MDA-MB-435 Human Breast Carcinoma Cell Homo- and Heterotypic Adhesion under Flow Conditions Is Mediated in Part by Thomsen-Friedenreich Antigen-Galectin-3 Interactions*

Sophia K. KhaldoyanidiDagger §, Vladislar V. Glinsky§, Lyudmila SikoraDagger , Anna B. Glinskii||, Valerie V. Mossine, Thomas P. Quinn, Gennadi V. Glinsky||**, and P. SriramaraoDagger **DaggerDagger

From the Dagger  Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California 92121,  Department of Biochemistry, University of Missouri, Columbia, Missouri 65201, and || Sidney Kimmel Cancer Center and Metastat, Inc., San Diego, California 92121

Received for publication, September 18, 2002, and in revised form, November 14, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The importance of Thomsen-Friedenreich antigen (T antigen)-galectin-3 interactions in adhesion of human breast carcinoma cells to the endothelium under conditions of flow was studied. Highly metastatic cells (MDA-MB-435) expressing high levels of both galectin-3 and T antigen demonstrated significantly increased adhesion to monolayers of endothelial cells compared with their non-metastatic counterpart (MDA-MB-468) in vitro. Within minutes of adhesion, the highly metastatic cells acquire the ability of enhanced homotypic adhesion, leading to the formation of multicellular aggregates at sites of attachment to endothelial cells in vitro. Treatment of cells with lactulosyl-L-leucine, a synthetic T antigen antagonist that targets galectin-3 by mimicking T antigen, caused a 60-80% inhibition of both homo- and heterotypic adhesion of MDA-MB-435 cells. Confocal microscopy and fluorescence-activated cell sorter analysis revealed redistribution of endothelial galectin-3 to the site of heterotypic intercellular contacts, whereas galectin-3 in MDA-MB-435 cells accumulated at sites of homotypic interaction. MDA-MB-435 cells also exhibited increased adhesion and intravascular retention within the microvessels of transplanted lung allografts in nude mice. T antigen and galectin-3-mediated interactions of metastatic cancer cells with endothelium under conditions of flow are characterized by a unique adhesion mechanism that qualitatively distinguishes their homo- and heterotypic adhesive behavior from other cell types such as leukocytes.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Understanding cellular and molecular mechanisms of tumor metastasis is critically important for the development of new approaches to cancer treatment. One of the rate-limiting steps in metastatic dissemination is the adhesion of circulating cancer cells to the microvascular endothelium (for review, see Ref. 1). Recent experimental evidence identified endothelium-attached blood-born tumor cells as the seeds of secondary tumors (2). In the lung, early metastatic colonies were entirely within the blood vessels, and hematogenous metastases originated from the intravascular proliferation of tumor cells anchored to the endothelia (2). These results underscored the significance of intravascular intercellular adhesion in cancer metastasis.

Although there is a substantial body of evidence demonstrating the role of various adhesion molecules in tumor cell adhesion (1, 3), the molecular and cellular mechanisms of cancer cell adhesion are still often modeled after the dynamics of the leukocyte adhesion cascade. Despite the many physical similarities, interaction of leukocytes and circulating malignant cells with the vascular endothelium are likely to be driven by distinct molecular mechanisms. For example, it is well documented that under conditions of shear force, circulating leukocytes participate in a multi-step cascade of sequential adhesion events involving rolling, adhesion, and transmigration across the vascular wall, where rolling is the first and rate-limiting step ultimately required for stable leukocyte adhesion to the endothelial cells (EC)1 (4). However, in contrast to leukocytes, published data regarding the rolling and adhesion of tumor cells on vascular endothelium suggest a non-leukocyte-like mechanism (5-9). Furthermore, it is also not clear whether this step is required for stable adhesion of tumor cells to the endothelium. Leukocyte rolling is mostly mediated by the interaction of the members of C-type lectin family, selectins, with their cognate carbohydrate ligands (4, 10). Studies from our laboratories (11, 12) as well as other investigators (13) have recently shown that another lectin, galectin-3, plays a key role in initiating the adhesion of human breast and prostate cancer cells to the endothelium by specifically interacting with the cancer-associated carbohydrate, T antigen. However, these studies were carried out under static conditions, and the relevance of galectin-3-T antigen interactions in mediating cancer cell adhesion under conditions of flow has not been investigated.

Shear forces have an important influence on cell adhesion and other cellular functions, and malignant cell lines appear to possess different adhesive properties under static and dynamic conditions (14, 15). To elucidate the molecular mechanisms of intercellular adhesive interactions relevant to breast cancer metastasis, we examined the adhesive behavior of two human breast carcinoma cell lines exhibiting distinct metastatic potential under conditions of flow in vitro and in vivo. Our studies have led to the identification of a novel sequence of T antigen-mediated adhesive events with galectin-3 that occur between breast carcinoma cells (homotypic) as well as between breast carcinoma cells and the vascular endothelium (heterotypic) under conditions of flow. This rapid activation of adhesive properties, resulting in the formation of multicellular aggregates at sites of primary attachment, appears to be unique to highly metastatic breast carcinoma cells, which qualitatively distinguishes their interaction with the endothelium from cells such as leukocytes and other less metastatic cells. These results underscore the potential significance of T antigen-galectin-3 interactions in mediating both homotypic and heterotypic intercellular adhesion of metastatic breast carcinoma cells under conditions of flow. To the best of our knowledge, this phenomenon, which could play an important role in the establishment of breast cancer micrometastasis, has not been described previously.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 5-(and 6)-(((4-Chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR), carboxyl fluorescein diacetate (CFDA), and goat Texas Red conjugated anti-rat antibody were obtained from Molecular Probes, Eugene, OR.

Cell Culture-- The previously described MDA-MB-435 and MDA-MB-468 human breast carcinoma cell lines of distinct metastatic potential in nude mice were kindly provided by Dr. Janet E. Price, M. D. Anderson Cancer Center, Houston, TX (16-19). Tumor cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and gentamycin (Invitrogen). Cells were grown to 70-80% confluency, detached from culture flasks using Enzyme Free Cell Dissociation Solution (Specialty Media, Phillipsburg, NJ), washed in serum-free Ultraculture media (Specialty Media), and used in adhesion experiments immediately. Simultaneous identification of live and dead cells was performed using acridine orange and ethidium bromide staining (20). Viability of cells used in adhesion experiments was 95% or greater. Human umbilical vein EC (HUVEC) and human lung microvasculature EC (HLMVEC) were obtained from Clonetics (Walkersville, MD). EC were grown on coverslips pre-coated with poly-L-lysine/fibronectin (10 µg/ml) until 100% confluent in cell type-specific media (Clonetics). To ensure phenotypic stability of cells, only breast carcinoma cells having undergone less than five passages in culture were utilized for the in vitro and in vivo adhesion assays.

In Vitro Laminar Flow Assay-- Rolling and adhesion of infused MDA-MB-435 and MDA-MB-468 cells were assessed in an in vitro parallel plate laminar flow chamber as previously described (21, 22). Briefly, glass coverslips were coated with poly-L-lysine (10 µg/ml) overnight at 4 °C and washed with phosphate-buffered saline twice. EC (HUVEC or HLMVEC) were grown on the poly-L-lysine-coated glass coverslips till 100% confluent. The glass coverslips with the EC were then positioned in the bottom of a parallel plate flow chamber (100-µm thickness), where the coverslips were exposed to different flow conditions. Defined levels of flow (increasing wall shear stress) were applied to the coverslip in the flow chamber by perfusing warm media (RPMI containing 0.75 mM Ca2+ and Mg2+ and 0.2% human serum albumin) through a constant infusion syringe pump (Harvard Apparatus, Holliston, MA). The flow chamber was next perfused with a single cell suspension of the MDA-MB-435 or MDA-MB-468 cells (5 × 104 cells/ml) for a period of 5 min. The interactions of the injected cells with the EC layer were observed using a Leitz Wetzlar inverted microscope, and the images were video-recorded for subsequent offline video analysis to manually determine the number of interacting cells. Rolling cells demonstrate multiple discrete interruptions and flow slowly, whereas adherent cells remain stationary at a given point for extended periods of time (>30 s) (22). Results are expressed as the number of rolling or adherent cells/field (average of four fields). In some experiments, MDA-MB-435 cells were incubated for 15 min with a synthetic low molecular weight non-toxic T-antigen mimicking galectin-3 antagonist lactulosyl-L-leucine (LL) (23) or its non-active isomer, lactitol-L-leucine (LT) at a final concentration of 1 mM and infused through the laminar flow chamber at a shear stress of 4 dyn/cm2. In addition, MDA-MB-435 cells were also preincubated with undiluted culture supernatant of monoclonal rat anti-human galectin-3 antibody hybridoma (TIB 166 from ATCC, Manassas, VA) or normal rat IgG (10 µg/ml in RPMI) for 15 min before infusion through the laminar flow chamber. Because galectin-3 belongs to the lactose binding (beta -galactosidase) group of proteins/lectins, the ability of lactose (Sigma) to inhibit binding of MDA-MB-435 cells to EC was also tested in this assay. Maltose (Sigma) was used as a control, and both sugars were tested at a final concentration of 10 mM.

Lung Allograft Technique and Intravital Microscopy-- Dorsal skin-fold chambers in nude mice were prepared as previously described (21, 24). Briefly, 10-week-old nude mice (25-30 g) were anesthetized (a mixture of xylazine 10 mg/kg and ketamine 200 mg/kg body weight, (Western Medical Supply, Arcadia, CA)), and one pair of identical titanium frames was implanted into a dorsal skin-fold parallel to the dorsum of the animal so as to sandwich the stretched double layer of skin. One layer of the dorsal skin was completely removed in a circular area of 15-mm diameter, and the remaining underlying skin layer was covered with a coverslip incorporated into one of the frames, after which the animals were allowed to recover from anesthesia. After an additional convalescence period of 2-3 days, sections of murine lung derived from female nude mice were labeled with 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) (a fluorescent dye that enables visualization of the lung allograft under an intravital video fluorescence microscope) and carefully placed into the chamber over the subcutaneous tissue. The transplanted murine lung allografts demonstrated complete revascularization within the skin chamber after 10-15 days post-transplantation2 (25). The ability of MDA-MB-435 versus MDA-MB-468 human breast carcinoma cells to interact with the revascularized lung microvasculature within the skin-fold chamber was investigated by intravital fluorescent microscopy. Briefly, 2.5 × 106 MDA-MB 435 or MDA-MB-468 cells labeled with CFDA (22) were injected into the tail vein of nude mice. Adhesive interactions of the CFDA-labeled cells within the lung microvasculature in the skin-fold chamber were visualized by stroboscopic epi-illumination using a video triggered xenon lamp and a Leitz Ploemopak epi-illuminator employing an I2 filter block. The images were recorded through a silicon-intensified tube camera (SIT68, Dage MTI, Michigan, IN) using a X10/0.13 water immersion objective (Nikon, Tokyo, Japan) and a SVHS video recorder HC-6600 (JVC, Tokyo, Japan).

Western Blot Analysis-- Equal amounts (30 µg) of total cellular protein extracted from MDA-MB-435 and MDA-MB-468 cells were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes. After blocking with a 2% solution of bovine serum albumin in Tris-buffered saline, galectin-3 was detected by incubation with monoclonal rat anti-human galectin-3 antibody hybridoma (ATCC) for 1 h at room temperature followed by alkaline phosphatase-conjugated goat anti-rat IgG (2 h at room temperature) and developed with Sigma FAST 5-bromo-4-chloro-indolyl phosphate/nitro blue tetrazolium (Sigma). The membranes were washed 3 times for 10 min each time in a 2% solution of bovine serum albumin in Tris-buffered saline between steps.

Immunofluorescence and Laser-scanning Confocal Microscopy-- Samples for the analysis of the cellular distribution of T antigen were prepared as detailed in our previous studies (11). T antigen expression was analyzed based on binding of biotinylated T antigen-specific peanut agglutinin lectin (Sigma) and detected with neutravidin-Texas Red conjugate (Molecular Probes). Labeled cells were mounted under cover glass and examined by fluorescent microscopy. Samples for the analysis of galectin-3 cellular distribution were prepared as previously described (12). Briefly, HUVECs were grown to confluence directly on microscope slides using the 4-well Lab-Tec II chamber slide system (NalgeNunc, Naperville, IL). MDA-MB-435 cells (5 × 104 cells/chamber) were added to the monolayer of EC and allowed to adhere for 1 h at 37 °C. At the end of the incubation, samples were gently rinsed with phosphate-buffered saline, fixed, and permeabilized in 4% formaldehyde solution in phosphate-buffered saline for 24 h. Monoclonal rat anti-human galectin-3 (ATCC) and goat Texas Red-conjugated anti-rat antibodies (Molecular Probes) were used to visualize galectin-3. The laser-scanning confocal microscopy was performed on a Bio-Rad MRC 600 confocal system. The Z stacks were prepared by obtaining serial sections with 0.5-µm increments and analyzed in orthogonal projections (Y-Z and X-Z sections) using the MetaMorph Imaging System software (Universal Imaging, Hallis, NH).

Flow Cytometry-- MDA-MB-468 or MDA-MB-435 cells (5 × 104 cells/ml) were passed through the laminar flow chamber containing HUVEC-coated coverslips at 0.8 dyn/cm2. EC and adherent tumor cells were harvested from the coverslips using a non-enzymatic cell dissociation reagent (Specialty Media), and the cells were used for fluorescence-activated cell sorter analysis. For galectin-3 expression, 5 × 105 cells were incubated with monoclonal antibodies (mAb) directed against galectin-3 (BD Biosciences) at a concentration of 10 µg/ml for 30 min at 4 °C and washed 2 times with fluorescence-activated cell sorter buffer (2% fetal calf serum, 0.1% bovine serum albumin, 0.01% NaN3 in phosphate-buffered saline). This was followed by incubation with fluorescein isothiocyanate-labeled secondary antibodies. For CD31 expression, phycoerythrin-labeled mAbs against CD31 (Ancell, Bayport, MN) were used. Cells incubated with appropriate isotype-matched, nonspecific phycoerythrin- or fluorescein isothiocyanate-labeled antibodies served as the negative control. Fluorescence was analyzed on a FACScan flow cytometer (BD Biosciences) according to standard procedures.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human Breast Carcinoma Cells Adhere to EC under Conditions of Physiological Shear Stress in Vitro-- The ability of two human breast carcinoma cells of differing metastatic potential to roll and subsequently develop firm adhesive interactions with EC under conditions of flow was examined using a parallel plate laminar flow chamber. Single cell suspensions of MDA-MB-435 and MDA-MB-648 cells were passed through flow chambers containing a monolayer of primary HUVEC adhered to coverslips. Interaction of these cells with HLMVEC was also examined in addition to HUVEC since the MDA-MB-435 cells, which were originally isolated from a pleural effusion of a breast cancer patient (16), are known to develop spontaneous lung metastasis in vivo in nude mice (17). There was no significant difference in the ability of the highly metastatic MDA-MB-435 and the poorly metastatic MDA-MB-468 cell lines to roll on HUVEC or HLMVEC under conditions of flow in vitro (data not shown). In contrast, MDA-MB-435 cells were observed to be 2-4-fold more adhesive to HLMVEC compared with MDA-MB-468 cells (Fig. 1). Strikingly, highly metastatic MDA-MB-435 cells demonstrated a unique ability to undergo a quick transition from rolling to stable adhesion on EC followed by rapid formation of multicellular tumor cell clumps at the site of firm adhesion, apparently due to enhanced homotypic aggregation. We observed that within a few minutes (<1.5 min) of firm adhesion of a single tumor cell to the EC, it frequently captured other rolling and floating MDA-MB-435 cells and initiated rapid formation of homotypic multicellular tumor cell aggregates at the site of primary attachment (Fig. 2). In contrast, poorly metastatic MDA-MB-468 breast carcinoma cells failed to significantly adhere to the EC and form multicellular aggregates on the EC layer under conditions of flow (data not shown). Thus, homotypic aggregation and multicellular clump formation appears to be a novel phenomenon restricted to the highly metastatic MDA-MB-435 cells.


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Fig. 1.   Adhesive properties of highly metastatic (MDA-MB-435) and poorly metastatic (MDA-MB-468) human breast carcinoma cells with human EC under conditions of flow in vitro. The adhesion of MDA-MB-435 and MDA-MB-468 to HLMVEC under conditions of varying shear stress (0.8-4 dyn/cm2) was studied. A single cell suspension (>95% viability; 5 × 104 cells/ml) was infused through a parallel plate laminar flow chamber, and the number of adherent cells per field was calculated. Data are presented as the mean ± S.D. of four observation fields from one representative experiment of three independent experiments.


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Fig. 2.   Formation of multicellular homotypic tumor cell aggregates by MDA-MB-435 cells. A single cell suspension of MDA-MB-435 cells (5 × 104 cells/ml) was infused through the laminar flow chamber at 0.8 dyn/cm2. The formation of homotypic multicellular clumps by MDA-MB-435 cells on HLMVEC was monitored under an inverted microscope and video-recorded as described under "Experimental Procedures" (n = 6). Soon after the adhesion of a single MDA-MB-435 cell to the EC, it frequently captured other rolling and floating MDA-MB-435 cells and initiated rapid formation of homotypic multicellular tumor cell aggregates at the site of primary attachment. The time taken for the formation of a representative homotypic multicellular MDA-MB-435 cell aggregate is shown on the right.

Role of T Antigen-Galectin-3 Interactions in Mediating the Adhesion of Metastatic Human Breast Carcinoma Cells to EC under Conditions of Flow-- Next, attempts were made to determine the molecular mechanisms involved in the adhesion of highly metastatic human breast carcinoma cells to EC. Because rolling and stable adhesion of granulocytes to vascular endothelium is mediated by the engagement of L-selectin and multiple cell surface integrins (alpha 4 or beta 2 integrins) (4, 10, 22), we attempted to determine if these receptors contribute to the adhesive interactions between metastatic human breast carcinoma cells and EC under conditions of flow. Our experiments revealed that pretreatment of MDA-MB-435 cells with function blocking anti-L-selectin, anti-alpha 4, or anti-beta 2-integrin mAbs failed to inhibit adhesion of these cells to EC under conditions of flow (data not shown). These results suggest that leukocyte rolling and adhesion receptors are unlikely to play a role in mediating adhesive interactions between MDA-MB-435 and EC. Our previous results from static adhesion assays demonstrated that the adhesion of MDA-MB-435 to EC is mediated by the interaction of cancer-associated carbohydrate, T antigen, with the endothelium-expressed beta -galactoside-binding protein galectin-3 and could be efficiently blocked by a synthetic low molecular weight compound LL (12). LL exerts its biological activity by interfering with the function of galectins (26), particularly galectin-3, by mimicking the essential structural features of T antigen and specifically blocking T antigen-galectin-3 interactions (12). Therefore, we investigated whether LL could also interfere with the adhesive interactions of MDA-MB-435 cells with human EC under conditions of flow. The incubation of MDA-MB-435 cells with LL at a final concentration of 1 mM before infusion into the flow chamber resulted in a significant inhibition (60-80%, p < 0.05) of tumor cell rolling and adhesion to EC (Fig. 3). Furthermore, treatment with LL also inhibited the homotypic multicellular aggregation and clumping of MDA-MB-435 cells on the EC monolayer (data not shown). In contrast, the non-active isomer, LT, which failed to inhibit T antigen-mediated adhesive interactions under static conditions (12) and was used as a control, did not affect either heterotypic or homotypic adhesion of MDA-MB-435 cells under conditions of flow. In addition, to confirm the involvement of galectin-3 in mediating tumor cell interactions with the endothelium, MDA-MB-435 cells were pre-incubated with anti-galectin-3 mAb (TIB 166) or lactose, including appropriate controls, before infusion in the laminar flow assay (Fig. 3). Anti-galectin-3 mAb and lactose were found to significantly inhibit (p < 0.05) MDA-MB-435 cell rolling and adhesion to EC, whereas normal rat IgG or maltose, which served as the corresponding controls, failed to alter these interactions. Overall, the flow studies demonstrate that T antigen-galectin-3 interactions may play an important role in intercellular adhesion of metastatic human breast carcinoma cells to human EC. Taken together with previous results that demonstrate that LL efficiently inhibits human breast cancer metastasis (up to 75%) in vivo in nude mice (26), these findings strongly suggest that T antigen-galectin-3 interactions could be important in metastatic dissemination of human breast cancer.


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Fig. 3.   The effect of LL on rolling and adhesion of MDA-MB-435 cells to human EC under conditions of flow. A single cell suspension of MDA-MB-435 cells (104 cells/ml) was incubated for 15 min with LL or LT (control non-active isomer) at a final concentration of 1 mM, anti-galectin-3 (gal-3) mAb (TIB 166, culture supernatant) or normal rat IgG (10 µg/ml), and lactose or maltose at a final concentration of 10 mM before infusion through the laminar flow chamber containing HUVEC-coated coverslips. The number of rolling and adherent tumor cells was calculated and expressed as the mean ± S.D. of four observation fields from one representative experiment of three independent experiments.

Expression of T Antigen and Galectin-3 by MDA-MB-435 and MDA-MB-468 Cells-- Because the adhesion of metastatic MDA-MB-435 cells to EC appeared to be dependent, at least in part, on T antigen-galectin-3 interactions, we examined whether MDA-MB-435 and MDA-MB-468 cells exhibit differences in expression of T antigen and galectin-3. Western blot analysis of whole cell extracts with anti-galectin-3 antibody (Fig. 4A) demonstrated that highly metastatic MDA-MB-435 cells express high levels of galectin-3, whereas poorly metastatic MDA-MB-468 cells were deficient in galectin-3. Furthermore, fluorescence microscopy using biotinylated T antigen-specific peanut agglutinin lectin followed by neutravidin-Texas Red conjugate showed that the highly metastatic MDA-MB-435 cells express high levels of T antigen (Fig. 4B), in contrast to the poorly metastatic MDA-MB-468 cells (Fig. 4C). The levels of T antigen expression on these cells was visualized in the pseudocolored images of the fields shown in Fig. 4, B and C (Fig. 4, D and E, respectively), where the different colors (purple, blue, green, and yellow) correspond to the level of T antigen expression, viz. with purple being the lowest, and yellow being the highest. These results further support the possible role for T antigen and galectin-3 in defining the metastatic potential of breast carcinoma cells.


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Fig. 4.   Differences in expression of galectin-3 and T-antigen on highly metastatic MDA-MB-435 and poorly metastatic MDA-MB-468 human breast carcinoma cells. A, Western blot analysis of galectin-3 expression in MDA-MB-468 and MDA-MB-435 cells. Total cell extract (30 µg) from each cell line was resolved by SDS-PAGE and subjected to Western blotting using rat anti-galectin-3 antibodies. B and C, expression of T antigen by poorly metastatic MDA-MB-468 (B) and highly metastatic MDA-MB-435 (C) cells visualized by fluorescence microscopy. D and E, pseudocolored images of the fields shown in B and C, respectively. Different colors (purple, blue, green, and yellow) correspond to different levels of T antigen expression (purple being the lowest, and yellow being the highest).

Cellular Distribution Pattern of Galectin-3 during MDA-MB-435 Cell-EC Interactions by Confocal Microscopy and Fluorescence-activated Cell Sorter Analysis-- Recent studies have shown that galectins support heterotypic intercellular adhesion such as the adhesion of human cancer cells to EC in static adhesion assays (11-13). Based on findings from the present study, galectin-3 appears to be an adhesion molecule that can conceivably participate in homotypic and heterotypic adhesive interactions under conditions of flow. We utilized confocal microscopy to study the intracellular distribution of galectin-3 during homo- and heterotypic intercellular adhesion of MDA-MB-435 and EC. Confocal microscopy of a homotypic aggregate of the MDA-MB-435 breast carcinoma cells adhered to a HUVEC monolayer using rat anti-galectin-3 followed by goat Texas Red-conjugated secondary antibodies is shown in Fig. 5A. Galectin-3 molecules in the MDA-MB-435 and EC were found to exhibit distinct intracellular redistribution. Pseudocolored images of the orthogonal sections through the YZ and XZ planes of the field shown in Fig. 5A revealed a rapid redistribution of endothelial galectin-3 to the site of heterotypic intercellular contacts (Fig. 5B), whereas galectin-3 in MDA-MB-435 cells accumulates at the site of homotypic intercellular contacts (Fig. 5C). Different colors represent different concentrations of galectin-3 (with purple being the lowest, and yellow being the highest). A clustering of the galectin-3 on EC at the sites of their contact with MDA-MB-435 cells (yellow arrows) and on MDA-MB-435 cells at the sites of homotypic contact with other MDA-MB-435 cells (red arrows) was observed. In contrast, a monolayer of EC before adhesion was found to show a uniform, low level basal distribution of galectin-3 (Fig. 5D). This adhesion-dependent galectin-3 activation appears to be specific since no changes were detected in the intracellular distribution as well as cell surface expression of galectin-1 (data not shown).


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Fig. 5.   A three-dimensional reconstruction of a homotypic aggregate of HUVEC-adhered MDA-MB-435 cells. A, expression of galectin-3 on MDA-MB-435 cells adhered to HUVEC by immunofluorescent staining with rat anti-galectin-3 antibody followed by Texas Red-conjugated secondary antibody. B and C, pseudocolored images of the orthogonal sections through YZ (B) and XZ (C) planes of the field shown in A. Different colors (purple, blue, green, and yellow) correspond to different levels of galectin-3 expression (purple being the lowest, and yellow being the highest). Note clustering of galectin-3 on EC at the sites of their contact with cancer cells (yellow arrows) and on cancer cells at the sites of homotypic adhesion (red arrows). D, pseudocolored image of an orthogonal section through the XZ plane showing the distribution of galectin-3 on a monolayer of EC before adhesion to MDA-MB-435 cells.

In the next set of experiments, we examined the cell surface expression of galectin-3 on EC before and after interaction with highly or poorly metastatic human breast carcinoma cells under conditions of flow. Breast carcinoma cells were infused in the flow chamber at low shear rates to facilitate maximal interaction with the endothelial monolayer immobilized on coverslips. Thereafter, both EC and adherent tumor cells were harvested from the coverslip, and the cell surface expression of galectin-3 was determined by flow cytometry. The expression of galectin-3 on breast carcinoma cells remained relatively unaltered before and after interaction with EC (data not shown). In contrast, the expression of galectin-3 on EC (CD31 positive population) increased significantly after interaction with highly metastatic MDA-MB-435 cells but not with the poorly metastatic MDA-MB-468 breast carcinoma cells (Fig. 6). These results support our previously reported observation that T antigen is able to induce galectin-3 mobilization to the cell surface in human EC (12).


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Fig. 6.   Cell surface expression of endothelial galectin-3 before and after exposure to breast carcinoma cells. MDA-MB-468 or MDA-MB-435 cells (5 × 104 cells/ml) were passed through the laminar flow chamber containing HUVEC-coated coverslips at 0.8 dyn/cm2. Thereafter, both EC and adherent tumor cells were harvested from the coverslips and stained for cell surface galectin-3 expression (gated square) before (panels A and C) and after (panels B and D) adhesive interactions in the flow chamber. Expression of galectin-3 was detected using a rat anti-human galectin-3 mAb followed by fluorescein isothiocyanate-conjugated secondary antibody. The expression of endothelial marker CD31 was detected using phycoerythrin-conjugated mouse anti-human CD31 mAb.

In Vivo Adhesion of MDA-MB-435 Human Breast Carcinoma Cells to the Lung Microvascular Endothelium as Revealed by Intravital Fluorescence Microscopy-- To corroborate that the distinct intercellular adhesive interactions observed in vitro are relevant in vivo, we visualized the adhesive interactions between human breast carcinoma cells of high and low metastatic potential and murine lung microvasculature in vivo by intravital microscopy. To examine this, MDA-MB-435 or MDA-MB-468 cells stained with 5-CFDA were injected into the tail vein of nude mice transplanted with lung allografts derived from female nude mice. Passage of the fluorescently labeled tumor cells within the re-vascularized blood vessels of the transplanted lung allograft in the skin chamber was monitored by intravital fluorescence video microscopy. Analysis of recorded images revealed that metastatic MDA-MB-435 cells have a greater propensity to engage in stable adhesion with the lung microvasculature, whereas only a few non-metastatic MDA-MB-468 cells remained adherent in the revascularized lung microvessels (Fig. 7). The observed differences between MDA-MB-435 and MDA-MB-468 cell lines in their mode of interaction and retention within the lung microvascular endothelium in vivo suggests that the attachment of MDA-MB-435 to microvascular endothelium could be relevant to metastasis and is mediated by specific adhesion mechanisms differentially manifested by the MDA-MB-435 and MDA-MB-468 cells.


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Fig. 7.   In vivo adhesion of human breast carcinoma cells to the lung microvascular endothelium. A single cell suspension (2.5 × 106 cells) of CFDA-labeled MDA-MB-468 (A) or MDA-MB-435 (B) cells (black arrows) were injected intravenously into the tail vein of nude mice, and their ability to interact within blood vessels of revascularized lung allografts derived from female nude mice transplanted into dorsal skin-fold chamber was examined by intravital microscopy (n = 3). Note the increased firm adhesion and retention of the highly metastatic MDA-MB-435 carcinoma cells within lung microvessels (white arrows) compared with the MDA-MB-468 cells. Magnification 250×.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adhesive interactions between human cancer cells (breast and prostate) and EC under static conditions have previously been shown to be mediated at least in part by the galectin-3 expressed on EC and T antigen expressed on cancer cells (12). In this study we have further expanded on these findings by describing the adhesive interactions of breast carcinoma cells and EC under conditions of flow as well as in vivo in a mouse skin-fold chamber model. Results presented indicate that T antigen-galectin-3 interactions mediate both homotypic intercellular adhesion of metastatic MDA-MB-435 human breast carcinoma cells as well as the heterotypic adhesion of these cells to human EC under conditions of flow. Several independent lines of experimental evidence suggest that the T antigen-mediated adhesion mechanisms described herein may be relevant to the metastatic process in vivo. This suggestion is supported by the increased adhesion of the highly metastatic MDA-MB-435 cells, which expresses high levels of T antigen and galectin-3 (Fig. 4, A-E), to lung mirovasculature in the mouse skin-fold chamber model in vivo in contrast to the poorly metastatic MDA-MB-468 cells (Fig. 7). LL, the synthetic T antigen-mimicking molecule that has previously been shown to interfere with adhesive interactions between EC and MDA-MB-435 cells in static adhesion assays in vitro (12) as well as anti-galectin-3 mAb and lactose were found to inhibit adhesive interactions between EC and MDA-MB-435 cells under conditions of flow, further establishing the functional role of galectin-3 in these interactions. Furthermore, LL is also known to inhibit MDA-MB-435 human breast carcinoma metastasis in mice (26). Taken together, these results strongly suggest that the adhesion mechanisms mediated by T antigen-galectin-3 interactions may play an important role in the spread of human breast carcinoma cells from primary orthotopic sites to secondary metastatic sites such as the lungs.

This type of carbohydrate-lectin interaction involving certain cancer-associated glycoantigens may play a significant role in promoting adhesion between metastatic cancer cells and human EC under flow conditions as well. Consistent with this idea, poorly metastatic MDA-MB-468 breast carcinoma cells that express significantly reduced levels of both galectin-3 and T antigen (Fig. 4, A-E) failed to induce galectin-3 expression on EC and did not demonstrate enhanced homotypic aggregation after adhesion to EC under flow conditions (data not shown). Based on our results, we propose a model describing the molecular mechanisms involved in T antigen-mediated homo-and heterotypic adhesive interactions of MDA-MB-435 cells and EC under conditions of flow that could be highly relevant to breast cancer metastasis (Fig. 8). According to this model, the initial adhesive interactions mediated by the T antigen expressed on cancer cells and galectin-3 expressed on EC causes a rapid increase in the cell surface expression and intracellular redistribution of endothelial galectin-3 to the sites of heterotypic intercellular contact, whereas galectin-3 in cancer cells accumulates at the site of homotypic intercellular interactions (Fig. 5). Within minutes of the initial adhesive interactions between metastatic cancer cells and the endothelium, the "primed" adhesive hot spots on the endothelium acquire dramatically enhanced adhesive properties and are able to "capture" rolling and floating carcinoma cells. This process results in rapid formation of multicellular homotypic aggregates of cancer cells at the multiple sites of primary attachment on EC under conditions of flow. Initiated by the primary adhesive interaction with the endothelium, homotypic aggregation of cancer cells could facilitate local microcirculatory aberrations and promote intravascular mechanical trapping. In addition, because enhanced homotypic aggregation of metastatic cancer cells has been shown to correlate with their ability to survive and resist apoptosis (18, 19, 27), the homotypic aggregates of endothelium-attached cancer cells may promote intravascular survival of metastatic cancer cells and serve as the seeds of early metastatic colonies. Consistent with this idea, recent experiments show that metastatic cancer cells exhibit increased resistance to circulatory stress-induced apoptosis in vivo in contrast to poorly metastatic cancer cells (28).


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Fig. 8.   A schematic representation of the T antigen-mediated galectin-3 redistribution in endothelial and metastatic cancer cells during intercellular adhesive interactions under conditions of flow. A, primary rolling cancer cells (R1) interact with the endothelial monolayer and induce increased cell surface expression of the endothelial galectin-3, thus creating an adhesive "hot spot." B, secondary rolling cancer cells (R2) encounter the adhesive hot spot on endothelium and develop firm heterotypic adhesive interactions constituting stable attachment under conditions of flow. C, endothelium-attached cancer cells (R2) and adhesion-primed EC (adhesive hot spot) capture other rolling (R3) and floating (F2) cancer cells, thus inducing the formation of homotypic cancer cell aggregates at the site of their primary attachment to the endothelium. Differential intracellular redistribution of galectin-3 in cancer cells and the endothelium provides the molecular basis for this metastasis-promoting adhesion mechanism under conditions of flow. The endothelium-expressed galectin-3 is clustered at the site of contact with cancer cells. Galectin-3 on the cancer cells attached to the endothelium (F2, R2, R3) is clustered at the sites of homotypic interaction with other cancer cells.

Presented here is a novel adhesion mechanism adopted by breast carcinoma cells under conditions of flow that is unique to metastatic breast cancer cells, such as MDA-MB-435 cells, and distinct from the adhesive behavior of other cancer cells that have thus far been studied or even leukocytes. Analysis of the adhesive behavior of six human tumor cell lines of different histological origin under conditions of flow indicated that none of the tumor cells rolled on venular endothelium in contrast to the rolling behavior of leukocytes (5). Interestingly, three of the tumor cell lines tested (HT-29, DLD-1, and HCT-8) were strongly positive for the oligosaccharides Lewis(x), sialyl-Lewis(x), and sialyl-Lewis(a), which are recognized by the endothelial selectins that support leukocyte rolling. Initial microvascular arrest of metastasizing tumor cells was found to be dependent primarily on mechanical factors rather than on receptor-mediated leukocyte-like adhesive interactions (5). In other studies, HT-29 colon carcinoma cells were found to roll on E-selectin-coated surfaces without subsequent adhesion but did not exhibit rolling or adhesion to vascular cell adhesion molecule-coated surfaces under physiological flow conditions. A375M melanoma cells, on the other hand, massively adhered to vascular cell adhesion molecule-coated surfaces but not to surfaces coated with E-selectin (6). More recent studies showed that HT-29 cells also adhere to collagen I using the beta 1 integrins under flow conditions (8). Rolling of KS breast carcinoma cells on vascular endothelium in vivo is mediated by CD24, a small mucin-type glycophosphatidylinositol-linked cell surface molecule, in a P-selectin-dependent manner (7). Analogous to the differential expression of galectin-3 and T antigen by metastatic (MDA-MB-435) versus non-metastatic (MDA-MB-468) cells observed in the present study, CD24 is also differentially expressed in breast carcinomas (cytoplasmic pattern) versus benign breast lesions (apical pattern), and this differential expression is thought to constitute an important adhesion pathway in cancer metastasis (29). More recently, a cell surface variant rather than the standard form of CD44 has been shown to support in vitro lymphoma rolling on hyaluronic acid substrate and its in vivo accumulation in the peripheral lymph nodes (30). Therefore, under flow conditions, cells from different tumor types appear to interact with the endothelial surface by different mechanisms, depending on adhesion molecules expressed on the tumor and endothelial cell surface.

Hematogenous spread of tumor cells and metastasis formation in secondary organs are insidious aspects of cancer. The two major concepts describing cancer metastasis are based either on the adhesion of cancer cells to the blood vessel endothelia (seed and soil hypothesis) or homotypic cancer cell aggregation (mechanical trapping theory) as a key component of the metastatic cascade (1). Here we have shown a unique sequence of homo- and heterotypic adhesive interactions of metastatic cells involving a rapid activation of both homo- and heterotypic adhesion of cancer cells after initial attachment to the endothelial layer under conditions of flow that are dependent on the engagement of galectin-3 and T antigen. These findings provide a mechanistic basis for the potential unification of the two major concepts of cancer cell metastasis (seed and soil hypothesis and mechanical trapping theory). Our data provide further justification for application of the anti-adhesion cancer therapy for intravascular targeting of cancer metastasis.

    FOOTNOTES

* This research was supported in part by NCI, National Institutes of Health (NIH) Grants 1R43CA72284 and 1RO1CA89827-01 (to G. V. G.) and CA86290-01 (to V. V. G.), a grant from MetaStat, Inc. (to G. V. G. and A. B. G.), Breast Cancer Research Program Grant 4JB-0164, and NIH Grant 1RO1AI35796 (to P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this study.

** These authors share senior authorship.

Dagger Dagger To whom correspondence should be addressed: Div. of Vascular Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Dr., San Diego, CA 92121. Tel.: 858-587-8788 (ext. 101); Fax: 858-587-6769; E-mail: rao@ljimm.org.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209590200

2 L. Sikora, A. Johansson, S. P. Rao, G. K. Hughes, D. H. Broide, and P. Sriramarao, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: EC, endothelial cells; T antigen, Thomsen-Friedenreich antigen; HUVEC, human umbilical vein endothelial cells; HLMVEC, human lung microvascular endothelial cells; LL, lactulosyl-L-leucine; LT, lactitol-L-leucine; CFDA, 5-carboxyfluorescein diacetate; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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