An Electron Microscope Study of Sarcoma I in a Homologous
Host. I. The Cells of the Growing Tumor*
0
VELMAC. CHAMBERSAND RUSSELL,S. WEISER
(Department of Microbiology, University of Washington, School of Medicine, Seattle, Washington)
SUMMARY
The fine structure of Sarcoma I cells from growing ascites tumors of C57BL/6K
mice (homologous strain) was compared with the fine structure of cells from the ascites
tumors of A/Jax mice (isologous strain). No differences were observed in the tumor
cells grown in either strain of mouse. The following ultrastructural features were
demonstrated: numerous free ribosomes generally occurring in clusters; a relatively
small amount of rough-surfaced endoplasmic reticulum; a fine filamentous component
of the cytoplasm; large, spherical lipide bodies; occasional annulate lamellae; and oc
casional doughnut-shaped, virus-like bodies within cisternae of endoplasmic reticulum.
Similar ultrastructural features have been described by other workers in studies of
various kinds of tumor cells.
Sarcoma I is a transplantable tumor that originated in
1947 in a mouse of the A/Jax strain after treatment with
dibenzanthracene.
The ascites form of the tumor grows
in the A/Jax mouse, producing death on the 12th to the
14th day. After transplantation to the peritoneal cavity
of the homologous C57BL/6K mouse the sarcoma cells
proliferate for about 6 days, at which time an immune
response begins and the tumor is rejected completely
between the 10th and the 14th day.
The cytology of Sarcoma I cells obtained from the
peritoneal cavity of the A/Jax and the C57BL/6K mouse
was described by Baker, Weiser, Jutila, Evans, and Blandau (1). Their report contributed valuable information
concerning the cytological features of the tumor cells
revealed by light microscopy, phase-contrast microscopy,
and phase-contrast cinematography.
In the present study the ultrastructural features of the
Sarcoma I cell during growth of the tumor in the homol
ogous C57BL/6K mouse are described. These features
are compared with those of the sarcoma cell from the
susceptible A/Jax mouse.
injected intraperitoneally into C57BL/6K mice for the
studies in the homologous host.
Electron microscopy.—Cells were removed from the
peritoneal cavity by means of a 25-gauge needle attached
to a tuberculin syringe. The needle was removed from
the syringe, and the cells were transferred immediately
to cold osmium tetroxide buffered with s-collidine. The
concentration of osmium was 2.67 per cent and that of
s-collidine was 0.067 M before the addition of cells. Fixa
tion time was between 40 minutes and 1 hour. The cells
were rinsed briefly in distilled water or in 0.067 M s-colli
dine buffer, dehydrated in alcohol and in propylene oxide,
and embedded in epoxy resin (14).
Sections were cut with a glass knife or with a Dupont
diamond knife mounted on an LKB Ultratome or on a
Porter-Blum microtome. Thin sections were stained
with lead hydroxide (16) or with 2 per cent uranyl acetate
and were viewed with an RCA EMU-2C, -3E, or -3G
electron microscope.
RESULTS
The ascitic fluid removed from C57BL/6K mice 6 days
MATERIALS AND METHODS
after transplantation of Sarcoma I intraperitoneally con
Sarcoma I cells.—SarcomaI cells were obtained in 1958 tained numerous sarcoma cells and a few host macro
phages. Although the presence of host macrophages
from the Roscoe B. Jackson Memorial Laboratory, Bar marked the onset of rejection, the majority of tumor cells
Harbor, Maine. The tumor has been maintained in our at this time appeared unchanged. The number of host
laboratory by weekly serial transfer in the peritoneal cells increased during the 7th and 8th days, and by the
cavity of A/Jax mice, the strain of origin. Sarcoma cells 9th day the cells were predominantly macrophages. Tu
were removed from the peritoneal cavity of an A/Jax
mor rejection was complete by the 10th to the 14th day.
mouse on the 7th or 8th day of tumor growth and were
The cellular component of the ascitic fluid from tumorbearing
A/Jax mice was almost entirely sarcoma cells
* Supported in part by United States Public Health Service
throughout the course of the tumor, which terminated in
Grant CA-05698.
death of the mice. A comparison of sarcoma cells from
Received for publication December 16, 1963.
693
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694
Cancer Research
a C57BL/6K mouse on the 6th day and an A/Jax mouse
on the 8th day is seen in low magnification electron micro
graphs in Figures 1 and 2, respectively.
At higher magnification, the fine structure of Sarcoma
I cells from the C57BL/6K mice during growth of the
tumor (Fig. 3) was similar to that of sarcoma cells from
the A/Jax mice (Fig. 4). The cells were usually spherical,
with microvilli of varying shape, length, and number pro
truding from the cell surface. Small vesicles often oc
curred within the microvilli or at their bases (Fig. 5).
In addition to the outfolding of the cell surface to form
microvilli, small invaginations of the cell surface were seen
(Fig. 5). It is not possible to distinguish between in
vaginations that represent an intermediate step in the
formation of pinocytotic vesicles for the uptake of mate
rial into the cell and those that are formed as the result
of vesicles discharging their contents into the extracellular
medium.
One of the outstanding features of the Sarcoma I cells
was the abundance of ribosomes in the cytoplasm. Many
of these ribonucleoprotein particles occurred free and were
usually arranged in small clusters (Fig. 6). Others were
attached to membranes of the endoplasmic reticulum.
Although the section presented in Figure 6 contains con
siderable endoplasmic reticulum, this cytoplasmic com
ponent was often rather sparse (Fig. 3).
The Golgi complex was prominent in some sections
(Figs. 6-8). It consisted of parallel cisternae bound by
smooth membranes and associated with numerous, small,
smooth-surfaced vesicles.
The mitochondria were seen in spherical, oval, and long
profiles with cristae extending from one side to the other,
from end to end, or diagonally (Fig. 6).
Vol. 24, May 1964
The Sarcoma I cell often contained large, spherical
bodies which, in electron micrographs, were characterized
by a homogeneous appearance and low electron density
(Fig. 6). They were frequently bound by a narrow margin
of increased density which appeared beaded or granular.
In l-p sections of epoxy-embedded cells stained with
Azure II and méthylène
blue (20) these bodies were a
light green color, which is the characteristic reaction for
lipide-containing structures. In smears of whole cells
fixed with formalin or osmium vapor and stained with
Oil Red 0. they were bright red. The fine structure of
these bodies, together with their staining properties, es
tablished that they were lipide. They were undoubtedly
identical to the "refractile bodies or spherules" described
by Baker et al. in earlier light microscope studies (1).
Lipide bodies were observed in association with mito
chondria, smooth-surfaced vesicles, endoplasmic reticu
lum, and ribosomes (Fig. 6). An electron micrograph
presented in an earlier publication (6) showed a lipide
body in contact with the membranes of annulate lamellae.
Annulate lamellae were observed in a number of sarcoma
cells from both A/Jax and C57BL/6K mouse tumors
(Fig. 9). They occurred in the region of the Golgi com
plex more frequently than in other positions in the cell
as described in an earlier publication (6).
Bundles of fine filaments were observed frequently at
high magnification (Figs. 7, 8). They were seen in the
cytoplasm near the nuclear membrane, in the region of the
Golgi complex, associated with mitochondria, and among
ribosomes and profiles of rough-surfaced endoplasmic
reticulum.
FIGS. 1-11.—Abbreviations:
= annulus of nuclear enveM = mitochondria
lope
Mv = microvilli
An = annulate lamellae
N = nucleus
ER = endoplasmic reticulum
Nu = nucleolus
F = cytoplasmic filaments
P = pore
G = Golgi complex
R = ribosomes
I
= invagination
V = vesicles
K = membranous profile in nu- VP = virus-like particles
cleus
L = lipide body
FIG. 1.—Electron micrograph of a thin section of Sarcoma I
cells from a 6-day tumor in a C57BL/6K mouse. X 3000.
FIG. 2.-—Electron micrograph of sectioned Sarcoma I cells
from an 8-day tumor in an A/Jax mouse. X 3200.
A
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FIG. 3.—Sarcoma I cell from a 6-day tumor in a C57BL/6K
mouse. Microvilli (Mr) protrude from the cell surface. The
cytoplasm contains an abundance of free ribosomes (/Õ)and a small
amount of endoplasmic reticulum (Eli). Note the membranous
profile (A') in the nucleus (A7). X 12,000.
690
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FIG. 4.—SarcomaI cell from an 8-day tumor in an A/Jax mouse.
Note
the present
similarity
cell in (A').
Fig. 3.Nuclear
Two membranous
profiles
(K) are
in to
thethe
nucleus
pores (P) occur
at
frequent intervals along the nuclear envelope. X 11,000.
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Fio. 5.—Sarcoma I cell from a 6-day tumor in a C57BL/GK
mouse. Microvilli (Mv) protrude from the cell surface. Smoothsurfaced vesicles (V) are seen within the microvilli and at their
base. Invaginations
of the plasma membrane are also present
(/). Ribosomes (R) are numerous.
X 35,(KM).
FIG. 6.—Aportion of a healthy-appearing
Sarcoma I cell from
an 8-day tumor in a C57BL/(>K mouse. Lipide bodies (Lt , L2 ,
and L3) are closely associated with mitochondria.
All four lipide
bodies have several small vesicles in contact with their surface.
Lipide body (L¡)is in close proximity to ER. X 23,000.
700
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CHAMBERSANDWEISER—SarcomaI in Homologous Host
Small, dense, doughnut-shaped bodies were occasionally
seen in electron micrographs of Sarcoma I cells from both
A/Jax and C57BL/6K mice (Fig. 10). These bodies were
approximately 90 m/u in diameter and usually occurred
within the cisternae of rough-surfaced endoplasmic reticulum. They resembled the type A virus-like bodies
described by Dalton and co-workers (7, 8) and others
(10, 27).
The nucleus was usually spherical and was frequently
located in a slightly eccentric position in the cell (Figs. 1,
2). The nucleoplasm was granular and often contained a
membranous profile which probably represents the in
vagination of the nuclear envelope. A suggestion of this
is seen in Figure 8. The nuclear envelope was composed
of the usual two membranes, interrupted at irregular inter
vals by pores (Figs. 4 and 10). A section showing the
characteristics of the pore wall or annulus described by
Wischnitzer (28) is shown in Figure 11. A layer of granu
lar material was observed along the inner surface of the
nuclear membrane (Fig. 10). The granules were approxi
mately the same size as the cytoplasmic ribosomes, but
appeared less dense. This layer gave the impression of a
very thick membrane in low-magnification electron micro
graphs and in photomicrographs of stained sections.
Sections through nuceloli showed irregular masses of
coarse, granular material of greater density than the sur
rounding nucleoplasm (Figs. 1, 2).
DISCUSSION
The electron microscope study of Sarcoma I cells during
the growth of the tumor in isologous and homologous hosts
shows certain aspects of fine structure which supplement
the observations made with the light microscope (1). In
comparing the cells from the two strains of mice during
tumor growth, no significant differences in fine structure
were noted.
Microvilli varying in size and number were commonly
found distributed irregularly over the surface of the cells.
These have been described in MCiM cells (2, 3), in Ehrlich
ascites cells (27), Walker tumor (15), and in normal cells
that have grown in peritoneal fluid or in tissue culture (23).
The small vesicles seen at the base or within the microvilli
are apparently related to the uptake of material by pinocytosis or to the release of substances from the cell. Micro
villi increase in number and in length during incubation
in vitro, as described by Baker et al. (1).
The numerous ribosomes in Sarcoma I cells caused high
electron density of the cytoplasm in electron micrographs
and a strong basophilia in smears and thick sections stained
with basic dyes. Many of the ribonucleoprotein particles
occurred free in the cytoplasm of Sarcoma I cells and were
usually arranged in clusters. Others were attached to
membranes of endoplasmic reticulum which was present in
relatively small amounts. Abundant free ribosomes and
poorly developed systems of endoplasmic reticulum are
common features of many kinds of tumor cells (2, 4, 7, 11,
13, 17, 18, 27). Howatson and Ham (11) suggested that
these features may be related to cell growth, whereas or
ganized endoplasmic reticulum may be related to special
707
ized cellular activities of differentiated cells. Birbeck and
Mercer (5) pointed out that free ribosomes are generally
associated with protein synthesis in cells that retain their
protein, and an abundance of ribosome-studded mem
branes is associated with protein synthesis in secretory
cells. The occurrence of numerous free ribosomes in Sar
coma I cells is probably related to the rapid rate of growth
and reproduction of these cells.
Lipide bodies were observed frequently in Sarcoma I
cells. They increased in number, not only in the sarcoma
cells but also in the host peritoneal cells, as the disease
progressed in the A/Jax mouse and during tumor rejection
in the C57BL/6K mouse.
The accumulation of lipide deposits in cells is a wide
spread phenomenon. Among the cells in which large
amounts of lipide have been described are cells grown in
tissue culture (23), pancreas cells of starved guinea pigs
(19), liver cells of rats during carbon tetrachloride intoxi
cation (25), fibroblasts of skin wounds of scorbutic guinea
pigs (22), Sarcoma 37 cells (9), and MCiM cells (2, 3, 12).
Palade (19) described a close association of lipide bodies
with mitochondria in the cells of the pancreas of starved
guinea pigs. He speculated that the lipide bodies repre
sent fat that has been mobilized from adipose tissue and
that their association with mitochondria was for the
purpose of fatty acid oxidation by the oxidative enzymes of
the mitochondria. Smuckler et al. (25) attributed the
accumulations of lipide in the liver cells of carbon tetrachloride-intoxicated rats to the lack of protein with which
triglycéridemay be coupled for transport from the cell.
This theory was also invoked by Ross and Benditi (22)
as the most plausible explanation for the development of
lipide bodies in fibroblasts of healing wounds of scorbutic
guinea pigs. Whether the development of lipide bodies
in Sarcoma I cells growing in either the susceptible A/Jax
mouse or the resistant C57BL/6K mouse is due to one of
the above alterations in metabolism or to some other cause
is not yet known.
The fine filaments in the cytoplasm of the Sarcoma I
cells resemble those seen in MCiM sarcoma cells (2, 3, 12),
in Sarcoma 37 cells (9), in fibroblasts (21), and the tonofilaments of epithelial cells (24, 26). The electron density
of the cytoplasmic matrix of Sarcoma I cells was partially
due to this fine filamentous component.
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An Electron Microscope Study of Sarcoma I in a Homologous
Host. I. The Cells of the Growing Tumor
Velma C. Chambers and Russell S. Weiser
Cancer Res 1964;24:693-708.
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