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Tumor Biology |
Departments of Pathology [E. M. J. B., M. V., W. N. M. D., T. H. V. D. K.] and Immunology [R. v. d. B.], Erasmus University, 3000 DR Rotterdam, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients with carcinoma in situ in the flat peripheral urothelium adjacent to tumors have a higher probability of tumor recurrences and/or invasion (4 , 5) . Clinical and experimental data suggest that bladder tumor recurrences could be the consequence of an IEE of the transformed cells from the original tumor or shedding and subsequent reattachment of bladder cells, particularly to traumatized areas in the bladder mucosa (6, 7, 8, 9, 10, 11) . Lateral expansion of the attached tumor cells can then lead to the replacement of normal urothelium by cancer cells. The mechanisms involved in lateral (i.e., intraepithelial) expansion of bladder tumor cells are only partially understood. A cocultivation model established recently in our laboratory permits the direct visualization of attachment and subsequent IEE of bladder tumor cells at the expense of surrounding normal urothelium (10 , 11) . In this assay, a tumor cell suspension is inoculated on confluent mouse urothelial cultures containing de-epithelialized areas of a standard size. Implantation of tumor cells occurs predominantly in these de-epithelialized areas, and the time course of IEE can be assessed by selective immunostaining of the tumor cells. Previously, we have shown in this model that exposure to growth factors and culture on substrates coated with particular extracellular matrix proteins could influence IEE (10 , 11) .
E-cadherin is a member of a family of transmembrane glycoproteins
involved in intercellular adhesion. E-cadherin function is mediated by
the interaction with the cytoplasmatic 
-, ß-, and 
-catenins.
These catenins connect E-cadherin with the cytoskeleton. In model
systems, loss of E-cadherin expression is associated with the gain of
the invasive phenotype in tumors (12
, 13
, 14)
. Similarly,
it was reported that loss of the invasion suppressor molecule
E-cadherin or catenins is associated with deeply invasive bladder
cancer and is predictive for poor survival of patients with bladder
cancer (15, 16, 17)
. The E-cadherin/catenin complex also
contributes to a variety of physiological functions like cell growth,
differentiation, wound healing, cell motility, morphogenesis, and
organogenesis (18)
. However, its role in the expansion of
carcinoma in situ of the bladder has not yet been studied.
In this study, initial experiments comparing E-cadherin-positive and -negative human TCC cell lines suggested a potentially enhancing role of E-cadherin in IEE. By use of T24 cells, stably transfected with an E-cadherin cDNA construct, we could confirm that this molecule indeed contributes to IEE, most likely by conveying increased cohesiveness to the TCC cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary Explant Cultures of Murine Urothelium.
Murine urinary bladders were dissected from female C3H/He mice, 6–8-
weeks of age, and cut into halves. The mucosa of the bladder was
stripped from its underlying muscle layer and subsequently spread on a
collagen type IV (25 µg/ml human collagen type IV)-coated Cyclopore
membrane (Becton Dickinson Labware, Bedford, MA) with the
submucosa facing the culture support. Standard culture medium consisted
of a 1:1 mixture of DMEM and Ham’s F10, supplemented with 10%
heat-inactivated FCS, 10 µg/ml insulin, 5 µg/ml transferrin, 5
ng/ml selenite, 10 µM HEPES, 50 nM
hydrocortisone, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The
explant cultures were grown at 37°C in a humidified atmosphere of 5%
CO2. These murine explant cultures on porous
membranes mimic the in vivo situation; the cultured
urothelium shows a polarized multilayering and differentiation into
umbrella cells (22)
.
IEE Assay.
The cocultivation model to study IEE of bladder carcinoma cells was
described previously (11)
. Briefly, in confluent murine
explant cultures, four standardized circular areas were denuded in the
periphery of the primary cultures by cautious imprinting with a 3-mm
diameter biopsy punch (Stiefel, Offenbach am Main, Germany). The
urothelium in the injured areas was scraped away from the Cyclopore
membrane with a micropipette tip. Subsequently, the cultures were
washed twice with PBS, followed by seeding of 105
tumor cells in 1.5-ml standard medium on the murine urothelial explant
cultures. The bladder tumor cells were allowed to attach to the injured
areas in the explant cultures for 24 h; nonadherent cells were
then washed away with PBS, and the cultures were either terminated or
continued for another 4, 7, or 14 days in standard medium. Each
experiment was performed twice in triplicate. The cultures were
terminated by fixing them in 70% ethanol and stored at 4°C until
immunohistochemistry; selective identification of the human bladder
carcinoma cells was performed.
Monoclonal, human-specific antibodies, RCK108 (Eurodiagnostica, Arnhem, the Netherlands) or DC10 (Beckman Coulter, Fullerton, CA), directed against cytokeratin 19 were used to distinguish the human bladder carcinoma cells from murine urothelium (10) . Monoclonal antibody DC10 was used for staining J82 cells, whereas the other cell lines were stained with monoclonal antibody RCK108. Nearly 100% of the bladder carcinoma cells of each cell line was labeled with RCK108 or DC10. The secondary antibody was horseradish peroxidase-conjugated goat antimouse immunoglobulins (Dako, Glostrup, Denmark). Peroxidase activity was visualized with 0.03% H2O2 and 0.02% 3,3,-diaminobenzidine tetrahydrochloride (Fluka, Basel, Switzerland) diluted in PBS.
The immunostained areas were quantitated with a Hitachi CCTV camera equipped with the KS400 image analysis software package (Kontron Elektronik, Eching, Germany) and expressed in mm2.
Construction of Stable Transfectants.
The E-cadherin-negative cell line T24 was cotransfected with plasmids
pBATEM2 and pSVneo, which harbors the neomycin resistance gene.
Marion Bussemakers (Urological Research Laboratory, University Hospital
Nijmegen) generously provided plasmid pBATEM2, containing the
full-length mouse E-cadherin cDNA, originally constructed by
Nagafuchi et al. (23)
. For transfection,
cultures were cotransfected with a total of 20 µg of DNA
(pBATEM2:pSVneo, 20:1) using the DNA-calcium phosphate method
(24)
, and transfected cells were selected with 800 µg/ml
G418. E-cadherin expression in these transfected cell lines was
detected as follows. Clones were grown to confluence on multichamber
slides (Nunc, Naperville, IL) and fixed in methanol at -20°C.
Cells were incubated overnight at 4°C with monoclonal antibody
DECMA-1 (Sigma Chemical Co., St. Louis, MO), followed by a biotinylated
rabbit antirat immunoglobulins (Dako) and fluorescein-conjugated
streptavidin (Dako). The different clones retained their resistance to
G418 and were stable in their E-cadherin expression during the entire
course of the described experiments.
Flow Cytometric Analysis of E-Cadherin and Integrin Expression.
Cells were harvested by a short trypsinization of confluent monolayers.
Cell suspensions were made in PBS containing 0.5% BSA, 0.1%
NaN3, 1 mM
CaCl2, and 0.5 mM
MgCl2 at a concentration of 1 x 106 cells/ml. E-cadherin expression of a
selection of nine different, transfected T24 clones was examined with
the DECMA-1 antibody, followed by biotinylated rabbit antirat
immunoglobulins and fluorescein-conjugated streptavidin. Integrin
expression was studied with the use of specific antibodies against
1-integrin (HP2B6; Beckman Coulter, Fullerton, CA), 2-integrin
(NCL-CDW49b; Novacastra, Newcastle upon Tyne, United Kingdom),
3-integrin (NCL-CDW49c; Novacastra), 4-integrin (P4G9; Dako),
5-integrin (P1D6; Dako), 6-integrin (NCL-CDW49f; Novacastra),
ß1-integrin (TDM29; Sanbio, Uden, the Netherlands), and ß3-integrin
(NCL-CD61; Novacastra). As a negative control, the primary antibody was
omitted and replaced by a PBS/BSA/azide solution. Data acquisition and
analysis were performed on duplicate samples on a FACScan flow
cytometer using CELLQuest software (Becton Dickinson, San Jose, CA).
Immunoblotting.
Confluent cultures were lysed in 2x sampling buffer [4% SDS, 200
mM DTT, 100 mM Tris (pH 6.8), 20% glycerol,
and 2% Triton X-100], and equal quantities of protein (30 µg) were
run on a 7.5% SDS-PAGE. The MDCK cell line was used as a positive
control for E-cadherin expression. After electroblotting, blots were
immunostained with the DECMA-1 antibody, followed by biotinylated
rabbit antirat immunoglobulins. Next, an alkaline
phosphatase-conjugated streptavidin label (Biogenex, San Ramon, CA) was
applied. Finally, bound antibody was visualized by histochemical
staining with nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt (Roche
Diagnostics, Basel, Switzerland).
In Vitro Invasion Assay.
Chicken heart invasion assays were performed as described by Mareel
et al. (26)
. Briefly, a selection of
E-cadherin-transfected T24 clones was confronted with
precultured rounded fragments of embryonic chicken heart on soft agar
for 24 h. Next, fragments with attached bladder tumor cells were
kept in suspension culture under gyrotory shaking (120 rpm; 37°C; 5%
CO2) for 6 days in MEM REGA 3 medium (Life
Technologies, Inc., Breda, the Netherlands) containing 10% FCS.
Fragments were fixed in 4% phosphate buffered formalin and embedded in
paraffin. Invasion was scored on serial histological sections stained
with H&E. The tumor cells were distinguished from the heart tissue by
their morphological features.
Wound Colonization Assay.
T24 Clones were grown to confluence in 10-cm diameter culture
dishes. With a plastic pipette tip, cells were scraped away in the
shape of a cross. The width of the lesion was 
5 mm. After 16 h,
the movement of cells into the wound was monitored and photographed.
Expansion of T24 Clones on Collagen Type IV-coated Cyclopore
Membranes.
With a 3-mm diameter biopsy punch (Stiefel, Offenbach am Main,
Germany), a superficial circular imprint was made on the cell culture
inserts, coated with collagen type IV. A cell suspension of
105 cells/2 µl of standard medium was pipetted
within the borders of the circular imprint with a 10-µl micropipette.
The cells in the drop of medium attached into the circular area within
24 h. Every 2 days, medium on top of the membranes was refreshed,
taking care that the medium only covered the area of tumor cells.
Medium underneath the membrane was refreshed twice a week. The
circumferences of the outgrowing sheet of tumor cells were drawn daily.
From these drawings, the area (mm2) of outgrowth
was determined.
[3H]Thymidine Incorporation.
To assess the potential differences in proliferation of the different
T24 clones, a cell kinetic study was performed as described earlier
(11)
. Briefly, T24 clones grown in
75-cm2 culture flasks were synchronized in
serum-free culture medium for 24 h. Subsequently,
105 cells/were seeded in collagen type IV-coated
96-well dishes and were cultured in standard medium for 4 consecutive
days. Proliferative activity was determined at 24, 48, 72, and 96 h. During the final 16 h of culture, cells were incubated with 0.5
µCi of [3H]thymidine/well. The incorporated
[3H]thymidine in harvested cells was counted in
a BetaPlate scintillation counter (LKB-Pharmacia, Woerden, the
Netherlands).
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RESULTS |
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, whereas bladder tumor cell lines with a homogeneous (SD,
RT112, and JON) expression of E-cadherin displayed a sharp demarcation
between the tumor cells and the normal urothelium (Fig. 1B)
.
The six tumor cell lines further differed with respect to their
subsequent IEE rate (Fig. 2
and Table 1
). The IEE of bladder carcinoma cells was most pronounced for the SD
cells and less for the 1207 and RT112 cells, whereas T24, JON, or J82
displayed hardly any or no expansion. Therefore, three of four
E-cadherin-positive bladder carcinoma cell lines had a better IEE rate
than the two E-cadherin-negative bladder carcinoma cell lines. These
results suggested a positive effect of E-cadherin on intraepithelial
expansion.
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ß-catenin (25)
. We obtained 19 G418-resistant T24 cell
clones, with membrane-bound E-cadherin expression (Fig. 3A)
. Clones varied with regard to their levels of
immunohistochemically detected E-cadherin expression. Expression levels
of E-cadherin of nine different T24 transfectants were analyzed by flow
cytometry. Clones T24H9+, T24 2D4+, T24D4+, and T24H5+ had low to
moderate expression levels (Fig. 4B)
, whereas the clones T24H3++ and T24D10++ had a high
expression level of E-cadherin (Fig. 4C)
. As a negative
control for E-cadherin expression, we used the clones (T24C1-,
T24F1-, and T24G1-) that were G418 resistant but had no detectable
expression of E-cadherin, either by immunofluorescence, immunoblotting,
or flow cytometry (Fig. 4A)
. Immunoblotting with the DECMA-1
antibody on different cell lysates of T24 clones showed a
characteristic band at Mr 120,000 that
was typical of the E-cadherin-positive clones (Fig. 3B)
. At
the ultrastructural level no difference in the presence of desmosomes
between the nontransfected (E-cadherin-negative) and transfected,
E-cadherin-positive T24 cells was noted (data not shown).
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. SD and the T24 clones with a high or moderate E-cadherin
expression filled up the lesions by moving as a cohesive sheet (Fig. 5B
and Table 2
). However, no difference in time required for the repair of the lesions
was seen.
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Untransfected T24 cells exhibited the strongest invasive capacity.
After successful adhesion, T24 tumor cells infiltrated nearly all
embryonic chicken heart fragments (87%). SD cells were unable to
invade the embryonic chicken heart fragments at all. Transfection of
E-cadherin in T24 cells resulted in a significant reduction of invasion
for T24D10++ and T24H3++ (Table 2)
. Both cell lines had a high
expression of E-cadherin. Clones T24H9+, with a moderate expression of
E-cadherin, T24C1- and T24G1-, with absent E-cadherin expression,
exhibited a moderate to high invasive capacity into the embryonic
chicken heart fragments. These results revealed that the levels of
E-cadherin expression of the transfected T24 clones corresponded with
their functionality.
IEE of Transfected T24 Cells.
The SD cell line was used as a positive control, because it had the
highest expansion rate of the TCC cell lines (Fig. 2)
. Transfection of
T24 cells with mouse E-cadherin cDNA stimulated IEE of two
independently obtained clones, T24D10++ and T24H3++, both with a high
expression of the E-cadherin protein (Fig. 6)
. Clones with a moderate/low expression level of E-cadherin
(e.g., T24H9+) did not show a significant stimulation of
IEE. The implantation (defined by the tumor area on day 1) of the
different T24 transfectants was comparable, indicating that possible
differences in IEE could not be attributed to differences in attachment
of the T24 transfectants to the wounded area. The expression of
integrins was determined by flow cytometry in T24, T24H3++, T24D10++,
and T24C1-. In the four tested clones, no differences were observed in
expression levels of 1-, 2-, 3-, 4-, 5-, 6-, ß1-,
or ß3-integrins (data not shown).
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. These experiments revealed no differences in the outgrowth or in the
[3H]thymidine incorporation (data not shown) of
the various T24 clones. The moderately E-cadherin-expressing T24 2D4+
had a lower expansion rate.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we have shown that the pattern of infiltration of TCC cells into the surrounding normal urothelium is determined by E-cadherin expression (10 , 11) . E-cadherin-negative TCC cells infiltrate the normal urothelium as individual cells, whereas tumor cells with a homogeneous expression of E-cadherin exhibit a sharp demarcation with the normal urothelium. The 1207 TCC cell line with a heterogeneous expression of E-cadherin in vitro also displays the capacity to infiltrate the normal urothelium as individual cells. Staining of these cocultivations with a human specific E-cadherin antibody revealed that the infiltrative single cells of the 1207 cell line had a reduced or absent expression of E-cadherin as compared with the high homogeneous expression of the primary tumor at the implantation site (data not shown). These results indicate that a reduced or absent expression of E-cadherin results in infiltration of the surrounding normal urothelium as single cells.
Comparison of the six TCC cell lines revealed that the two constitutively E-cadherin-negative bladder carcinoma cell lines (T24 and J82) hardly showed any IEE, whereas three of four E-cadherin-positive cell lines (SD, RT112, and 1207) did. We hypothesized that a functional E-cadherin-catenin complex is required for effective IEE of TCC cells. To further test this hypothesis, we established stable transfectants of T24 cells expressing the full-length mouse E-cadherin cDNA. Several clones of E-cadherin-expressing T24 cells were obtained. Functionality of the transfected T24 clones was analyzed with the embryonic chicken heart in vitro invasion assay (12 , 26) and a wound colonization assay (27) .
The E-cadherin-negative cell line T24 was generally capable of invasion
into the embryonic chicken heart, whereas only the T24 transfectants
with high expression of E-cadherin (T24H3++ and T24D10++) had a
significantly reduced invasive capacity, implying a functional
E-cadherin-catenin complex. The T24 clones with a moderate/low but
homogeneous expression of E-cadherin (e.g., T24H9+) retained
part of their invasiveness (Table 2)
. This observation is in accordance
with those of Vleminckx et al. (12)
, who
suggested that a threshold expression of E-cadherin has to be reached
to prevent in vitro invasion into the embryonic chicken
heart fragments. Similarly, in IEE assays a significant enhanced
expansion rate was only observed for the two T24 clones with high
E-cadherin expression but not for the T24 clones with a moderate or low
expression level, suggesting that the same E-cadherin-mediated
mechanism induces both suppression of in vitro invasion and
promotion of IEE. The enhanced E-cadherin-mediated expansion rate
became manifest only under the cocultivation conditions of IEE. Thus,
increased expression of E-cadherin by transfected T24 cells was neither
associated with an increased expansion rate on collagen type IV-coated
membranes nor with an increased proliferative activity or wound repair.
Several reports documented E-cadherin-mediated suppression of cell
motility and inhibition of proliferation (27, 28, 29)
. This
discrepancy with our findings on T24 cells may be explained by the use
of fibroblast cells in these studies rather than epithelial cells.
Furthermore, the motility assays used in these reports
(27, 28, 29)
were based on single cell assays, whereas our
expansion assay (Fig. 7)
monitors the outgrowth of a cohesive sheet of
cells.
Recently, Pignatelli (30)
speculated on the possibility of
a molecular cross-talk between cadherins and integrins in cancer cells.
Because integrins are involved in motility and could be a regulator in
IEE (7)
, we examined whether E-cadherin expression levels
in transfected T24 cells would correlate with expression levels of
integrins. Flow cytometric analysis of 1-, 2-, 3-, 4-,
5-, 6-, ß1-, or ß3-integrin expression levels did not point
to such a mechanism in E-cadherin-transfected T24 cells. It
could still be argued that E-cadherins could lead to an altered
localization or affinity of integrins on the cell membrane. Because we
did not find any altered migratory behavior of the transfected T24
cells in our expansion assay in the absence of surrounding normal
urothelium, E-cadherin-integrin cross-talk in our transfected T24 cells
seems an unlikely mechanism for the observed IEE. We strongly feel that
cohesion of transformed T24 cells is the most important
E-cadherin-mediated determinant for IEE.
Our study suggests that E-cadherin is an important molecule for IEE of TCC in vitro, but it does not prove that this view holds true for bladder carcinoma in situ. Although a number of studies analyzed E-cadherin immunoreactivity during different stages of bladder cancer progression, none reported on the E-cadherin expression in carcinoma in situ of the bladder in detail (15, 16, 17 , 31) . In a preliminary study, we stained paraffin sections with carcinoma in situ of 10 patients for E-cadherin. All of these 10 lesions had a normal, homogeneous, membranous expression of E-cadherin, confirming our hypothesis on the contributory role of E-cadherin in the intraepithelial propagation of bladder carcinomas.
For carcinogenesis in vivo, our observations would imply that on the one hand, E-cadherin promotes expansion of carcinoma in situ and on the other hand, opposes invasiveness of the transformed cells. Generalizing, E-cadherin-mediated cohesiveness may represent a major property of transformed clones, allowing carcinoma in situ to expand at the expense of surrounding normal epithelial cells in vivo.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Pathology, Josephine Nefkens Institute,
Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, the
Netherlands. Phone: 3-10-4087924; Fax: 31-10-4089487; E-mail: vanderkwast{at}path.fgg.eur.nl 
2 The abbreviations used are: TCC, transitional
cell carcinoma; IEE, intraepithelial expansion.
Received 7/ 6/99. Accepted 11/ 1/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, ß-, -catenins and p120cas) in bladder tumors. Cancer Res., 56: 4154-4158, 1996.This article has been cited by other articles:
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  C. Patriarca, P. Colombo, A. Pio Taronna, J. Wesseling, G. Franchi, F. Guddo, R. Naspro, R. M. Macchi, P. Giunta, M. Di Pasquale, et al. Cell Discohesion and Multifocality of Carcinoma In situ of the Bladder: New Insight From the Adhesion Molecule Profile (e-Cadherin, Ep-CAM, and MUC1) International Journal of Surgical Pathology, April 1, 2009; 17(2): 99 - 106. [Abstract] [PDF]
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 A. Margulis, W. Zhang, A. Alt-Holland, H. C. Crawford, N. E. Fusenig, and J. A. Garlick E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs Cancer Res., March 1, 2005; 65(5): 1783 - 1791. [Abstract] [Full Text] [PDF]
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C. Y. Sasaki, H. Lin, P. J. Morin, and D. L. Longo Truncation of the Extracellular Region Abrogrates Cell Contact but Retains the Growth-suppressive Activity of E-cadherin Cancer Res., December 1, 2000; 60(24): 7057 - 7065. [Abstract] [Full Text]
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