Ehrlich ascites tumour cells show tissue-specific adherence and modify
their shape upon contact with embryonic fibroblasts and myotubes
BEATA W6JCIAK and WLODZIMIERZ KOROHODA
Department of Cell Biology, The J. Zurzycki Institute of Molecular Biology, Jagiellonian University, al. Mickiewicza 3, 31-120
Krakdw, Poland
Summary
Adhesiveness of Ehrlich ascites tumour (EAT) cells
to glass, to mouse peritoneal membrane, living and
aldehyde-fixed mouse embryo fibroblasts and chick
embryo fibroblasts, myoblasts and myotubes was
investigated. The ascitic EAT cells (and leukaemia
L1210 cells) did not adhere to glass and peritoneum
but readily adhered to embryo fibroblasts, myoblasts
and myotubes. The attachment was followed by cell
spreading and migration. Fixation of fibroblasts or
myogenic cells with aldehydes did not prevent ascitic
cells from attaching but reduced the rate of spread-
ing. Only direct interaction of ascitic cells with
embryo myoblasts or fibroblasts induced changes in
tumour cell adhesiveness followed by cell spreading
and locomotion. These results are discussed in
relation to an observation that ascitic cells growing
as a cell suspension intraperitoneally grow as a solid
tumour when injected subcutaneously.
Introduction
suggest that the cell-cell interactions depend upon the
tissue character of cells and not on the species origin of
cells, and that the adhesiveness and morphology of cancer
cells can be strongly influenced by interactions with
neighbouring cells.
The shape and surface properties of cancer cells are
commonly postulated to be associated with their neoplastic character and their ability to produce metastases
(Vasiliev, 1985; Raz and Ben-Ze'ev, 1987; Hochman et al.
1984; Leighton and Tchao, 1984). Cells of ascitic
transplantable tumours, such as Ehrlich ascites tumour
(EAT) or leukaemia L1210, can be propagated in vivo by
growing them as cell suspensions in the intraperitoneal
cavity of mice. In this microenvironment individual cells
are suspended in an ascitic fluid and do not adhere to the
peritoneal membrane (Whur et al. 1973). When cultured in
vitro, these cells are anchorage-independent, grow in
suspension, do not adhere to glass or plastic, and maintain
a spherical shape not spreading on the substratum.
(CieSlak and Korohoda, 1978; Kajstura et al. 1986). On the
other hand solid tumours develop when a mouse is injected
with a suspension of these cells subcutaneously instead of
intraperitoneally. Mice bearing ascitic EAT cells in the
intraperitoneal cavity survive 22(±2) days, whereas
animals with solid tumours survive over 40 days, with a
great dispersion of the survival time of individual
specimens (some of the animals survived over 60 days).
The aim of the experiments presented in this paper was,
therefore, to examine: (1) whether the EAT cells interact
with epithelial cells (mesothelial cells) of the peritoneal
membrane differently from fibroblasts or myogenic cells,
and (2) whether such an interaction influences the EAT
cell adhesiveness and morphology. Additionally, some
experiments were repeated with leukaemia L1210 cells to
check whether the observed EAT cell responses are
confined to one type of cancer cell or whether other cancer
cells react similarly. The results presented in this paper
Journal of Cell Science 97, 433-438 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
Key words: Ehrlich ascites cells, co-cultures, cell shape,
spreading, adhesiveness, cell-to-cell interactions.
Materials and methods
Cell cultures
EAT cells were maintained in vivo intraperitoneally in female
Swiss albino mice (Camura Breeding Laboratories, Cracow,
Poland), recovered and washed before incubation in vitro as was
previously described (Cieslak and Korohoda, 1978). Then the cells
were suspended in Eagle's minimal essential medium (EMEM)
supplemented with: 1% chick serum, 4% calf serum (56 °C heatinactivated sera) lOmmoll" 1 Hepes buffer (iV-2-hydroxyethylpiperazine W-2-ethanesulphonic acid, pH7.4), 100 units ml" 1
penicillin, lO/igml" streptomycin. For experiments with mice
fibroblasts, the incubation medium containing 5 % calf serum was
used. The EAT cells were plated at a cell density of 7xl0 4
cells ml" 1 into Leighton tubes with cultures of myoblasts or
fibroblasts or into Rose chambers with peritoneal membrane. Cocultures were maintained at 37 °C for 4-24 h.
Murine leukaemia cells (L1210/V) were maintained by weekly
intraperitoneal transplantation by injecting 0.2ml of a 5%
dilution of ascites tumor in 0.9% NaCl to female mice of the
DBA/2W strain as described by Dowjat and Kawiak (1979). In the
experiments the same co-culture procedure was used for both EAT
and L1210 cells.
Primary cultures of fibroblasts isolated from 9- to 11-day-old
chick embryos (White Leghorn or Astra S) were carried out in
Legroux glass flasks as described previously (Korohoda, 1971).
Cells from 48-h cultures were rinsed with EMEM, incubated for
5min in 0.25% trypsin solution, centrifuged (100 #, 5min) and
resuspended in trypsin-inactivating solution (EMEM with 10%
433
CHICK serum;, men uie cens were again conceniraieu oy
centrifugation and resuspended in the culture medium: EMEM
supplemented with 5% chick serum, O.Smgml"1 L-glutamine,
lOmmoll"' Hepes buffer, lOOi.u.ml"1 penicillin, 10/igml
streptomycin. For secondary cultures fibroblasts were plated at
an initial cell density of 1.5 xlO 5 cells/Leighton tube. For some
experiments Chance glass coverslips were inserted into the
Leighton tubes before cell plating.
Mice fibroblasts were isolated from the skin of 18- to 19-day-old
embryos of Swiss albino mice. The skin was cut into small pieces
with scissors in EMEM and trypsinized for 10 min in 0.25%
trypsin solution. The cell suspension obtained was filtered
through 100 ;/m nylon mesh, centrifuged (100 g, 5 min), and
resuspended in trypsin-inactivating solution (EMEM supplemented with 10 mM Hepes, 20 % calf serum). Then the cells
were again centrifuged and resuspended in culture medium:
EMEM supplemented with 10 % calf serum, 10 mM Hepes buffer
0.5mgml
L-glutamine, lOOi.u.ml"1 penicillin, 10;<gml
streptomycin. Primary cultures of mice fibroblasts were carried
out in Legroux flasks at 37 °C (Yamada and Okigaki, 1983).
Secondary cultures were prepared in the manner described for
chick fibroblasts. The cells were plated at initial cell density of
1.5xlO6 cells/Leighton tube and were grown at 37°C in the same
culture medium as primary cultures.
Chick myoblasts were isolated from the pectoral muscle of 9- to
11-day-old chick embryos (White Leghorn or Astra S) and
prepared for primary culture as described (Reiss and Korohoda,
1988). The culture of myogenic cells was carried out in Leighton
tubes coated with gelatin as recommended by Mashuko and
Ishikawa (1983). The initial cell density was 105 cells ml" 1 . To
induce formation of myotubes, cells were incubated at 35°C in a
culture medium of EMEM, 2 % chick serum, 10 % horse serum,
10 mM Hepes and antibiotics. For some experiments, Chance glass
coverslips were inserted into culture vessels prior to the cell
plating.
Peritoneal membrane was isolated from Swiss albino mice,
washed twice for 15 min in PBS (Dulbecco's phosphate-buffered
saline) and put onto the bottom of a Rose chamber. Only those
membranes that were undamaged and big enough to cover the
bottom of the Rose chamber were used for experiments. In the
chamber membranes were spread carefully so that only the
surface covered with mesothelium was exposed to EAT cells. The
EAT cell suspension was plated into the Rose chamber and
incubated at 37 °C for 4h. Since the area of the bottom of this
chamber and that in the Leighton tube were approximately the
same, the number of EAT cells used for this experiment was the
same as for myoblasts and fibroblasts.
Adhesion of EAT cells to the surface of fibroblasts and
myoblasts
Fibroblasts and myoblasts in cultures were grown to confluency
and then were washed once with EMEM. EAT cells suspended in
the incubation medium at a density of 7xl0 4 cellsml~ 1 were
seeded gently onto the surface of monolayers. All subsequent
steps were carried out at 37 °C. At the end of the attachment
period (15min, 30min, In, 2h, 4h) the EAT cells that did not
adhere to the substratum were harvested and counted in a Biirker
haemocytometer. The percentage of cells attached in an experiment was calculated as the starting number of cells in an
incubation minus the number of non-attached cells divided by the
starting number (st. no.) of cells (Grinnell, 1984):
%Adh.EAT=(st.no.EAT-no. non-attached EARxlOO/st.no.EAT
Adhesion of EAT cells to the surface of fixed fibroblasts
and myoblasts
Cell monolayers were washed with EMEM and fixed for 5 min in
3% glutaraldehyde or 4% formaldehyde solution in PBS
(Dulbecco's phosphate-buffered saline). Afterwards, the aldehyde
solution was removed and cultures were washed three times with
PBS (3x10 min incubation in PBS). Before the suspension of EAT
cells was added, fixed cells were washed once again with
incubation medium (EMEM supplemented with serum). The
number of adhering EAT cells was estimated as described above.
434
B. Wdjciak and W. Korohoda
spreading area assays
Chance glass coverslips with fibroblasts or myoblasts were
transferred from culture vessels into a glass chamber of the
following size: 3 cm x 2 cm x 2 mm. Then the suspension of EAT
cells in incubation medium was plated into this chamber. All
subsequent steps were carried out at 37 °C.
Changes in cell morphology were followed under a Pluta
differential interference contrast microscope (Biolar, PZO,
Warszawa, Poland) and phase-contrast microscopy (PZO,
Warsaw, Poland). Microphotographs of subsequent steps of cell
spreading were taken every 30 min for 2 h. The area of spread cells
was calculated planimetrically from microphotographs. For
determining the area and shape of the spread EAT cells, we also
used glass coverslips taken at the end of incubation time from
culture vessels. The cells were fixed with 3% glutaraldehyde,
washed three times with distilled water and used for planimeter
analysis. For planimetry a Microplan II, computer-driven planimeter, Don Santo Corp. USA, in conjunction with a Nikon
microscope 104, Japan, was used. The two methods gave
consistent results.
Microphotography and SEM
Microphotographs were taken under a Pluta differential interference contrast microscope (Biolar, PZO, Warsaw, Poland), a
phase-contrast microscope (PZO, Warsaw, Poland) and a JSM-35
scanning electron microscope. The cell-fixing procedure and
preparation for SEM have been described (Kajstura et al. 1986).
For photography, Chance glass coverslips were inserted into
culture vessels. The cells were fixed in 3 % glutaraldehyde for
5 min, washed twice in distilled water and dried.
Fluorescent visualization of EAT cells spread on living
fibroblasts
Coverslips with fibroblast—EAT cell co-cultures on them were
taken out of Leighton tubes, washed with PBS and stained with
fluorescein diacetate solution in PBS (S/igmP 1 ). Preparation of
fluorescein diacetate solution was as described (Kajstura and
Reiss, 1989). Observations were made 50-60 s after staining, in
phase-contrast and epifluorescence under a Leitz Orthoplan
microscope (Wetzlar, FRG).
Chemicals
Eagle's Minimal Essential Medium (EMEM) with Earle's salts
from Gibco Laboratories (Grand Island, NY, USA), horse, bovine
sera, trypsin solution and Dulbecco's phosphate-buffered saline
(PBS) from Wytwornia Surowic i Szczepionek (Lublin, Poland),
Hepes, L-glutamine and fluorescein diacetate (FDA) from Sigma
(Sigma Chemical Company, St Louis, MO, USA), gelatin from
Loba (Loba Chemie Wien-Fischamed, Vienna, Austria), penicillin
and streptomycin from Polfa (Tarchomin, Poland), glutaraldehyde and formaldehyde from Reanal (Budapest, Hungary). Chick
serum was made from fresh Leghorn chicken blood, inactivated
for 30min at 56°C before storage at -18°C.
Results
In the first series of experiments the attachment of EAT
cells to the peritoneum and surface of glass was assessed in
vitro. As shown in Fig. 1, only a negligible fraction of cells
(less than 8 %) attached to the surface of the peritoneum or
to glass within 4h of incubation at 37 °C. This shows that
the situation in vitro corresponds to the behaviour of EAT
cells in vivo. In the following experiment the fractions of
EAT cells adhering to mouse embryo fibroblasts were
determined (Fig. 1). Fast and relatively stable adhesion of
EAT cells to monolayers of fibroblasts was observed. In the
case where EAT cells were seeded over the surface of a
fibroblast monolayer, over 80 % of EAT cells had already
adhered during the first ten minutes of incubation. What
is more, attachment of ascitic EAT cells to the surface of
80-
60-
-r
T
T
i
r
40-
20•
—3:
•
0-
^!*Trf,
0
i i i •¥•<
1
2
i
i
i
|
3
Time (h)
I
i
i
ft:
i
1
4
t
i
i
i
5
Fig. 1. Time of EAT cell attachment to the surface of glass, to
the peritoneum and to living mouse embryofibroblasts.The
data show the percentage of EAT cells attached to a glass
substratum (-O—O-), the peritoneal membrane (—•—•—),
and livingfibroblasts(—A —A — ). A suspension of EAT cells
(7xlO4 cells ml"1) was incubated for 4h at 37°C in Leighton
tubes with or withoutfibroblasts,and in Rose chambers with
mouse peritoneal membrane. Cell attachment was measured at
the end of the incubation (details in Materials and methods).
Vertical bars represent standard deviation.
fibroblasts was followed by changes in EAT cell shape,
production of cell surface processes and eventually by cell
spreading and locomotion on and under the fibroblast
layer. The scanning electron microphotographs of spreading EAT cells after attachment to fibroblasts and myoblasts are shown in Fig. 2.
The process of cell flattening continued for the next two
to three days, and by then the morphology of EAT cells did
not differ significantly from the morphology of surrounding fibroblasts. Nevertheless, the flattened EAT cells were
easily visualized by fluorescent microscopy after a short
staining with FDA.
In the next series of experiments adhesion of EAT cells
to living and fixed fibroblasts and myoblasts isolated from
chick embryos was followed. As shown in Fig. 3, the EAT
cells attached as well to chick embryo fibroblasts and
myoblasts as to mouse embryo fibroblasts. Fixation of
chick fibroblasts with formaldehyde instead of the commonly used glutaraldehyde (Matuoka and Mitsui, 1981;
Oesch et al. 1987) increased the fraction of attached EAT
cells (Fig. 3). Whereas attachment of EAT cells to living
mouse or chick embryo fibroblasts and myoblasts was
followed by fast cell flattening and spreading, the EAT
cells attached to fixed cells showed significantly reduced
rate of cell spreading (Fig. 4).
In Fig. 4 the kinetics of EAT cell flattening after
adhesion to chick embryo fibroblasts is shown. During the
first 2 h there was already more than a doubling of the
area of the EAT cell's cross-section. The faster spreading
on living than on fixed fibroblasts could result from the
metabolic cooperation of contacting cells or from the
effects of diffusible proteins secreted by living cells and
then covering the surface of ascitic cells. As it is known,
macromolecules attached to the surface of cancer cells
(fibronectins, high molecular weight dextrans, proteoglycans) can induce the cells attachment to glass and their
spreading on glass or plastic (Lipkin and Knecht, 1976;
Mallucci and Wells, 1976; Yamada et al. 1976; CieSlak and
Korohoda, 1978). Therefore we observed the behaviour of
EAT cells seeded to Leighton or Rose culture vessels into
which fibroblast-covered cover glass was inserted. Only
EAT cells in direct contact with fibroblasts attached to
them and changed their morphology, whereas the EAT
cells in close vicinity but without contact with fibroblasts
maintained spherical morphology.
An additional experiment was done in which conditioned medium from cultures of fibroblasts was separated and used for EAT cells, but this did not support the
attachment of EAT cells to glass and spreading.
The experiments described above were carried out with
EAT cells. Some of them were repeated in a preliminary
fashion with spherical leukaemia L1210 ascitic cells.
These cells adhered to living chick embryo fibroblasts and
to fixed fibroblasts in a similar fashion to EAT cells.
Discussion
Results presented in this paper show that the ascitic cells
of EAT or leukaemia L1210, which do not attach to
mesothelial cells of peritoneal membranes in vivo and in
vitro and to glass in vitro, adhere strongly to embryonic
fibroblasts, myoblasts and myotubes. The adherence is
only slightly decreased when fibroblasts are fixed with
formaldehyde and more significantly decreased if glutaraldehyde is used for fixation. The attachment does not
depend upon species of fibroblast origin. The EAT cells are
shown to adhere equally well to fibroblasts from mouse
embryos (homogeneous, heterotypic adhesion) and to
fibroblasts from chick embryos (heterogeneous, heterotypic adhesion). Differences in the attachment of EAT cells
to mesothelial cells and to mesenchymal cells seem to
correspond to unknown differences in the chemical
composition of cell surfaces among cells from different
tissues (Weiss, 1975; Grady and McGuire, 1976; Nicolson,
1984). The observation that cancer cells react differently
upon contact with cells of various tissue origins than when
in contact with glass points to the need for more extensive
research on cancer cell behaviour in co-culture with other
cell types, and also shows that it is difficult to extrapolate
from the results of research concerning the adhesion of
cancer cells to solid substrata to situations in vivo.
Co-cultures of cancer cells with normal cells have been
studied in a few laboratories in near monolayer, but
emphasis has been on the effects of cell-to-cell contacts
upon neoplastic cell proliferation (Holzer et al. 1986;
Mehta et al. 1986; Sargent et al. 1988) or on the
modification of normal tissue cells by neoplastic cells
(Benke et al. 1984). The results of experiments reported
here demonstrate that the neoplastic cell surface properties and morphology are strongly modified upon contact
with normal tissue cells. Observed and often reported
changes in the rate of proliferation of neoplastic cells
interacting with various tissues represent, rather, the
final effect of these interactions.
The observation that fixation of fibroblasts does not
inhibit the adhesion of ascitic cells to their surfaces shows
that adhesion is determined by the chemical composition
of the cell surface. We have previously shown that chick
embryo fibroblasts colliding with aldehyde-fixed neighbouring cells show contact inhibition of movement
(Korohoda et al. 1988). Matuoka and Mitsui (1981) and
Oesch et al. (1987) have shown that the addition of
glutaraldehyde-fixed cells to sparsely seeded cells resulted
in a strong inhibition of growth. This indicates that
Cell contacts change morphology of EAT cells
435
Fig. 2. (A) Differential interference image of EAT cell flattening on chick embryo fibroblasts after 24 h of incubation. Bar, 10 /mi.
(B) Scanning electron microscope photographs of EAT cell flattening on the surface of chick embryo fibroblast after 24 h of
incubation. Bar, 2 ;im. (C) EAT cell migrating along chick embryo myotube after 4 h of coculture. Scanning electron microscope.
Bar, 2 /im. (D) EAT cells spread on the surface of mouse embryo fibroblasts after 24 h of coculture. Scanning electron microscope.
Bar, 2
436
B. Wojciak and W. Korohoda
fixation with aldehydes of one partner engaged in cell-tocell interaction does not always exclude the responses of
the second partner.
The direct attachment of EAT cells to living embryo
fibroblasts (of mouse or chick origin), and to myoblasts or
myotubes, was followed by changes in tumour cell shape,
lOO-i
80-
60o 40<
s? 2 0 -
0
1
2
3
Time (h)
4
5
Fig. 3. Kinetics of EAT cell attachment to living chick embryo
fibroblasts (—•—•—), living chick embryo myoblasts
(—O—O—), formaldehyde-fixed chick embryo fibroblasts
(—A —A—), glutaraldehyde-fixed chick embryo fibroblasts
(—• — • — ) and glutaraldehyde-fixed myoblasts ( —D —D—).
Vertical bars represent standard deviation.
260-1
flattening, spreading, and locomotion over and between
normal cells. The need of direct contact between cells was
obvious when ascitic cells were seeded into cultures in
which fibroblasts covered only a limited area of glass, as in
cultures in which fibroblasts growing on coverglasses were
inserted into a larger culture vessel. Only those ascitic
cells that were in direct contact with fibroblasts changed
their morphology. The conditioned medium from fibroblast cultures did not induce changes in EAT cell
morphology and adhesive properties. Direct cell-to-cell
contact has been shown to stimulate secretion of matrix
proteins (Aggeler et al. 1982; Little and Chen, 1982) known
to regulate many cell functions (Ben-Ze'ev, 1984; Bissell
and Barcellos-Hoff, 1987; Nakagawa et al. 1989).
Cell surface adhesive characteristics are often treated as
fairly stable cell features. Curtis (1987, 1988), however,
stressed that the adhesiveness of leucocytes and blood
platelets can be changed from a state of non-adhesion to
very strong adhesion to each other, and to many surfaces,
in a very short time. Our results strongly support the view
that cell adhesiveness cannot be considered as a stable cell
feature. They show that ascitic cells can respond to contact
with embryonic fibroblasts or myoblasts by changes in
their adhesiveness and shape. The influence of cell shape
upon cell proliferation and differentiation has been
documented (Castor, 1970; Folkman and Moscona, 1978;
Ben-Ze'ev, 1985; O'Neill et al. 1986; Bereiter-Hahn, 1990).
The results discussed here indicate that cancer cell shape
might be dependent upon tissue-specific cell-to-cell interactions. Research carried out on co-cultures of neoplastic
and normal cells can add new data concerning often
reported growth regulation in vivo of metastatic cancer
cells by host organs (Auerbach et al. 1987; Sargent et al.
1988; Benke et al. 1988; Honn et al. 1989).
The authors gratefully thank Professor A. S. G. Curtis
(Glasgow University) for stimulating discussion and correction of
this manuscript. We also thank Professor W. Kilarski (Cracov
University) for kind help with SEM, Professor J. Stachura
(Medical Academy in Cracow) for arranging the use of the
computer-driven Microplan II planimeter and Professor J.
Kawiak (Medical Center of Postgraduate Education, Warszawa)
for providing L1210 cells.
References
AGGELER, J., KAPP, L. N., TSENG, S. C. G. AND WERB, Z. (1982).
Regulation of protein secretion in Chinese hamster ovary cells by cell
cycle position and cell density. Expl Cell Res. 139, 275-283.
AUERBACH, R., LU, W C , PARDON, E., GUMOWSKI, F., KAMINSKA, G. AND
KAMINSKI, M. (1987). Specificity of adhesion between murine tumor
cells and capillary endotheliunv an in vitro correlate of preferential
metastasis in vivo. Cancer Res. 47, 1492-1496
BENKE, R., LANG, E., KOMITOWSKI, D., MUTO, S AND SCHIRRMACHER, V.
(1988). Changes in tumor cell adhesiveness affecting speed of
dissemination and mode of metastatic growth. Invasion Metast. 8,
159-176.
BENKE, R., WERLING, H. O. AND PAWELETZ, N. (1984). Studies on
Time (h)
Fig. 4. Time course of EAT cell spreading on a substratum
with living (—•—•—) and formaldehyde-fixed (—O—O—)
chick embryofibroblasts.The area of spread EAT cells was
measured planimetrically at the end of the incubation time
(see Materials and methods) and is expressed as the percentage
of an average EAT cell area measured before incubation.
Vertical bars represent standard deviation. Cross-section of
spherical EATcells=65/<m2±12 (100%).
confrontation of different tumor cells with human diploid fibroblasts.
Antwancer Res. 4, 241-246.
BEN-ZE'EV, A. (1984). Differential control of cytokeratins and vimentin
synthesis by cell—cell contact and cell spreading in cultured epithelial
cells. J Cell Biol 99, 1424-1433.
BEN-ZE'EV, A. (1985). The cytoskeleton in cancer cells. Biochim. biophys.
Ada 780, 197-212.
BEREITER-HAHN, J. (1990). Spreading of trypsinized cells1 cytoskeletal
dynamics and energy requirements. J. Cell Sci. 96, 171-188.
BISSELL, M. J. AND BARCELLOS-HOFF, M H. (1987). The influence of
extracellular matrix on gene expression: is structure a message? J.
Cell Sci. Suppl 8, 327-343
CASTOR, L. N. (1970). Flattening, movement and control of division of
epithehal-like cells. J. cell. Physiol. 75, 57-64.
Cell contacts change morphology of EAT cells
437
CIESLAK, J. AND KOROHODA, W. (1978). Dextran T500 induction of
spreading in Ehrlich ascites tumour cells on glass surface. Cytobiol.
16, 381-392.
CURTIS, A. S. G. (1987). Cell activation and adhesion. J. Cell Sci 87,
609-611.
CURTIS, A. S G (1988). Cell-cell interactions: activation or specific
adhesion. In Eukaryote Cell Recognition: Concepts and Model Systems
(ed. G. P. Chapman, C. C. Ainsworth and Chatham). Cambridge:
University Press.
DOWJAT, K. AND KAWIAK, J. (1979). Karyotypic analysis of two L1210
murine Leukemia lines growing in vivo and in vitro. Cytologia 44,
927-934.
FOLKMAN, J. AND MOSCONA, A. (1978). Role of cell shape in growth
control. Nature 273, 345-349.
GRADY, S. R. AND MCGUIRE, E. J. (1976). Intercellular adhesive
selectivity. J. Cell Biol. 71, 96-106.
GRINNELL, F. (1984). Manganese-dependent cell-substratum adhesion. J.
Cell Sci. 65, 61-72.
HOCHMAN, J., LEVY, E., MADOR, N., GOTTESMAN, M. M., SHEARER, G. M.
AND OKON, E. (1984). Cell adhesiveness is related to tumorigenicity in
malignant lymphoid cells. J. Cell Biol. 99, 1282-1288.
HOLZER, C , MAIER, P. AND ZBINDEN, G. (1986). Comparison of exogenous
growth stimuli for chemically transformed cells: growth factors, serum
and cocultures. Expl Cell Biol. 54, 237-244.
HONN, K. V., GROSSI, I. M., DIGLIO, C. A., WOJTUKIEWICZ, M. AND
TAYLOR, J. D. (1989). Enhanced tumor cell adhesion to the
subendothelial matrix resulting from 12(S)-HETE-induced endothelial
cell retraction. Fedn. Am. Socs exp. Biol. J. 3, 2285-2293.
KAJSTURA, J., KILARSKI, W. AND KOROHODA, W. (1986). Experimentally
of myogenic cells during the cell cycle, fusion and myotube
formations. Dev. Growth and Differ. 25. 56-73.
MATUOKA, K. AND MITSUI, M. (1981). Involvement of cell surface sulfate
in the density-dependent inhibition of cell proliferation. Cell Struct.
Fund. 6, 23-33.
MEHTA, P. P., BERTRAM, J. S. AND LOEVENSTEIN, W. R. (1986) Growth
inhibition of transformed cells correlates with their junctional
communication with normal cells. Cell 44, 187-196.
NAKAGAWA, S , PAWELEK, P. AND GRINNELL, F. (1989). Extracellular
matrix organization modulates fibroblast growth and growth factor
responsiveness. Expl Cell Res. 182, 572-582.
NICOLSON, G. L. (1984). Cell surface molecules and tumor metastasis.
Regulation of metastatic phenotypic diversity. Expl Cell Res. 150,
3-22.
OESCH, F., JANIK-SCHMITT, B., LUDWIG, G , GLATT, H. AND WIESER, R. J.
(1987). Glutaraldehyde-fixed transformed and non-transformed cells
induce contact-dependent inhibition of growth in non-transformed
C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrenetransformed cells. Eur. J. Cell Biol. 43, 403-407
O'NEILL, C, JORDAN, P. AND IRELAND, P. J. (1986). Evidence for two
distinct mechanisms of anchorage stimulation in freshly explanted
and 3T3 Swiss mouse fibroblasts. Cell 44, 484-496.
RAZ, A. AND BEN-ZE'EV, A. (1987). Cell-contact and -architecture of
malignant cells and their relationship to metastasis. Cancer Metast.
Rev. 6, 3-21.
REISS, K AND KOROHODA, W. (1988). The formation of myotubues in
cultures of chick embryo myogenic cells in serum-free medium is
induced by the insulin-pulse treatment. Folia histochem. cytobiol. 26,
133-142.
induced modifications in surface morphology of EAT cells. Cell Biol.
Int. Rep. 10, 727-733.
KAJSTURA, J. AND REISS, K. (1989). Measurement of cell swelling in a
hypotonic medium as a rapid and sensitive test of cell injury. Folia
histochem. cytobiol. 27, 39-48.
KOROHODA, W. (1971). Interrelations of motile and metabolic activities
in tissue culture cells. Respiration of chicken embryo fibroblasts
actively locomoting, contact-inhibited, and suspended in a fluid
medium. Folia biol. (Cracow) 19, 41-51.
SARGENT, N. S. E., OESTREICHER, M., HADNICK, H. M. AND BURGER, M.
KOROHODA, W., KAJSTURA, J. AND REISS, K. (1988). Chick embryo
inhibitor on the adhesion of Ehrlich ascites cells to host cells in vivo.
Br. J Cancer 28, 417-428.
YAMADA, M AND OKIGAKI, T. (1983). Promotion of epithelial cell
adhesion on collagen by proteins from rat embryo fibroblasts. Cell
Biol. Int. Rep. 7, 1115-1120.
fibroblasts show contact inhibition of movement when colliding with
glutaraldehyde fixed cells. Cell Biol. Int. Rep. 12, 477-482.
LEIOHTON, J. AND TCHAO, R. (1984). The propagation of cancer, a process
of tissue remodeling. Cancer Metast. Rev. 3, 81-97.
LITTLE, C. D. AND CHEN, W. T. (1982). Masking of extracellular collagen
and the co-distribution of collagen and fibronectin during matrix
formation by cultured embryonic fibroblasts. J. Cell Sci. 55, 35-50.
LIPKIN, G. AND KNECHT, M. E. (1976). Contact inhibition of growth is
restored to malignant melanocytes of man and mouse by a hamster
protein. Expl Cell Res. 102, 341-348.
MALLUOCI, L. AND WELLS, V. (1976). Determination of cell shape by a
cell surface protein component. Nature 262, 138-141.
MASHUKO, S. AND ISHIKAWA, Y. (1983). Changes in surface morphology
438
B. Wojciak and W. Korohoda
M. (1988). Growth regulation of cancer metastases by their host
organ. Proc. natn. Acad. Sci. U.S.A. 85, 7251-7255.
VASILIEV, J. M. (1985). Spreading of non-transformed and transformed
cells. Biochim. biophys. Ada 780, 21-65.
WEISS, L. (1975). Some biophysical aspects of cell contacts in metastasis.
V. Cell Membr. Tumor Cell Behavior, pp. 361-382. Baltimore:
Williams & Wilkins Co.
WHUR, P., ROBSON, R. T. AND PAYNE, N. Y. (1973). Effect of a protease
YAMADA, K. M., YAMADA, S. S. AND PASTAN, I. (1976). Cell surface
protein partially restores morphology, adhesiveness, and contact
inhibition of movement to transformed fibroblasts. Proc. natn. Acad.
Sci. U.S.A. 73, 1217-1221.
(Received 8 June 1990 - Accepted 13 August 1990)