From the 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
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ABSTRACT |
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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.
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.
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 ( 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.
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.
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 ( 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.
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).
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).
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.
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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
View larger version (56K):
[in a new window]
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.
4 or
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-
4, or
anti-
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
-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.
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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).
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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.
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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.
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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
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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 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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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.
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