The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

Abstract

Background: The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).

Results: We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.

Conclusion: C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.

Figure 1

Diagram of core 2 β1,6 O-glycan synthesis. (A) Core 1 O-glycans are synthesized by addition of β1,3 galactose to N-acetylgalactosamine. (B) The C2GnT1 enzyme converts an unsubstituted core 1 O-glycan to a core 2 β1,6 O-glycan. (C) Core 2 can be further modified by α2,3 sialyltransferase and α1,3 fucosyltransferase, forming a sLe^x terminus (dotted box). These modifications result in the synthesis of the sLe^x-modified core 2 β1,6 O-glycan (C2-O-sLe^x) structure. The figure is simplified and some enzymatic steps are omitted for clarity. GalNAc, N-acetylgalactosamine; Gal, galactose; GlcNAc, N-acetylglucosamine; NeuAc, sialic acid; Fuc, fucose.

Figure 2

Human well differentiated colorectal adenocarcinomas expressed low levels of C2-O-sLe^x. (A) Photomicrograph of a tissue section of a well differentiated colorectal adenocarcinoma stained with hematoxylin and eosin, scale bar = 500 μm. (B) A serial section of the same tissue as shown in (A) stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 500 μm. In (A) and (B) the arrow indicates adjacent normal colorectal mucosa. The arrowhead indicates nests of neoplastic cells in the tunica submucosa and muscularis. (C) Another well differentiated colorectal adenocarcinoma stained with CHO-131 mAb. Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 100 μm. (D) Increased magnification of the boxed area shown in (C) demonstrating red stained CHO-131⁺ cells. Arrows in (D) indicate cytoplasmic and luminal reactivity with CHO-131 mAb, scale bar = 50 μm. (E) Mucosa of normal colorectal epithelium stained with CHO-131 mAb. Note the absence of red color, indicating a lack of reactivity with the CHO-131 mAb, scale bar = 150 μm. (F) A serial section of the same tissue as in (C) and (D), stained with CEA mAb (1.6 μg/ml). Brown color indicates positive reactivity with CEA mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 20× magnification, (C) 200× magnification, (D) 400× magnification, (E) 100× magnification, and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.

Figure 3

Human colorectal adenocarcinomas, but not colorectal adenomas, expressed high levels of C2-O-sLe^x. (A) Photomicrograph of a tissue section of a moderately differentiated colorectal adenocarcinoma stained with CHO-131 mAb (15 μg/ml). Red color indicates positive cytoplasmic and luminal reactivity with CHO-131 mAb. (B) A poorly differentiated colorectal adenocarcinoma stained with CHO-131 mAb. Notice the red color indicating positive cytoplasmic reactivity of neoplastic cells (arrow). (C) A colorectal adenoma stained with hematoxylin and eosin. (D) A serial section of the same tissue as in (C), stained with CHO-131 mAb. Notice the absence of red color. (E) Mucosa of normal colorectal epithelium stained with CHO-131 mAb. (F) A serial section of the same tissue as in (C) and (D), stained with CEA mAb (1.6 μg/ml). The brown color indicates positive reactivity with CEA within the lumens of glands and in the cellular cytoplasm. All tissue sections were 4 μm thickness; scale bars = 100 μm, 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.

Figure 4

Moderately differentiated human colorectal adenocarcinomas expressed high levels of C2-O-sLe^x. (A) A graph of the number of neoplastic cells in well, moderately, and poorly differentiated colorectal adenocarcinomas that were positive for expression of CHO-131 mAb at low (0+ to 1+ staining intensity) and high (2+ to 3+ staining intensity) levels. (B) A graph of the percentage of colorectal adenocarcinomas that were highly positive for C2-O-sLe^x compared to sLe^x; n = 22, n = 83, and n = 8 for well, moderately, and poorly differentiated colorectal adenocarcinomas stained with CHO-131 mAb that detected C2-O-sLe^x; n = 11, n = 112, and n = 11 for well, moderately, and poorly differentiated colorectal adenocarcinomas stained with CSLEX1 mAb that detected sLe^x. *Moderately differentiated colorectal adenocarcinomas expressed significantly higher levels of C2-O-sLe^x than well differentiated carcinomas by the Pearson's chi-squared test, p = 0.04.

Figure 5

Human primary colorectal adenocarcinomas and metastatic liver tumors expressed sLe^x. (A) Photomicrograph of a serial tissue section of the same well differentiated colorectal adenocarcinoma as shown in Figure 2A stained with CSLEX1 mAb (15 μg/ml). Brown color indicates positive reactivity with the CSLEX1 mAb. The arrow indicates adjacent normal colorectal mucosa that lacks reactivity with CSLEX1 mAb, scale bar = 500 μm. (B) A moderately differentiated colorectal adenocarcinoma stained with CSLEX1 mAb. Brown color indicates positive reactivity with CSLEX1 mAb, scale bar = 100 μm. (C) A serial tissue section of the same poorly differentiated colorectal adenocarcinoma as in Figure 3B stained with CSLEX1 mAb (15 μg/ml). Brown color indicates positive reactivity with CSLEX1 mAb, scale bar = 100 μm. (D) A moderately differentiated metastatic colorectal adenocarcinoma in liver stained with CSLEX1 mAb. The cytoplasm of neoplastic cells reacted positively with CSLEX1 mAb as indicated by the brown color, scale bar = 50 μm. (E) Mucosa of normal colorectal epithelium stained with CSLEX1 mAb. Note the absence of brown color, indicating a lack of reactivity with CSLEX1 mAb, scale bar = 100 μm. (F) Normal liver stained with CSLEX1 mAb. Note the absence of brown color, indicating a lack of reactivity with the CSLEX1 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figure (A) 20× magnification, (B) and (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain. Representative sections from multiple stained tissues are shown.

Figure 6

Human metastatic liver tumors of primary colorectal adenocarcinomas expressed C2-O-sLe^x. (A) Photomicrograph of a tissue section of metastatic colorectal adenocarcinoma tumors in liver stained with hematoxylin and eosin, scale bar = 150 μm. (B) A serial section of the same tissue as shown in (A), stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 150 μm. (C) A different tissue section of metastatic colorectal adenocarcinoma in liver stained with CHO-131 mAb, scale bar = 100 μm. (D) A higher magnification of the same tissue section as in (C), stained with CHO-131 mAb (15 μg/ml), scale bar = 50 μm. (E) A serial section of the same tissue as shown in (C) and (D), stained with CEA mAb (1.6 μg/ml) as a positive control. Brown stained cells are positive for CEA mAb, scale bar = 100 μm. (F) Normal liver stained with CHO-131 mAb (15 μg/ml). Note the absence of red color, indicating a lack of reactivity with CHO-131 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 100× magnification, (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.

Figure 7

C2GnT1 mRNA levels were upregulated in human colorectal adenocarcinomas compared to normal tissues. (A) Graph of amplification plots obtained by the Applied Biosystems 7500 Real-Time PCR system for C2GnT1 mRNA expression. (B) A box-plot showing the results of semi-quantitative RT-PCR analysis. The distribution of mRNA gene expression is shown as a fold change in C2GnT1 mRNA expression in normal colorectal tissues and in well, moderately, and poorly differentiated colorectal adenocarcinomas. A Kruskal-Wallis non-parametric test was performed for statistical comparison among the groups. ^a Denotes a significant difference (p = 0.015) between normal and well groups; ^b Denotes a significant difference (p = 0.025) between normal and moderate groups.