(CANCER
RESEARCH
28, 2115-2125, OctOber 1968]
Ultrastructure
of Human'
Robert A. Erlandson, Bernard Tandler,2 Philip H. Lieberman, and Norman 1.Higinbotham
Division of Cytology, Sloan-Kettering Institute for Cancer Research, and Department of Pathology and the Bone Service of the De
partment of Surgery, Memorial Hospital for Cancer and Allied Diseases, New York, New York 10021
published
SUMMARY
(5, 15, 29) ; each deals with a single case. The present
article describes the fine structure
Three sacrococcygeal chordomas, two of which were recur
rent, were examined by electron microscopy. The three tumors,
which were quite similar, consisted of cords, lobules, and clusters
of cells embedded in a mucinous matrix. The so-called stellate
and physaliferous cells represented the extremes of a develop
mental sequence of tumor cells and were separated by a spec
trum of morphologically
intermediate
cells. The physaliferous
cells appeared to arise from the stellate cells by a process of
progressive cytoplasmic
vacuolization.
Some of the resulting
vacuoles were so large that the nucleus and the cytoplasmic or
ganelles were displaced to the cell periphery. Many vacuoles
contained deposits of glycogen.
The most striking feature of the tumor cells was the consist
of three sacrococcygeal
chordomas, with special emphasis on a unique complex of cyto
plasmic organelles which was observed in each case.
MATERIALS
AND METHODS
Specimens of sacrococcygeal chordomas were obtained from
three patients by aspiration biopsy. Their case histories are
briefly summarized below:
Case 1. The patient was a 78-year-old
white Jewish male
who in 1950 underwent partial sacrectomy because of chordoma.
He remained
symptom-free
until
necessitated cobalt-60 therapy
tissue specimen
was obtained
1965, when recurrent
tumor
(6300 made, tumor dose) . The
late in 1966 about
three
hours
ent association of mitochondria and endoplasmic reticulum in a
structurally ordered complex. In such complexes, single mito
after a single treatment of 300 mads.
chondria alternated with single cistemnae, each organelle being
separated from its neighbor by an interval of constant width.
The central portions of the mitochondria were so flattened that
the limiting membranes of opposite sides almost met ; at their
ends, the mitochondria
were bulbous and possessed a few
angulated cristae. Beyond the lateral boundaries of the complex,
the cisternae abruptly became smooth surfaced. The function
of these unusual complexes is unknown.
who had undergone a partial sacrectomy for chordoma after
INThODUCTION
Chordoma, a tumor which usually appears in middle or late
adult life, arises from remnants of the embryonic notochord
(20, 27) . The majority of these tumors occur at sites corre
sponding to the ends of the notochord, 45 to 56% of reported
cases occurring in the sacrococcygeal region (25, 32) . While of
low malignancy, chordomas have a tendency to be locally in
vasive and destructive. A recent report by Higinbotham et at.
(19) has shown that following surgical removal, local recur
rences and metastases commonly occur.
Although
the light microscopic
characteristics
of human
chordomas have been described in numerous articles (6, 10, 12,
14, 17, 31 ) , few studies of their ultrastructural
organization
have appeared. To date, only three such works have been
Case 2. The patient was a 60-year-old
white Jewish female
six months of rectal pain. The present specimen was from a
recurrent lesion removed in 1966. No radiotherapy
had been
given.
Case 3. The patient was a 65-year-old
white Jewish male
who in 1966 underwent partial sacrectomy because of the
presence of a chordoma. The specimen was obtained without
antecedent radiotherapy.
For electron microscopy, small blocks of tissue were fixed
for one hour in a mixture of 2% acrolein and 6% glutaraldehyde
(28) and postfixed for one hour in 2% osmium tetroxide (24)
with added sucrose (7) . Both fixing solutions were buffered
with veronal acetate. After dehydration,
the specimens were
embedded in Maraglas-Dow
Epoxy Resin 732 (11) . Thin see
tions were doubly stained with methanolic uranyl acetate (30)
and with lead citrate (26) , and were examined in a Siemens
Elmiskop I electron microscope.
Specimens for routine light microscopy were fixed in phos
phate-buffered
formalin, embedded in paraffin, and stained with
hematoxylin
and eosin. Half-micron
thick sections of epoxy
embedded tissue were stained with toluidine blue using the
method of Björkman (2) . Photomicrographs
were taken with
a Zeiss standard WL microscope.
OBSERVATIONS
1 This
investigation
from the National
2 Present
address
was
supported
Cancer Institute,
: School
in part
by
Grant
of Dentistry,
Case
Western
University, Cleveland, Ohio 44106.
Received December 20, 1967; accepted June 13, 1968.
OCTOBER
CA-08748
NIH.
Reserve
Light
Microscopy
In hematoxylin and eosin-stained paraffin sections, the tumors
appeared to consist of two distinct cell types. The larger cell
type contained prominent vacuoles, and it is because of this
1968
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2115
Robert A. Erlandson, Bernard Tandler, Philip H. Lieberman, and Norman L. Higinbot ham
feature that they are called “physaliferouscells.―The smaller,
The most striking feature of all the tumor cells was the con
spindly cells, which appeared to be nonvacuolated,
are usually
referred to as “stellatecells.―Both cell types, intermixed, were
arranged in cords, lobules, and clusters (Figs. 1, 2) . The rela
tive proportion of the two cell types varied from tumor to tu
mom and also in different sites within each specimen. The cell
sistent association of mitochondria
and endoplasmic reticulum
in a structurally ordered complex (Fig. 11) . In such a complex,
single mitochondria
alternated
with single ergastoplasmic cis
ternae, each organelle being separated from its neighbor by an
interval of approximately
500 A. As many as 10 layers of mito
groupings were separated by a homogenous, mucinous matrix,
chondria and 10 cistemnae were observed in some arrays. The
and in one specimen they were surrounded by fibrous connec
tive tissue as well as matrix.
Much greater detail could be discerned in the thinner, tolu
mitochondmia
idine blue-stained epoxy sections (Figs. 3—5)
. Not only physalif
usually
were long (about
2.3 @z)and dumbbell
shaped. In their central portions, they were so attenuated that
membranes of opposite sides almost met. At such points the
total thickness of the mitochondria measured as little as 340
A. The bulbous ends of the mitochondria were 10 times as
erous and stellate cells were apparent,
but a spectrum of
morphologically intermediate cells as well. While the latter cells
thick, measuring about 3400 A. These end portions frequently
resembled stellate cells in form, they also possessed small cyto
contained a few angulated cristae. The cistemnae were of the
plasmic vacuoles. Many of the physaliferous
cells contained
rough-surfaced variety (RER), but at the lateral boundaries
such large vacuoles that they assumed a typical “signetring― of the complex, which can be defined as the point where the
mitochondria end, the membranes abruptly became smooth
shape (Fig. 3) . Some of the larger vacuoles contained a floe
culent material (Figs. 4, 5).
(Figs. 13, 14) . In many cells, especially the physaliferous cells,
the smooth endoplasmic reticulum (SER) ramified throughout
The nuclei of the tumor cells frequently were highly irregular
the cytoplasm (Figs. 7, 13).
in size and contour. Many were multilobulated
or had deep
clefts. Occasional cells appeared to be binucleate. Intranuclear
Solitary mitochondria, as well as solitary elements of RER,
inclusions were present in many of the tumor cells, irrespective
were infrequently
seen. In contrast, a single distended cistern
of cell type (Fig. 5) . These inclusions consisted of either a
was frequently
observed with several mitochondria
in close
dense spherical structure delimited by a light halo or a large
relation (Fig. 12) . In such cistemnae, ribosomes were present
mass of intermediate
density surrounded by a very dense rim.
primarily where mitochondria
were apposed to the cistemnal
The matrix, which appeared to be structureless, was stained
membrane ; at other sites, the membranes usually were smooth.
pink by the metachromatic
toluidine blue, a tinctorial response
Golgi complexes were present in many of the tumor cells,
which was not shared by any other component of the tumors.
being most abundant
in the physaliferous
cells, where they
were usually situated near to or contiguous with the cytoplasmic
Electron Microscopy
vacuoles (Fig. 15) . The Golgi complexes consisted of closely
Specimens from each of the three tumors studied were very
similar at the ultrastructural
in composite. The electron
level and are therefore described
microscope observations confirmed
that stellate and physaliferous cells are not disparate cell types,
but are related
by a continuum
of intermediate
cells. Never
theless, for the sake of convenience, this terminology is retained
in this report.
The aggregations
magnifications
of tumor cells were easily recognized
at low
(Fig. 6) . The cells were irregular in outline,
frequently
being locked together by interdigitating
cellular
processes (Fig. 6, 7) . These processes often extended deep into
adjacent cells. Desmosomes occasionally were present at such
sites.
The nuclei of the tumor cells also were extremely irregular
in form. Deep incursions of cytoplasm gave many of the nuclei
a multilobulated
appearance
(Figs. 8—10). These cytoplasmic
intrusions, when viewed in cross-section, appeared as intra
vesicles
were numerous,
characterized
density
with no adjacent
empty
rare.
2116
appearance
condensed chromatin
inclu
and usually had an
(Fig. 10) . Such inclusions
were relatively
instead
by the presence
than the vacuolar
and
by a single membrane
Small
were
of bundles
of tonofila
contents,
exclusive
of the glycogen.
At higher magnifications fine fibrils and granular material could
be observed scattered throughout the matrix.
velope, hence were delimited by two membranes.
(b) They
frequently
were encompassed
by condensed chromatin.
(c)
Nearly all contained cytoplasmic
components .such as mito
hand, were bound
that
ments.
The extracellular matrix, at lower magnifications, appeared
structureless, but it exhibited considerably greater electron
DISCUSSION
chondria and endoplasmic reticulum. True intranuclear
and some were observed
either budding or fusing with the Golgi cistemnae (Fig. 16).
Similar vesicles were sometimes joined to the limiting membrane
of the vacuoles.
Many of the cytoplasmic vacuoles, large and small, con
tamed glycogen (Figs. 6, 11) . Clusters of glycogen also were
abundant in both intermediate and physaliferous cells in those
cytoplasmic areas which were rich in SER (Fig. 7) . Stellate
cells contained little glycogen or SER, their cytoplasm being
nuclear inclusions (Figs. 8, 9) . Such pseudoinclusions could be
readily distinguished from true intranucleam inclusions for the
following reasons : (a) They were bounded by the nuclear en
sions, on the other
@
packed, smooth cistemnae showing many fenestrations.
The mitochondrial-endoplasmic reticulum (MER) complexes
described in the prescnt study constitute the most prominent
striking
ultrastructumal
feature
of the
cells of human
chordoma. These structures, which consist of alternating layers
of RER and attenuated mitochondria, have not been described
previously in any vertebrate cell type. A similar-appearing
complex was illustrated, but not discussed, in a report on the
fine structure of the corpus allatum of the moth of the silk
worm, Bombyx
mon
(16).
CANCER
RESEARCH
VOL.28
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Chordoma
While among vertebrates the MER complexes appear to ‘be
limited to chordoma cells, several examples of MER arrays,
albeit of a somewhat different configuration, have been recog
nized in other cells. These occurred in the hepatic cell.@of rats
subjected to several different experimental procedures, includ
ing prolonged starvation
(22), copper intoxication
(1), and
partial hepatectomy (8) . Like the MER complexes of chordoma,
the hepatic arrays consist of elements of endoplasmic reticulum
alternating with elongated mitochondria. Although they are in
direct continuity with RER beyond the limits of the mito
chondrial stack, the cistemnal elements in the array are of the
smooth-surfaced
variety, the exact reverse of the situation in
chordoma. Furthermore,
instead of being separated from the
mitochondria
by an interval
of fairly constant
width, the
smooth membranes are closely applied to the outer surface of
both sides of the mitochondria.
Since the mitochondria
also
possess longitudinally
oriented cristae, the result is a densely
packed stack of membranes in which the precise identity of
individual membranes is not readily discernible.
At a simpler morphologic level, single cistemnae of endo
plasmic reticulum frequently have been observed in association
with single mitochondria
(13, 18). Such cistemnae parallel the
outer membrane of the mitochondria
but are separated from
the mitochondria by a gap equal to the intercisternal
distance
observed in parallel arrangements of RER, such as those found
in exocrine cells. Fawcett (13) has suggested that in normal
cells, closely
related
mitochondria
and RER
cisternae
may
signify a transient juxtaposition of the mitochondrion to a local
site of energy utilization. However, the paucity of solitary
mitochondria and solitary cistemnae in chordoma cells strongly
indicates that the MER complexes are not transient structures.
The
functional
significance
of the MER
complexes
is not
clear. It is obvious that stratification of the organel!es brings
the maximum
surface area of RER membranes
into juxtaposi
Ultrastructure
demonstrated
by the great difference in electron density be
tween the two sites. In addition, the vacuoles remain unstained
in 0.5-@&-thicksections stained with toluidine blue, while the
matrix is stained an obvious pink.
Previous studies with the light microscope demonstrated the
presence of glycogen in chordoma cell vacuoles by staining with
Best's carmine (6, 9) or by the periodic acid-Schiff technic with
diastase controls (9, 12) . These observations were confirmed
by the present study. In the electron microscope, the vacuolar
glycogen is momphologically similar to the glycogen in tie cyto
plasmic ground substance.
The latter glycogen is in close topo
graphic relation to the abundant
SER, but, as in liver cells
(21), it is lodged in the interstices of the tubular reticulum
and is not actually enclosed by membranes. In other cell types,
glycogen occasionally has been observed engulfed by autophagic
vacuoles (23) . However, the presence of a single unit membrane
delimiting the chordoma cell vacuoles shows that these struc
tures are not of the autophagic variety. If, in chordoma cells,
the two classes of glycogen—vacuolar
and cytoplasmic—are
identical, then the mode of entry of the cytoplasmic glycogen
into the vacuoles is unknown.
In addition to cytoplasmic vacuoles, intranuclear
also are present
in chordoma
vacuoles
cells. In the case described
by
Friedmann et at. (15), some nuclei contained “patchy―
in
clusions of unknown nature. We have also observed several
of these structures,
and they apparently
correspond to the
class of nuclear inclusions termed “sphaeridions―by Büttner
and Horstmann
(4), which are widely distributed
in many
species of normal and pathologic cells (3). In the chordoma
studied by Spjut and Luse (29), a different type of intra
nuclear inclusion was present ; this was described as an area
of cytoplasm trapped within the nucleus. In our material sun
ilar configurations were common. It was clearly evident, how
propinquity. It is possible, though no direct proof exists, that
in chordoma cells a high concentration of ATP is necessary to
ever, that such inclusions were in direct continuity with the
cytoplasm and that they in fact represented cross-sections of
villiform cytoplasmic extensions. In addition to these pseudo
inclusions, several examples of true intranuclear inclusions were
observed in the present study. These usually appeared
as
keep ribosomes
watery vacuoles bounded by a single membrane. Like the cyto
tion with mitochondria. Fig. 12 demonstrates that the presence
of attached
ribosomes
attached
is directly
related
to the membranes
to mitochondrial
of the endoplasmic
reticulum.
Whatever
their functional
status,
the MER
complexes
con
stitute convenient morphologic markers. It is highly improb
able that so unusual a structure could arise independently in
unrelated cell types. Their presence in almost every cell in the
three
cases studied
is compelling
evidence
that
the different
cell types are directly related and that they probably represent
a developmental sequence.
The progressive increase in the degree of vacuolization also
supports the concept of a single cell type in chordoma. Small
vacuoles, which are present in the stellate cells, increase in
dimension in the intermediate cells and may attain enormous
size in the physalifemous cells. There is no doubt, as Cancilla
et at. (5) have observed, that many of the larger so-called
vacuoles apparent in routine light microscopic preparations
are
in reality large extracellular
spaces demarcated by cell proc
eases. However, the vacuoles examined by us in the electron
microscope were predominantly
cytoplasmic. That these vac
uoles had no continuity
with the extracellular
matrix was
OCTOBER
plasmic
unclear.
vacuoles,
the cytogenesis
of the nuclear
vacuoles
is
ACKNOWLEDGMENTS
The expert technical assistance of Roy R. Keppie and Mona
Brandreth is gratefully acknowledged.
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Chordoma
Ultrastructure
Figs. 1—5are light micrographs. Figs. 1 and 2 are of paraffin-embedded material, while Figs. 3—5are of half-micron-thick sections
of epoxy-embedded tissue.
Fig. 1. An area of tumor showing strands of vacuolated physaliferous cells and small stellate cells separated by large areas of in
tracel!ular matrix. H & E, X 120.
Fig. 2. Typical tumor cells embedded in a dense, collagenous matrix. H & E, x 120.
Fig. 3. This section shows physaliferous cells displaying varying degrees of vacuolization. Some are of the “signet-ring―
type, with
the nucleus and cytoplasm displaced to the cell periphery by a large vacuole (arrows) . The stel!ate cells are smaller and have a denser
cytoplasm. The cells are stained various shades of blue, while the matrix is stained pink. Toluidine blue, X 400.
Fig. 4. A higher power micrograph showing numerous intracellular vacuoles, some of which contain flocculent material (Vi) . The
difference in staining between the clear vacuoles (V2) and the intercellular matrix is obvious. Note the small vacuoles in the stellate
cell at the upper right. Toluidine blue, X 1350.
Fig. 5. Two types of nuclear inclusions are present in this bibbed nucleus. One type is a transversely sectioned process of cyto
plasm, thus constituting a pseudoinclusion (P1). In this connection, note the deep clefts in the nuclear membrane of the larger lobe.
Two true intranuclear inclusions (INI) are demarcated by a halo of low density. The darkly stained nuclear structures are nucleoli.
The adjoining cell contains a large vacuole (V) filled with flocculent material. The dense structures (@) in the cytoplasm are mito
chondrial endoplasmic reticulum complexes. Toluidine blue, X 1880.
Figs. 6—16are electron micrographs of thin sections stained serially with umanyl acetate and lead citrate.
Fig. 6. Survey micrograph of chordoma cells. A physaliferous cell containing a huge vacuole (V) is at the upper left. The other cells
are of the stellate type. They are characterized by a highly filamentous cytoplasm and by a relative paucity of vacuoles. At the
right is a series of cell processes, each of which is enwrapped by other cells. This cellular interdigitation is common among all types
of chordoma cells. Clusters of glycogen are distributed throughout the cytoplasm, being especially prominent in the cell process at
the upper right. x 7,500.
Fig. 7. A cytoplasmic process surrounded by portions of two other cells and bound to them by small desmosomes (D). The proc
ess contains numerous filaments (F) , which appear as dots or granules in transverse section. The surrounding cells have abundant
smooth endoplasmic reticulum (SER). x 24,000.
Fig. 8. A nucleus demonstrating two types of nuclear inclusions. The large central vacuole appears to be within the nucleus but
actually is contained in a cytoplasmic extension, here seen in cross-section. A narrow rim of cytoplasm that includes a mitochondrion
(arrow) envelops the vacuole. The cytoplasmic process is demarcated by nuclear envelope (E) with associated condensed chromatin.
A similar process (*), also in transverse-section but without vacuoles, is seen at the upper left. A true intranuclear inclusion (NB)
of the nuclear body type is present at the lower right of the nucleus. NU, nucleolus. X 11,000.
Fig. 9. A nucleus displaying many pseudoinclusions, some of which contain cytoplasmic vacuoles. X 21,000.
Fig. 10. A group of true intranuclear inclusions. These usually are clear vacuoles with a single limiting membrane. Unlike the
pseudoinclusions, no chromatin is condensed at their periphery. A nucleolus is at the lower right. x 21,600.
Fig. 11. A typical mitochondria! endoplasmic reticulum complex. The regular alternation of organelles is well demonstrated. The
mitochondria are attenuated in their central regions and bulbous at their ends. Elements of smooth endoplasmic reticulum and Golgi
complex are present in the cytoplasm. Small clusters of glycogen are scattered through the cytoplasm, and a deposit of this substance
is present in a vacuole (arrow). A portion of the nucleus is at the left. X 59,000.
Fig. 12. Distended endoplasmic reticulum cisternae with associated mitochondria. Note that ribosomes are present on the cistemnal
membranes primarily where mitochondria are closely apposed to the membrane. X 50,000.
Fig. 13. A mitochondrial endop!asmic reticulum complex in a region rich in smooth endoplasmic reticulum. Note the transition of
rough into smooth endoplasmic reticulum at the periphery of the complex (arrow). This area is shown at higher magnification in the
next micrograph. x 33,000.
Fig. 14. An enlarged portion of Fig. 13, showing the direct continuity of rough endoplasmic reticulum and smooth endoplasmic
reticulum. X 48,000.
Fig. 15. A Golgi complex displaying the fenestrated appearance typical of this organel!e in chordoma cells. A cytoplasmic vacuole
is present at the top of the micrograph. x 44,000.
Fig. 16. A grazing section of a fenestrated Golgi cistern. Several vesicles appear to be in continuity with the Golgi membrane
(arrow). X 55,000.
OCTOBER
1968
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2119
I
‘‘1
‘I
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2125
Ultrastructure of Human Chordoma
Robert A. Erlandson, Bernard Tandler, Philip H. Lieberman, et al.
Cancer Res 1968;28:2115-2125.
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