Volume 115, Issue 2, Pages 287-296 (August 1998) Metastasis of human colon cancer is altered by modifying expression of the β- galactoside-binding protein galectin 3  Robert S. Bresalier*,‡,§, Nachman Mazurek*, Lawrence R. Sternberg*, James C. Byrd*, Christopher K. Yunker*, Pratima Nangia- Makker§, Avraham Raz§  Gastroenterology  Volume 115, Issue 2, Pages 287-296 (August 1998) DOI: 10.1016/S0016-5085(98)70195-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Expression of galectin 3 in human colon cancer cell lines of differing metastatic capacity by Western blot analysis. Parental cell line LS174T has low metastatic potential, whereas cell lines LSLiM6 (LiM6) and HM7 have high metastatic potential in animal models (see text). Equal amounts of homogenate protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot analysis as described in Materials and Methods. (B) Reverse-transcription PCR analysis showing corresponding differences in the 756-bp galectin 3 RNA product in variant cell lines. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Expression of galectin 3 in human colon cancer cell lines of differing metastatic capacity by Western blot analysis. Parental cell line LS174T has low metastatic potential, whereas cell lines LSLiM6 (LiM6) and HM7 have high metastatic potential in animal models (see text). Equal amounts of homogenate protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot analysis as described in Materials and Methods. (B) Reverse-transcription PCR analysis showing corresponding differences in the 756-bp galectin 3 RNA product in variant cell lines. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of galectin 3 antisense on galectin 3 expression in LiM6- and HM7-derivative lines. (A) Changes in relative abundance of galectin 3 and β-actin mRNA resulting from stable introduction of pGal-3AS into LSLiM6- and HM7-derivative cell lines. The values represent mean individual density values generated from four identically prepared RNA dot blots containing 1 μg of total RNA. The results of analogously probed blots generated from serial dilutions of these RNAs (100 and 10 ng) were used in linear regression analyses that indicate multifold changes in galectin 3 mRNA in the absence of concomitant changes in β-actin mRNA (see text). (B and C) Hybridization by a galectin 3 mRNA–specific riboprobe to Northern blots of total RNA, indicating corresponding relative decreases in galectin 3–specific mRNA in derivative cell lines. Size in kilobases has been estimated from molecular weight markers. L6, LiM6; Gal-3, galectin 3; Asen, antisense. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of stable introduction of galectin 3 sense or antisense constructs on galectin 3 protein expression in human colon cancer cells (Western blot analysis). (A) Relative abundance of galectin 3 in cell line LS174T, a vector-transformed control (Cont) or LSSen, the galectin 3 sense-transformed derivative (Sense). (B and C) Relative abundance of galectin 3 in LSLiM6 (LiM6), HM7, vector-transformed controls, or galectin 3 antisense–transformed derivatives LSLiM6ASen and HM7ASen (ASense). Western blot analyses were performed using MAb TIB-166 as described in Materials and Methods. Data in A, B, and C represent separate experiments. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Stable integration of pGal-3S into the genome of LSSen. (A) Hybridization by a galectin 3–specific DNA probe to restriction-digested genomic DNA from LS174T (LS174) and LSSen (LS-Sen). Results show the presence of two endogenous galectin 3 bands (7.0 and 2.2 kb) and an additional 0.9-kb species in LSSen, consistent with stable integration of PGal-3S. (B) Hybridization by galectin 3 mRNA–specific (left) and human β-actin–specific (right) riboprobes to identically prepared Northern blots of total RNA. The results indicate the presence of a novel galectin 3–specific band in LSSen. The size in kilobases has been estimated from molecular weight markers (not shown). Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Alterations in cell surface galectin 3 after stable transfection as determined by flow cytometry. Single-cell suspensions of colon cancer cell lines were exposed to anti–galectin 3 MAb TIB-166 and submitted to flow analysis as described in Materials and Methods. Introduction of galectin 3 antisense into metastatic cell lines HM7 and LSLiM6 resulted in reductions in cell surface galectin 3, whereas introduction of galectin 3 sense into low-metastatic LS174T resulted in increased cell surface expression. Solid areas depict controls (second antibody only). Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Liver colonization by derivatives of human colon cancer cell line HM7 after splenic–portal injection. Livers were harvested from athymic nude mice 4 weeks after splenic-–portal injection of 106 cells. The top row depicts representative livers of animals inoculated with HM7 cells transfected with vector alone, whereas the bottom row depicts livers from animals injected with HM7ASen cells transfected with antisense to galectin 3. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Galectin 3 expression in liver metastases from (A) animals injected with HM7 control cells or (B) cells transfected with antisense to galectin 3 (HM7AS). Galectin 3 was detected using MAb TIB-166. Derivative colon cancer cell lines maintain their galectin 3 phenotype in vivo (H&E; original magnification 100×). Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Galectin 3 expression in liver metastases from (A) animals injected with HM7 control cells or (B) cells transfected with antisense to galectin 3 (HM7AS). Galectin 3 was detected using MAb TIB-166. Derivative colon cancer cell lines maintain their galectin 3 phenotype in vivo (H&E; original magnification 100×). Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Flow analysis of human colon cancer cells and derivatives transfected with galectin 3 antisense and sense constructs. Single-cell suspensions were allowed to aggregate for 30 minutes, and the formation of tumor cell aggregates was measured. Altered aggregation of transfected cells compared with control cells is indicated by a shift in forward-angle light scatter. Gastroenterology 1998 115, 287-296DOI: (10.1016/S0016-5085(98)70195-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions