS, Byrd JC, Raz A. Colon cancer mucin is a major ligand for the b-galactoside binding protein, galectin-3 (abstr). Proc Am Assoc Cancer Res 1996;37:81. 43. Takada A, Ohmori K, Yoneda T, Tsuyuoka K, Hasegawa A, Kiso M, Kannagi R. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res 1993; 53:354 – 361.

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Received July 5, 1995. Accepted December 14, 1995. Address requests for reprints to: Robert S. Bresalier, M.D., Henry Ford Health Sciences Center, 2799 West Grand Boulevard, Detroit, Michigan 48202. Fax: (313) 876-9487. Supported by the Research Service of the Henry Ford Health Sciences Center and Research Foundation (to R.S.B.), National Cancer Institute grant RO1 CA69480 (to R.S.B.), and the Research Service of the Department of Veterans Affairs (to R.S.B. and Y.S.K.).