Cell Surface Properties of B16 Melanoma

Transcription

[CANCER RESEARCH 40, 1645-1651,
May 1980]
Cell Surface Properties of B16 Melanoma Variants with Differing
Metastatic Potential1
A. Raz,2 W. L. McLellan, I. R. Hart, C. D. Bucana, L. C. Hoyer, B-A. Sela, P. Dragsten, and I. J. Fidler
Cancer Biology Program. National Cancer Institute Frederick Cancer Research Center, Frederick, Maryland 21701 [A. R., W. L. M.. I. R. H., C. D B., L. C. H., l. J.
F.]; Department of Biophysics, The Weizmann Institute of Science, Rehovot. Israel Â¡B-A. SJ; and National Cancer Institute, Laboratory of Theoretical Biology
Bethesda, Maryland 20205 [P. D.]
ABSTRACT
Several cell surface properties of three B16 melanoma var
iant lines, which exhibit differing lung-colonizing capacities,
have been examined. Equal numbers of cells from the B16F10Lr and B16-F1 lines, injected i.v. into syngeneic mice,
produced significantly fewer lung nodules than did cells of the
B16-F10 line. These wide differences in biological behavior
could not, however, be ascribed to major qualitative differences
in the cell surface properties studied.
The 3 cell lines displayed similar quantitative and qualitative
patterns of exposed surface proteins. The quantity of radioac
tive labeling of the major sialoglycoprotein (78,000 dallons)
was inversely proportional to the capacity of cells to produce
lung tumor colonies. Lectin-binding assays revealed that B16F10Lr cells bound 50% less concanavalin A than did the B16F1 or B16-F10 cells; the B16-F10 cells bound 40% less wheat
germ agglutinin than did the B16-F1 or B16-F10Lr cells. Levels
of 5'-nucleotidase activity were lower in B16-F10 cells than in
B16-F1 and B16-F10Lr cells. At the same time, there was no
reduction in levels of the cytoplasm-associated
enzyme, acid
phosphatase. No differences in membrane lipid fluidity among
the cell lines were detected by either fluorescent polarization
or photobleaching techniques. The kinetics of adhesion of B16F10Lr and B16-F10 to a variety of substrates were very similar.
The present data suggest that wide differences in metastatic
behavior of B16 melanoma cells need not be correlated with
major qualitative changes in a single cell surface biochemical
or morphological characteristic.
INTRODUCTION
The process of metastasis involves the release of cells from
the primary tumor, dissemination to distant sites, arrest in the
microcirculation of organs, extravasation and infiltration into
the stroma of those organs, and survival and growth into new
tumor colonies. The outcome of metastasis depends on both
host factors and tumor cell properties, which may vary among
tumor systems (15). Since the interaction of metastatic cells
with their environment is mediated to a considerable extent by
the cell surface, it seems likely that cell surface properties
could influence the metastatic potential of tumor cells. Changes
in the cell surface that accompany neoplastic transformation
have been studied in great detail. In fact, almost all character
istics of the transformed cell membrane appear altered when
1 Research sponsored by the National Cancer Institute under Contract NO1CO-75380 with Litton Bionetics, Inc.
2 To whom requests for reprints should be addressed.
Received August 20, 1979; accepted February 6, 1980.
compared to those of the untransformed counterpart (21, 24,
33, 47). The relationship of any specific cell surface change to
the outcome of experimental metastasis, however, is unknown.
In order to identify those properties of tumor cells that allow
them to interact favorably with their host and to establish
metastatic growths, it is advantageous to study variants of the
same tumor with differing capacities to form experimental mÃ©
tastases. The isolation of tumor variants can be achieved by
several methods. The first involves isolation and propagation
of variant lines from mÃ©tastases.In such diverse murine tumor
systems as melanoma (12), fibrosarcoma (44), methylcholanthrene-induced sarcoma (26), lymphosarcoma (7), Lewis lung
carcinoma (18), and lymphoma (27), variants with different
metastatic potentials have been selected. A second method for
isolation of tumor variants involves in vitro selection of cells for
specific properties, such as detachment from a monolayer (5),
resistance to lysis by syngeneic lymphocytes (14), and resist
ance to lectin-mediated toxicity (46), and then to determine
whether the selection affects metastatic behavior in vivo. A
third more recent and complementary approach has been to
establish in vitro a large number of clones or lines from parent
tumors such as the B16 melanoma (6, 12, 13, 16), a UVinduced fibrosarcoma (28), a mammary tumor (10), and a
methylcholanthrene-induced
fibrosarcoma (43). Using this ap
proach, the behavior in vivo and in vitro of such clones can be
studied.
In recent years, we have concentrated on the B16 melanoma,
syngeneic to the C57BL/6 mouse, as a tumor system. By
manipulating various host and tumor variables, we have at
tempted to determine the relative contributions of several fac
tors to the survival and growth of blood-borne tumor cells. We
have described the selection in vivo (12) and in vitro (14) of
variant sublines of the B16 melanoma with different capacities
for experimental metastasis. Several biochemical differences
between the cells of low and high lung-colonizing potential
have been reported. Among these were electrophoretic mobil
ity and surface glycoproteins (19). However, comparative anal
ysis of surface proteins labeled by lactoperoxidase iodination
and glycopeptides released by mild trypsin-pronase digestion
failed to demonstrate any significant differences between highl
and low-metastatic B16 cell variants (34, 48).
The present report concerns our analysis of the functional,
structural, morphological, and dynamic properties of the cell
membrane components and their correlation with the lungcolonizing behavior of 3 variants of the B16-F series. The
results indicate that no qualitative differences in major com
ponents of the cell membrane among the lines can be demon
strated. However, subtle quantitative and perhaps cumulative
changes in the cell surface may contribute to the success or
failure of blood-borne tumor cell survival and growth in vivo.
MAY 1980
Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1980 American Association for Cancer Research.
1645
A. Raz et al.
MATERIALS
AND METHODS
Animals
Specific-pathogen-free
C57BL/6 mice were obtained from
the Animal Production Area of the Frederick Cancer Research
Center. For each experiment, all mice were age and sex
matched.
Tumor Cell Variants
The B16-F1 cell line (low incidence of lung colonization) was
derived from pulmonary mÃ©tastasesproduced by i.v. injection
of B16 melanoma cells obtained from The Jackson Laboratory,
Bar Harbor, Maine. The B16-F10 cell line (high incidence of
lung colonization) was selected by successive passaging of
lung tumor colonies for 10 in vivo-in vitro selections as de
scribed in detail previously (12). B16-F10Lr (lymphocyte resist
ant, low incidence of metastasis to the lung) is a variant cell
line selected from B16-F10, after tumor cell monolayers were
subjected to cytotoxic lymphocytes 6 times in vitro (14).
Tumor Cultures
All cultures were maintained on plastic in Eagle's minimal
essential medium supplemented with 10% fetal bovine serum,
sodium pyruvate, nonessential amino acids, L-glutamine, and
2-fold vitamin solution (Flow Laboratories, Inc., Rockville, Md.).
Antibiotics were not included in the medium for routine main
tenance of the cultures. Cell cultures were grown at 37Â°in a
humidified atmosphere containing 5% CCv The cell lines were
examined for and found to be free of Mycoplasme and the
following murine viruses: reovirus type 3; pneumonia virus of
mice; K virus; Theiler's virus; Sendai virus; minute virus of
mice; mouse adenovirus; mouse hepatitis virus; lymphocytic
choriomeningitis virus; ectromelia virus; and lactate dehydrogenase virus (Microbiological Associates, Bethesda, Md.). The
cell lines were grown from frozen stocks and were maintained
in continuous cultures for periods not exceeding 60 days. The
lung colonization potential of the 3 variant lines was evaluated
several times during the studies, as described below. The cell
lines were coded for all in vitro assays.
Experimental Pulmonary Metastasis
Cells from semiconfluent monolayers were harvested with a
short (2-min) treatment of 2 rriM EDTA in a Ca2+ and Mg2+-free
PBS,3 pH 7.2. The cells were washed in PBS and pipetted
gently to dissociate all cell clumps. Only cell suspensions of
more than 90% viability, as measured by trypan blue exclusion,
and those free of all aggregates were used for in vivo or in vitro
studies. Unanesthetized mice were given i.v. injections of 0.2
ml containing 50,000 viable single tumor cells suspended in
Hanks' balanced salt solution via the tail vein. All mice were
killed 18 days after tumor cell injection, and their lungs were
removed, rinsed in water, and fixed in formalin. The number of
lung tumor colonies was determined by counting surface mÃ©
tastases under a dissecting microscope, since the majority of
3 The abbreviations used are: PBS. phosphate-buttered saline (0.01 M phos
phate buffer, pH 7.2-0.15 M NaCI); SBA, soybean agglutinin; UEA1, Ulex europeus agglutinin I; Con A. concanavalin A; WGA. wheat germ agglutinin; WBA,
wax bean agglutinin; DPH, 1,6-diphenyl-1,3,5-hexatriene;
DM,3,3'-dioctadecylindocarbocyanine.
1646
such experimental mÃ©tastases in mice are located near the
lung surface (11 ). MÃ©tastaseswere counted in a blind fashion
by 2 observers. All extrapulmonary mÃ©tastaseswere also re
corded.
Lectins
The following lectins were radioactively
labeled by the 125I-
chloramine-T method (23): SBA, UEA1 (Vector Laboratory,
Burlingame, Calif.), Con A, WGA (Miles Yeda, Rehovot, Israel),
and WBA. The radioactively labeled lectins were separated
from free iodine by gel filtration on a Bio-Gel P-2 column.
Specific radioactivity of the lectins ranged from 3 to 10 x 106
cpm/mg protein.
For quantitative studies with radioactively labeled lectins, 0.5
x 106 tumor cells were exposed to increasing concentrations
of a given lectin for 30 min at 24Â°. The specific binding of a
lectin to the cell surface was calculated by subtracting the
amount bound in the presence of a specific inhibitor (to lectin
binding) from the amount of lectin bound in the absence of its
inhibitor. The specific inhibitors were 0.1 M a-methyl-D-mannopyranoside for Con A, 0.1 M N-acetylglucosamine for WGA,
0.1 M A/-acetylgalactosamine for SBA, 0.1 M L-fucoseforUEA1,
and 0.1% fetuin for WBA. Lectin binding in the presence of
specific inhibitors constituted less than 10% of total binding.
Neuraminidase Treatment
B16 cells suspended in PBS were incubated for 30 min at
37Â°with protease-free Vibrio cholera neuraminidase (50 units/
ml) purchased from Calbiochem, La Jolla, Calif., and then
washed in buffer to remove the free sialic acid and the enzyme.
When comparison between untreated and neuraminidasetreated cells was desired, the untreated cells were incubated
in PBS for the same duration as the enzyme treatment. No loss
of cell viability could be detected following neuraminidase
treatment.
Surface Labeling of Glycoproteins
Galactose residues of cell surface glycoproteins were radiolabeled before or after neuraminidase treatment. Cells were
incubated with 50 jug of galactose oxidase for 180 min at 37Â°
and then centrifuged, washed in, and reduced with 0.5 mCi
NaB3H4 (New England Nuclear, Boston, Mass.) for 30 min at
24Â°. Cells were washed and processed for electrophoresis.
For surface labeling of sialic acid residues of glycoproteins, 2
x 106 cells suspended in 1 ml of phosphate-buffered
NaCI
(0.13 M NaCI in 0.02 M potassium phosphate, pH 7) were
incubated with 5 row NalO4 at 4Â°for 15 min (19); 0.2 ml of 0.1
M glycerol was then added, and the cells were incubated for
an additional 10 min. After being washed with the buffer, the
suspensions were reduced with [3H]borohydride, and the cells
were processed for electrophoresis.
not lead to decreased cell viability.
Labeling procedures
did
Electrophoresis
The labeled cells were solubilized with 1% Triton X-100, 0.4
M KI-0.05 M Tris-HCI, pH 9.0, and centrifuged at 10,000 x g
for 15 min to remove insoluble material. An aliquot was resolved
by electrophoresis separation on a 5 to 20% gradient of sodium
dodecyl sulfate-polyacrylamidegel
(29). Fluorography wascarCANCER
RESEARCH
VOL. 40
Surface
ried out by the method of Bonner and Laskey (3). Gels were
fixed, stained and destained, and then incubated with 22%
dimethyl sulfoxide in dimethyl sulfide, dried, and exposed to
RP Royal X-omat film (Kodak) at -70Â°. Interpretation of label
Properties
of Metastatic
Cells
ing could be made by superimposition of the developed fluorogram on the Coomassie blue-stained protein bands.
tively then, we are confident that in vitro studies of tumor
surface properties, which may correlate with lung colonization
in vivo, can be carried out on these cell lines incubated in vitro
for up to 12 consecutive weeks. Nonetheless, we maintained
the cell lines for no longer than 60 culture days before replacing
the cultures from frozen stocks.
Assay for Enzyme Activities
Labeling of Galactose
All assays were performed on attached monolayer cell lines
(38). The determinations of enzyme activity are expressed in
relation to mg protein. Cells of semiconfluent monolayers were
washed 3 times with 2-ml aliquots of 0.9% NaCI solution,
drained, and exposed to 0.6 ml of glass-distilled water. The
culture plates with lysed cells were frozen at â€”¿24Â°.
Acid Phosphatase. After thawing, buffer (0.4 M sodium
acetate, pH 5, 0.3 ml) and substrate (0.2 M ÃŸ-glycerophosphate, pH 5, 0.3 ml) (Fisher Scientific Company, Fairlawn, N.
J.) were added, and the plates were incubated at 37Â°for 2 hr.
The reaction was terminated by the addition of 0.4 ml of cold
20% trichloroacetic acid. After 15 min in the cold trichloroacetic acid, the solution was centrifuged, and an aliquot of the
supernatant fluid (0.5 ml) was analyzed for PÂ¡
release according
to the method of Ames and Dubin (1 ). A correction for free PÂ¡
in cells and for nonenzymic substrate hydrolysis was included.
5'-Nucleotidase.
Essentially the same procedure as adopted
for acid phosphatase was used. Buffer and substrate consisted
of 0.5 ml of 0.2 M Tris-HCI, pH 8.5, containing 20 mM MgCI2
and 20 mM AMP.
Protein Determination. Triplicate cell cultures treated and
washed identically to the method used for cultures in the
corresponding assays were dissolved in 0.1 N NaOH, and their
protein content was measured according to the method of
Lowry era/. (31).
Labeling of glycoprotein galactose residues was carried out
by borohydride reduction, before or after treatment of cells
with neuraminidase to remove sialic acid and oxidation with
galactose oxidase (Fig. 1). Only traces of labeled NaB3H4
incorporation could be detected in the absence of neuramini
dase treatment in all 3 cell lines. The glycoprotein profile on
the gel reveals that less radioactivity was incorporated into the
Table 1
Incidence of pulmonary mÃ©tastasesin C57BL/6 mice following the i.v. injection
of B16 melanoma cells3
of pulmonary mÃ©
no.612,500
lineB16-F1B16-F10B16-F10LrCell
Cell
tastases2
(1-1 0)0
3(2-13)
12(1-79)12(7-18)
25,000
50,00012.500
25,000
50,00050,000Median0
100(59-145)
170(125-204)2
(0-7)a
Ten mice/group.
Cells were harvested for i.v. injection from a semiconfluent culture with 2
mM EDTA.
c Pulmonary mÃ©tastasescounted with a dissecting microscope 18 days after
i.v. tumor cell injection.
Numbers in parentheses, range.
RESULTS
In Vivo Lung Colonization Properties
B16-F1, B16-F10,
and B16-F10Lr cell lines were grown in
vitro from frozen stocks. The lung colonization capacity of the
3 cell lines was tested several times over the period during
which the various in vitro studies were carried out. Data from
representative experiments show that the relative lung coloni
zation capacities of the 3 cell lines remained constant for 12
weeks of continuous in vitro cultivation (Table 1). In all tests,
cells from B16-F1 or B16-F10Lr formed fewer pulmonary mÃ©
tastases than did equal numbers of injected B16-F10 cells.
Moreover, cells from B16-F1 also formed extrapulmonary mÃ©
tastases in organs such as liver, lymph nodes, ovary, and
adrenal gland. At 18 days after i.v. injection, no extrapulmonary
mÃ©tastaseswere found in mice given injections of cells of B16F10orB16-F10Lr.
These findings are in agreement with data demonstrating
that the low lung colonization of B16-F1 and high lung coloni
zation capacities of B16-F10 cells were maintained in cell lines
even after 18 weeks of continuous incubation in vitro.'1 Simi
larly, cells obtained from B16-F1 or B16-F10 serially trans
planted s.c. for 12 months (35 s.c. transplants) retained their
respective low or high lung colonization capacities.5 Collec
4 G. Poste, personal communication.
5 l. Witz and M. Baniyash, personal communication.
F10
+
FlOLr
Fig. 1. i-iuorography pattern of sodium dodecyl sulfate-polyacrylamide
gel
electrophoresis of B16 melanoma variants labeled in galactose residues of cell
surface glycoproteins. +, after neuraminidase and galactose oxidase treatment;
-, galactose oxidase treatment without neuraminidase.
MAY 1980
1647
A. Paz et al.
B16-F10 major glycoprotein than into the B16-F1 and F16F10Lr major glycoprotein. The decrease in labeling is best seen
in the direct labeling of sialic acid.
with which the lectin can interact.
Enzyme Activities
Labeling of Sialic Acid
The selective labeling of cell surface sialoglycoproteins
achieved by mild periodate oxidation of the sialic acid and
reduction with [3H]borohydride is illustrated in Chart 1. Densitometric tracings of the gels show no qualitative differences in
the pattern of surface labeling among the 3 B16 melanoma cell
lines. On the other hand, the degree of labeling of the major
sialoglycoprotein
(78,000 daltons) appeared to be inversely
proportional to the degree of lung colonization in vivo, high at
B16-F10Lr and low at B16-F10. Similarly, the B16-F10Lr
78,000-dalton glycoprotein is more intensely labeled than that
of the B16-F10, following the galactose oxidase method of
labeling.
Lectin Binding
The composition
suggesting that the B16 melanoma has no exposed L-fucose
of carbohydrate-containing
receptors that
can specifically bind lectins could serve as an indicator of the
similarity of cell surface components. The binding of radioactively labeled Con A, WGA, SBA, and WBA to the surface of
B16-F, B16-F10, and B16-F10Lr is illustrated in Chart 2. The
number of Con A molecules bound to the surface of B16-F1
and B16-F10 cells is identical. In B16-F10Lr cells, 50% fewer
Con A molecules are bound. A different pattern of binding is
observed with WGA. The high-lung-colonizing
cell line B16F10 had about 40% fewer WGA-binding sites than did the 2
low metastatic cell lines B16-F1 and B16-F10Lr. Free SBAbinding sites on the cell surface were barely detectable, but a
30-min treatment with neuraminidase exposed the receptor
sites (Chart 2). Here again, the B16-F10 cells bound 2.5-fold
fewer SBA molecules than did the cells of B16-F1 or B16F10Lr. The number of WBA surface-bound molecules revealed
that the 3 cell lines had the same number of binding sites. No
binding of UEA1 could be detected with the 3 cell lines,
The 3 cell lines were analyzed for the specific activity of the
plasma membrane enzyme marker, 5'-nucleotidase,
and for
the specific activity of the lysosomal enzyme marker, acid
phosphatase. The results in Table 2 show a significant reduc
tion in the specific activity of 5'-nucleotidase in the B16-F10
cell line as compared with B16-F1 and B16-F10Lr cell lines.
The decrease in 5'-nucleotidase activity was not associated
with a similar decrease in cytoplasmic enzyme activities, since
acid phosphatase activity was similar in the 3 cell lines (Table
2).
Gangliosides
Gangliosides were extracted from lyophilized cell pellets and
identified as described previously (9, 45). The 3 melanoma cell
variants exhibited a very simple ganglioside pattern, which was
almost identical to that observed when the cells were separated
on thin-layer chromatography plates. They exhibited a very
prominent GM3 and only minor traces of GM? and GMi (not
shown).
Fluorescence Measurements
Fluorescence Polarization. The 3 cell lines were harvested
from confluent and semiconfluent conditions and were assayed
for membrane fluidity of lipid regions by means of fluorescence
polarization analysis of DPH embedded in membranes of intact
viable cells (25, 41). No differences were detected in the
degree of fluorescence polarization of the 3 DPH-labeled cell
lines, grown to the same cell densities.
Fluorescence Photobleaching Recovery. Fluorescence
photobleaching measurements (39) of diffusion of the lipid
probes DM and rhodamine-conjugated
WGA on the plasma
membrane of single cells of B16-F10 and B16-F10Lr demon
strated no differences
in either diffusion constants or mobile
i
200 150
100 70
50
30
20
FRONT
200 150
100
70
50
30
20
FRONT
i i 111 i i i i
200 150
100
70
50
i
30
20
FRONT
MOLECULAR WEIGHT ( x 10'3)
Chart 1. Densitometric tracing of a fluorogram of sodium dodecyl sulfate-polyacrylamide
were labeled in the terminal sialic acid. A, B16-F1; B, B16-F10; C, B16-F10U.
1648
gel electrophoresis
of B16 melanoma variant cell lines. Glycoproteins
CANCER
RESEARCH
VOL. 40
Surface
Properties
of Metastatic
Cells
ion
24
-x
20
0.
Ãœ 16
â€¢¿o
A
60
â€¢¿o
12
m
g 40
S
CM
20
10
20
30
40 50 60
70
125l-Con A(Mg/ml)
80
90
100
10
20
30
40
125|-WGA l^g/ml)
50
_ 12
01
b
Â«IO
Q.
.^
4
10
..
20
30
.
40
50
60
125l-WBA(Mg/ml)
70
80
100
Chart 2. Specific binding of iodinated lectins to B16-F1 (O, â€¢¿),
B16-F10 (A, A), and B16-F10Lr (O, â€¢¿)
in I25I-SBA binding curve. O, A, D, cells pretreated with
Ã¤uraminidase;â€¢¿,
A. â€¢¿
without neuraminidase
Bars, S.D.
Table 2
and acid phosphatase in B16 melanoma
DISCUSSION
cell lines
In this study, we have examined some aspects of cell surface
composition and properties of 3 B1 6 melanoma variant cell
lines with differing capacity to form experimental pulmonary
mÃ©tastasesfollowing i.v. injection. The incidence of pulmonary
metastasis produced by cells of B16-F10 line was at least 10fold that of B16-F1 or B16-F10Lr (Table 1). This difference in
Specific activities ol 5'-nuc/eotidase
CellsB16-F1
treatment. Experiments were done in quadruplicate.
activity
(nmol
protein)142.2
P,/mg
2.1a90.9
Â±
phosphatase activity
(nmol
protein)1066.6
P,/mg
B16-F10
Â±1.5
B16-F10Lr5'-Nucleotidase
176 Â±3.5Acid
1Mean Â±S.E. computed from 6 different samples.
Â±15.9
919.5 Â± 8.1
1046 Â±11.5
fractions within the 20 to 30% S.D. of the measurements. The
diffusion of the DMmolecules in the membrane plane was ~150
x 1010 sq cm/sec, while the diffusion of rhodamine-WGA was
~3 x 10'Â° sq cm/sec. These values were similar in both cell
lines.
Morphological Observation
Treatment of the cells with 2 HIM EDTA to prepare cells for
i.v. injection induced cell rounding and detachment from the
substratum. The surface of the detached cells is rough, and
the cells have a moderate number of flaps and ridges with
extended thin filopodia. The cells of the B16-F10Lr cell line
appeared to be somewhat smaller than the cells of the B16-F1
or B16-F10 lines. Thus, the differences in cell arrest in the lung
are not due to gross variations in the morphology or size among
the 3 B16 melanoma cell lines.
biological behavior in vivo was not reflected by comparable
major qualitative alterations in the cell surface characteristics
that we studied.
The process of metastasis is complicated, and the production
of visible mÃ©tastasesis dependent on the ability of malignant
cells to survive and complete a number of sequential steps.
The injection of tumor cells into the circulation bypasses the
first steps of the process, i.e., invasion and detachment. How
ever, all the subsequent steps such as transport and survival in
the circulation, arrest in the capillary bed of an organ, extra
vasation into the parenchyma, establishment of a microenvironment, escape from host defense mechanisms, and tumor
cell multiplication in the distant organ must all be completed
for clinical metastasis to occur. Failure to complete any one of
these steps will lead to the elimination of a tumor cell as a
potential progenitor of a secondary growth (42).
Reports from several laboratories have now demonstrated
that the in vivo metastatic capacity of the B16-F lines correlates
MAY 1980
1649
A. Raz et al.
with their ability to aggregate with platelets (20), a phenomenon
that could be due to differences in prostaglandin production
(1 7). The cell lines also differ in their ability to clump with
syngeneic lymphocytes (13) and endothelial cells (35, 48) and
in their response to hormones (36). Control of many of these
interactions probably resides at the tumor cell surface, and it
is not unreasonable to postulate that differences in cell surface
composition may account for the disparate behavior of the
variant cell lines. In fact, some isolated cell surface differences
between the cell lines have been described (4, 50), but no
correlation between these differences and divergent metastatic
behavior has been possible. Since no one property of tumor
cells may determine the eventual outcome of metastasis, we
attempted to survey a wide range of tumor cell surface char
acteristics and then compared the overall profile of one cell line
with the others. We wish to emphasize 2 points regarding the
experimental procedures: (a) this study represents a collabo
rative effort of 5 laboratories, in which each concentrated on
assays that they use routinely and with which they are most
familiar; (b) the cell lines were coded for all in vitro assays, and
thus the studies represent blind assays.
We have used several different techniques of labeling surface
constituents since most techniques, if used in isolation, have
some drawbacks (8). Protein composition of the cell surface
was studied by protein tyrosine iodination with lactoperoxidase
(32). Analysis of surface-labeled proteins failed to demonstrate
any appreciable difference in the surface-labeling pattern of
the 3 B16-F lines (data not shown). These results, which failed
to demonstrate any difference between B16-F1 and B16-F10
cell lines, are in agreement with an earlier report (34).
Studies of cell surface carbohydrates used 3 techniques: (a)
labeling of galactose residues; (b) surface labeling of sialic acid
residues; and (c) lectin binding. Analysis of cell surface glycoproteins by gel electrophoresis and fluorography prior to and
after neuraminidase treatment demonstrated that most of the
terminal galactose residues in the carbohydrate chains of glycoproteins are sialated, since no appreciable labeling was
detected prior to the cleavage of the sialic acid. At least 11
distinct bands of surface glycoproteins ranging from 14,000 to
200,000 daltons, with a major glycoprotein at 78,000 dallons,
could be demonstrated. Direct labeling of the sialic acid and
analysis of the labeled sialoglycoprotein by gel electrophoresis
gave a pattern similar to the one obtained with the galactose
oxidase method. Both techniques failed to demonstrate any
qualitative differences among the B16-F1, B16-F10, and B16F10Lr cell lines.
Lectin binding of the cell lines differed. B1 6-F1 and B16-F10
cells exhibited similar binding of Con A. This is in agreement
with studies reporting similar agglutination of these 2 cell lines
by Con A (34). However, the poorly metastatic cell line, B16F10Lr, had a significantly lower number of surface Con A
receptors than the 2 other lines. The B16-F10Lr cells are
resistant to in vitro lysis mediated by syngeneic lymphocytes,
which is presumably due to the deficiency of lymphocytebinding receptors (13). Whether the receptors for Con A and
cytotoxic syngeneic lymphocytes are related is unclear. The
B16-F10 cells were found to have about a 50% reduction in
WGA or SBA binding as compared with B16-F1 and B16-F10Lr
cells. SBA agglutinin, which binds to terminal galactose resi
dues, barely bound to the surface of the 3 cell lines prior to the
removal of sialic acid. This finding correlated with the results
1650
that we obtained by surface labeling with galactose oxidase.
Moreover, the fact that after neuraminidase treatment fewer
SBA-binding sites were exposed on the F10 cell membrane
than on the other 2 cell lines also agrees with the data obtained
by the galactose oxidase labeling. The latter technique dem
onstrated that surface galactose accessible for labeling was
reduced on B16-F10 cells and that B16-F10 cells also have
less surface sialic acid accessible for labeling. We did not find
that B16-F10 cells have a 2-fold increase in neuraminidaseaccessible sialic acid surface molecules (4, 50). The 3 cell
lines had the same number of WBA-binding sites, reflecting the
fact that the sugar residues, which are in proximity to the
protein core, are the same in all 3 cell lines.
Plasma membrane gangliosides are believed to be receptors
for numerous biologically active agents such as toxins and
hormones. Moreover, changes in ganglioside pattern may ac
company transformation (21). We found no qualitative differ
ences in the ganglioside composition of the 3 cell lines. The
ganglioside pattern was found to be a simple one, consisting
mainly of GM3with traces of GM2and GMi. These findings agree
with a previous report in which the ganglioside profile of B16F1 and B16-F10 cells was studied (50).
We next examined the relationship of protein dynamics to
membrane lipid fluidity. Fluorescence polarization analysis of
DPH when embedded in membrane lipids was used to monitor
the degree of microviscosity of the surface membrane of B16F10, B16-F1, and B16-F10Lr tumor cells. We detected no
differences in lipid microviscosity among cells of the 3 variant
lines. The fluorescence polarization analysis, which examines
populations of cells, is an averaging method. In contrast, fluo
rescence photobleaching recovery measures the diffusion of
the lipid probe DM and rhodamine-conjugated
WGA in the
plasma membrane of individual cells. The measurements were
performed at a given lectin concentration on the same day.
Nonetheless, no detectable differences in either diffusion con
stant or mobile fractions were observed between cells of B16F10 and B16-F10U lines.
The adenosine-generating
enzyme 5'-nucleotidase
is a
plasma membrane-bound enzyme of mammalian cells. Different
levels of this enzyme can distinguish among different subpopulations of cells both in normal and transformed systems (2,
30). The specific activity of 5'-nucleotidase in the highly met
astatic melanoma cell line (B16-F10) was decreased by about
50% as compared with the 2 low-metastatic cell lines (B16-F1
and B16-F10Lr). The observed reduction of enzyme activity
was limited to the plasma membrane, since it was not accom
panied by a similar reduction of an intracellular enzyme, acid
phosphatase. The B16 melanoma variants had a relatively low
specific activity of 5'-nucleotidase as has been observed in
other transformed cells (37).
Collectively then, the present data as well as that of others
(34, 48) are unable to demonstrate a major alteration in the
plasma membrane characteristics of B16 melanoma cells that
might be associated with their metastatic potential in vivo. The
production of a metastasis requires a cell to survive many
destructive events. It is entirely possible that what appears to
be a subtle variation in a cell surface property measured in
vitro could be responsible for a dramatic alteration in malignant
behavior in vivo. Certainly, our study illustrates some of the
problems inherent in evaluating a complex in vivo process such
as metastasis by using in vitro correlates which may measure
CANCER
RESEARCH
VOL. 40
Surface
only one cellular property (of many) requisite for the metastatic
cell. Nonetheless, it is possible that subtle variations in com
position (47) or arrangement of cell surface constituents could
indeed be responsible for the ability of tumor cells to interact
with their host, survive, and develop into mÃ©tastases.
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MAY 1980
1651
Cell Surface Properties of B16 Melanoma Variants with Differing
Metastatic Potential
A. Raz, W. L. McLellan, I. R. Hart, et al.
Cancer Res 1980;40:1645-1651.
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Cell Surface Properties of B16 Melanoma

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