A Structural Difference between the Surfaces of Normal
1
ri—'
•
1@'
ana or Larcrnomatous
•1
@piaermai Leiis
DALE REX COMAN AND THOMAS F. ANDERSON
(Department
of Pathology, and the Johnson Research Foundation, University
Philadelphia 4, Pennsylvania)
Hoffman
The behavior of the cancer cell is determined in
part by peculiarities
of its external surface. For
example, the cancer cell is unable adequately to
bind calcium (7), and, as a consequence, cancer
cells do not adhere
to one another
tightly
methods
ent
features
of cancer
may
(5, 10)
be attributed
largely to alterations of the cell surface.
It, therefore, becomes of interest to inquire
into the characteristics
of the cancer cell surface,
especially into its ultrastructure
and, if possible,
its
chemical
composition.
As
a first
step
in
this
direction, it would be pertinent to compare, on a
macromolecular
level, the appearance of the sur
face of a cancer cell with that of a normal cell. As
has been
suggested
calcium
may
recently
(6), inability
be correlated
with
Both
the cells chosen
of these
for the pres
cell surfaces.
prototype
ab
of the Vx2 cell is, therefore,
the normal
epidermal cell of the rabbit.
Normal squamous epithelial cells were obtained
from the skin. The back of the rabbit was shaved
under
and then
paste
depilated
for 30 seconds
by applying
a barium
sulfide
and
thoroughly
with
washing
Accordingly,
experiments
were planned to make
visible the ultrastructure
of the cell surface and to
water.
washed
compare in this regard a cancer cell with its
normal prototype.
Vx@2 carcinoma
cells were
sharp blade. The cells dislodged by scraping were
gathered in drops of Earle's physiologic salt solu
selected
tion lying on the skin while the scraping
for comparison
of the rabbit
@
erythrocytes.
The neoplastic squamous epithelial cells were
from the Vx@carcinoma. Pieces of the tumor were
forced through a stainless steel wire mesh into
physiologic
salt solution. The suspension was
allowed to settle for a few minutes until the super
natant contained mostly the single cells and small
clumps that were used in the experiments.
The
Vx92cancer is a malignant derivative of the Shope
rabbit papilloma, which in turn arises from a virus
infection of normal squamous epithelial cells. The
normality,
but, even aside from this possibility,
any structural
difference
from the normal might
well prove of significance to an eventual
standing of the behavior of cancer cells.
with
investigation.
shadow-cast
to bind
a surface
(9) with
failed
School of Medicine,
The method finally developed involved the
preparation and examination of carbon replicas of
but tend to separate, migrate (8), and to invade
the surrounding
tissues. Thus, the invasive or
malignant
of Pennsylrania
with
the epidermal
cells
Both normal
skin.
electron
microscopy.
However,
there are at least
three ways by which the cell surface might be
visualized.
The external
cell membrane
might be
stripped off and examined directly; or the contents
of the cell might be withdrawn, collapsing the en
veloping membranes, as was done by Hillier and
work
was
supported
by
Grant
C-731
from
for publication
5 minutes,
was done.
cells were washed
the
May 9, 1955.
after which interval
in five changes
of distilled
pension
cells
of
in
the
was
they were washed
water.
A cloudy
spread
on
clean
sus
glass
slides and allowed to dry in the air. (The slides
were cleaned in acid dichromate
solution, rinsed in
tap water
National
Cancer Institute
of the National
Institutes
of
Health, U.S. Public Health Service; Contract AT(30-1)-1684,
U.S. Atomic Energy Commission;
and by an Institutional
Grant from the Anna Fuller Fund.
Received
and cancer
five changes
of Earle's
physiologic
salt solution
and recovered
by centrifugation.
The cells were
then fixed in 1 per cent osmic acid for from
to
MATERIALS AND METHODS
Whole cells are too thick for high-resolution
* This
Twenty-four
hours
later the skin was
again and then scraped with a moderately
and distilled
oven.)
After experience
water,
and dried in an
had been gained
the cell types, mixtures
in identifying
of normal and neoplastic
cells were made before being spread on the slides,
so that they could be compared
directly
in the
same preparation.
541
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1955 American Association for Cancer Research.
Cancer Research
54@
Chromium-carbon
replicas of the air-dried
specimens were made, with equipment purchased
from the Optical Film Engineering
Company of
Philadelphia.
The slides were laid on a table in
the high vacuum apparatus where, at a pressure
of 0.15 is of mercury, they were lightly shadow
cast with chromium at an angle of 15°from the
horizontal.
To hold together the chromium cast, a film of
carbon was deposited over the specimen, accord
ing to a modification of a method developed by
Bradley
(3, 4). Three to 5 mg. of spectroscopic
carbon were evaporated
at an angle of 30°.The
source, the mean distance of which was 10 cm.
from the specimen, consisted of two carbon rods,
I inchin diameter,the endsofwhichwereheld
in contact by spring tension. The end of one rod
had been squared off, while the end of the other
had been turned down to a diameter of 1 mm. for
a length of @—4
mm., depending on the desired
thickness of carbon film. When a current of 60
amperes was passed through this assembly, the
1-mm. diameter carbon evaporated.
While the
carbon was being evaporated,
the table bearing
the specimen was rotated at a speed of 1 r/sec
by an externally mounted motor. This produced a
uniform deposit of carbon over the specimens.
Following deposition of the carbon films, the
specimens were removed from the vacuum and
placed in dishes containing 10 N sodium hydroxide;
they were covered and left overnight. The next
morning the chromium-carbon
films either were
found floating on the surface of the sodium
hydroxide or could be teased from the slides by
gentle manipulation.
Digestion of the cells was
allowed to continue for another hour while the
films floated on the alkali surface. This usually
resulted
in complete
disappearance
of the
cells,
while the carbon and chromium remained.
The chromium-carbon
film replicas thus ob
tamed were washed several times by successive
transfers
via glass slides to distilled
water
surfaces.
Finally, they were picked up on copper mesh
screens and air-dried for electron microscopy. In
some cases polystyrene particles (Dow Run No.
L.S. 040-A) with a size distribution of 880 ±80 A
( @)
wereaddedtothefinalrinsewaterto serveas
an internal index of magnification.
A Phillips
electron microscope equipped with a high-resolu
tion lens system was used to examine the speci
mens.
RESULTS
Erythrocytes (Fig. 1).—These cells occurred in
most of the preparations,
and advantage
was
taken of their presence to test the fidelity of the
replica method, since the ultrastructure
of the
erythrocyte
surface has been studied directly
by others, as noted below. The replicas of the
erythrocyte
surfaces were readily recognized by
their size and shape. Upon close inspection, the
fine surface structure appeared uniformly granu
lar, the particles or granules varying in size from
100 to 200 A. In the replica preparations the di
mensions of the particles correspond well with the
measurements
obtained
by Hillier
and
Hoffman
(9) in their direct observations
of collapsed
erythrocyte membranes.
Normal squamous epidermal cells.—These epi
thelial cells were easily distinguished, even at low
magnification,
by their
size
and
shape
and
by the
presence of hundreds of “pricides―
studding their
surfaces (Fig. 2). Some of the “prickles―
remained
erect or partially so during the preparation of the
specimens, while others collapsed; this seemed to
depend upon the thickness of the carbon films, the
thicker films preventing collapse.
A somewhat higher magnification
(Fig. 3 is
typical)
revealed
a uniformly
smooth
texture
in the
ultrastructure
of the cell surface, except for oc
casional strings of particles, approximately
150 A
in diameter, like those along the edge in the up
per right-hand
corner. A still higher magnifica
tion (Fig. 5) revealed granules measuring from
30 to 60 A in diameter. These uniform, fine par
tides were found to be characteristic
of the sur
faces of normal squamous epithelial cells. Some
times
they
were arranged
in short
chains
or small
clumps, but they were usually uniformly dis
tributed over the projecting “prickles―
as well as
over the areas between the “prickles.―
Thus, the
general appearance of the cell surface at high mag
nification was of a uniformly fine, sandy texture
throughout
due to the presence of the tiny par
tides composing it, and, other than the “prickles,―
the surfaces were generally smooth and regular.
Vx2 epiderinaid carcinoma celLi.—Differences
were apparent at once when the replicas of the
cancer cells were compared with their normal
prototypes.
At low magnification
the shapes of the cells
were seen to be more variable and their edges less
sharply defined. Numerous
nodular projections
covered the cell surfaces (Figs.
@,4). Probably
some of these represent aborted “prickles―
and
are indicative of the incomplete differentiation
of
the cancer cells. The surfaces, in general, were
made up of large and small hills and hollows. Many
of these no doubt reflect the presence of intra
cellular structures which, having different water
contents,
distort the cell's surface during air
drying.
of even greater interest, however, was the
nature of the finer ultrastructure
(Fig. 6) ; the
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1955 American Association for Cancer Research.
Fia. 1. (2-2--55.2).—€hromium-carbon
replica of an eryth.
rocyte. The granules comprising
the surface are apparent.
Measurements
indicate that these particles range from 100 to
200 A in diameter;
in general
they
but sometimes form small chains
X35,000. 100 kv.
are uniformly
or tiny
distributed
clusters.
Approx.
Fio. 2. (4-7-55 A. 2, 3).—Astereoscopic pair of micrographs
showing
a portion
squamous
epithelial
the right
lower corner.
“
prickles―
oil
the
of a chromium-carbon
cell, with
surface
replica of a normal
an overlying
The conspicuous
of
the
Vx2 cancer
cell in
projections
normal
cell
are the
which,
when
viewed with a stereoscope, are seen to he protruding
upward
toward the observer. The irregular surface contours of the
cancer
surface
cell and its lack of “prickles―contrast
structure
±6.5°.Approx.
FIG.
thelial
3.
(4-9-55
of its
normal
prototype.
sharply
with the
Stereoscopic
angle,
X 12,000. 60 kv.
E.
2)—Edge
cell. In this preparation
of a normal
squamous
a very thin carbon
epi
film was
used, and the “prickles―
are collapsed. The Dow latex par
tides on the background
average 880 A in diameter.
The
smooth texture of the ultrastructure
is apparent even at this
magnification.
X42,000. 60 kv.
Fia. 4. (4-9-55 B. 6).—Edge of a Vx@ carcinoma cell. The
irregularity
of the surface contours is quite evident and ap
pears in sharp contrast to the smooth texture of the normal
cell above. “Prickles―
are absent, with the possible exception of
the tongue-like
nodule to the right of center, which may
represent an aborted form.
Both of the cells shown in this plate were from a Inixed
preparation of normal and neoplastic cells and occurred on the
same specimen screen. X42,000. 60 kv.
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Fin. .5. (4-9-5.5 E. @2).—Normal S@UaflIOUSepithelial (-eli.
Au enlargement from an area of Figure 3, to sho@vmore clearly
the characteristics
of its ultrastructure.
The general impres
sioll
is of a flue
sandy
texture
resulting
from
the
myriads
of
tiny granules of quite uniform size, ranging in diameter be
tweeii 30 and 60 A. In some areas these particles are arranged
in more or less parallel rows, as in the central part of the upper
most tongue-like
process. Fine cracks can he seen in the
chromium where it has been deposited most heavily. X 170,000.
60 kv.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1955 American Association for Cancer Research.
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Fm. 6. (4-9-55 B. 6).—Vx@ carcinoma cell. An ealarge
ment from an area of Figure 4. The typical irregularities in the
surface ultrastructure
are shown. Tortuous valleys wind be
tweeii large and small peaks. The fine particles vary greatly
in diameter,
from
30 to 300 A or more.
In some
areas,
as on
the rounded elevation in right center, smaller particles pre
dominate, whereas in the lower left and central areas much
coarser
granules
are
is a wide scatter
of the granules,
111 the
cancer
right central
cell,
conspicuous.
Over
most
in particle size. Occasionally,
as described for the normal
as in the
upper
part
part of the photograph.
of the
of the
cell
there
parallel beading
cell, is seen also
elevation
in the
X 170,000. 60 kv.
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Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1955 American Association for Cancer Research.
COMAN
AND ANDERSON—Surfaces
of Normal
particles
composing
it were characterized
by
great diversity rather than uniformity
in size,
giving an appearance reminiscent of mixed coarse
and fine gravel and sand. The larger knobs and
protuberances
were covered with these smaller
granules or particles, as were the areas between
them. The particles presented a range in diameter
from about 30 to 300 A, although some of the
largest particles may be aggregates of smaller
ones.
The smallest particle size observed was the
same
in both
normal
and
cancerous
corresponding
to the degree
tamed. In the normal cell the
fairly uniform, whereas in the
was a tremendous variation in
cells,
no doubt
of resolution
at
particle size was
cancer cell there
particle size.
DISCUSSION
The foregoing observations indicate that when
a normal epidermal cell becomes carcinomatous
an alteration occurs in the ultrastructure
of its
surface. The significance of this difference in
surface structure to the neoplastic state of the
cells remains unknown.
Whether or not the difference in the surface
structure of the cancer cell bears any relevance to
its inability
further
groups
to bind
study.
which
calcium
The
is also a matter
carboxyl
presumably
bind
and
for
calcium
are
of the
two
cell
types
under
identical
con
543
CelL!@
SUMMARY
Methods have been devised for making high
resolution chromium-carbon
replicas of cell sur
faces.
The ultrastructures
of the external surfaces of
normal epidermal
cells and Vx@ epidermoid
cancer cells from the rabbjt have been observed
by making such replicas, which were examined
and photographed with an electron microscope.
Normal squamous epithelial cells revealed a
uniform ultrastructure
consisting mainly of par
tides ranging from 30 to 60 A in diameter.
The cancerous squamous epidermal cells pre
sented a strikingly apparent difference, in that
their surfaces were composed of particles ranging
from 80 to 300 A in diameter, in sharp contrast
to the uniformity of particle size of the normal
cell surface.
Transformation
of a normal rabbit epidermal
cell to a Vx@ carcinoma is accompanied
by an
alteration of the ultrastructure
of the cell surface.
REFERENCES
far
ditions demonstrated
differences in surface struc
ture. In other studies it might be better to avoid
the effects of drying by using the critical point
method of dehydration
(1). This was done with
some of our preparations, but then the chromium
Carcinomatous
carbon films almost entirely surrounded the un
collapsed cells, leaving only a tiny uncoated
aperture at the point of cell-glass contact, through
which it was difficult to dissolve the cell complete
ly with the alkali.
phosphate
below current limits of resolution
in electron
microscopy, but if the differences observed are
related to differences in calcium binding, it may
be possible to induce such structural changes in
normal cells by removing calcium from their sur
faces. Chelating agents, for example, by with
drawing the calcium from the surface may pro
duce abnormalities that might help in the eventual
explanation of the altered surfaces of Vx@?2
cancer
cells. The effect of carcinogens in this regard must
also be investigated.
Obviously more and different kinds of cancer
cells should be compared with their normal pro
totypes to determine whether or not the surface
differences are indeed characteristic
of neoplastic
cells in general.
The development
of methods for the study of
cell surfaces upon a macromolecular
level should
be of value in many fields of cytobiology. It should
be stressed that, in the investigation
reported,
air-dried cells were used. Even so, the direct corn
parison
and
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Three-Dimensional
Structure in Preparing Specimens for
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Carbon Replica Techniques for Use
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I. The Sig
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Neoplastic Tissue. Cancer, 3:718—21, 1950.
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H. T., and Co@,
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Motility of Human and Animal Neoplastic Cells. Cancer,
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9. HiLisun, J., and Hosvs@,
J. F. On the Ultrastructure of
the Plasma Membrane as Determined
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10. MCCUTCREON, M.; Coaw@, D. R.; and Moons, F. B.
Studies on Invasiveness
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Adhesiveness
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A Structural Difference between the Surfaces of Normal and of
Carcinomatous Epidermal Cells
Dale Rex Coman and Thomas F. Anderson
Cancer Res 1955;15:541-543.
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