[CANCER RESEARCH 38, 2003-2010,
0008-5472/78/0038-0000$02.00
July 1978]
Morphology and Metastatic Nature of Induced Hepatic Nodular Lesions
in C57BL x C3H F, Mice1
S. D. Vesselinovitch, N. Mihailovich, and K. V. N. Rao
Departments ol Pathology ¡S.D. V.¡and Radiology ¡S.D. V., N. M., K. V. N. R.], The Pritzker
Research Institute2 ¡S.D. V.¡,University ol Chicago, Chicago, Illinois 60637
School
of Medicine,
and the Franklin
McLean
Memorial
showed that 22% of animals bearing diethylnitrosamineinduced liver tumors had pulmonary metastatic foci (7).
The metastatic capabilities of well-defined nodular he Transplants of the primary liver tumors induced by ethylnipatic lesions induced by benzo(a)pyrene, ethylnitroso- trosourea grew expansively, invaded locally, and metastaurea, benzidine 2HCI, and diethylnitrosamine were evalu
sized to lymph nodes and lungs, killing recipients in a few
ated. Coded liver and lung tissues from 1264 treated weeks (8).
C57BL/6J x C3HeB/FeJ F, mice were assessed independ
For determination of whether metastatic behavior of ma
ently for the presence of primary nodular lesions and lignant neoplasia is exclusively and thus predictably asso
métastases,respectively. Primary lesions were classified ciated with a distinct morphological pattern, it was decided
according to their size, cell morphology, and growth pat
to reexamine a large number of liver and lung tissues from
terns into hyperplastic, adenomatous, and trabecular mice exposed to benzo(a)pyrene, ethylnitrosourea, benzi
nodules. None of the 126 mice bearing hyperplastic nod dine-2HCI, and diethylnitrosamine; classify the primary
ules had pulmonary métastases.Four of 291 (1.4%) mice hepatic lesions; identify pulmonary métastases;and corre
with adenomatous nodular lesions showed métastases. late these 2 parameters. The ultimate objective was to
In contrast, of the 733 mice bearing the trabecular type of determine whether it could be possible to diagnose cancer
nodular lesions alone or in combination with other lesions in the mouse liver solely by histological examination of the
266 (36%) showed pulmonary métastases.The pulmonary primary lesion.
métastaseswere first detected in mice dying between 51
and 60 weeks of age (5%). This rate increased as a
MATERIALS AND METHODS
function of age at death, reaching an incidence of 51% in
mice surviving more than 81 weeks. It was concluded that
The investigated liver and lung tissues originated from
nodules showing trabecular and the more anaplastic solid mice exposed to various carcinogens (6, 7, 21-23) accord
sheet type of growths represented bona fide hepatocellu- ing to protocols summarized in Table 1. In all of those
studies C57BL/6J x C3HeB/FeJ F, mice (hereafter called
lar carcinomas in the mouse.
B6C3F,) were utilized. Slides were prepared from buffered,
formalin-fixed tissues, which were stained with hematoxylin
INTRODUCTION
and eosin. To secure proper sample size, whenever possi
The mouse has been used extensively as a bioassay ble we sectioned the whole lung en bloc 5 ¿¿m
thick at 2
system for testing the potential carcinogenicity of drugs, different levels (7). Livers and lungs of 1264 mice were
food additives, and environmental pollutants. The malig
coded and examined separately for the presence of primary
nant nature of the commonly induced nodular liver lesions and metastatic lesions, respectively. Liver nodules were
in the absence of métastasesand even their neoplastic classified as hyperplastic, adenomatous, and trabecular
character have been discussed frequently (1, 4, 5, 10, 11, nodules, the morphology of which is detailed in "Results."
18). Because identical nodular lesions have been diagnosed Animals with more than 1 type of liver nodule were assigned
as separate morphological entities (14) and because several into a category according to the presence of the most
conditions such as strain, age, and sex of the animals have advanced lesion. Thus the trabecular nodules outranked
been identified as modulators of hepatocarcinogenesis,
adenomatous nodules; the adenomatous nodules out
some investigators have even suggested that the mouse ranked in turn hyperplastic lesions. Lungs were examined
for the presence of hepatic cell métastases.Metastatic rate
might not be an appropriate model for carcinogenicity
has been correlated with the primary nodular lesions.
testing.
Our interest in mouse hepatocarcinogenesis led us to
investigate metastatic behavior of induced nodular liver RESULTS
lesions in the primary (6, 7) and upon transplantation in the
secondary isogeneic hosts (8). Original histological studies Morphology of Primary and Metastatic Lesions
ABSTRACT
' The investigations have been supported in part by NIH Contracts NCI-E69-2087 and N01-CP-43317 from the National Cancer Institute, and the
evaluation study was made in part during S.D. Vesselinovitch's tenure of the
Alexander von Humboldt Senior U.S. Scientist Award in the Department of
Experimental Pathology at the School of Medicine. Hanover. Germany.
2 Operated by the University of Chicago for the U. S. Energy Research and
Development Administration.
Received May 26. 1977; accepted April 12, 1978.
JULY 1978
The primary nodular lesions of the liver were classified
into hyperplastic, adenomatous, and trabecular nodules.
Hyperplastic Nodules. These measured 1 to 3 mm in
diameter involving up to several liver lobules. On gross
inspection they were tan. Microscopically, the lesions were
characterized by focal variation in the staining aspects and
textural appearance of otherwise normal hepatic cells (Fig.
2003
S. D. Vesselinovitch
et al.
Table 1
Protocol for induction of nodular hepatic lesions in 86C3F, mice
Detailed information on material and methods has been presented in earlier publications as
referenced: benzo(a)pyrene (21), ethylnitrosourea (23), benzidine-2HCI (22), and diethylnitrosamine (7).
CarcinogenBenzo(a dose75-150
¿ig/g
(pyrene
60-120 fj.g/g
Ethylnitrosourea
Benzidine-2HCIDiethylnitrosamineTotal
50-250 ppm,
30 ¿/g
daily
6-12/ug/gNo.
1). The tinctorial
qualities
and type of
treatmentsSingle
Single
1, 15,42
ContinuousIntermittent42-630
7-27
1-16
(4
15-30
treatments)Age
42-57No.
of the cells were clear, eosino-
philic, and/or basophilic.
Normal liver architecture
was
preserved, as indicated by the presence of central veins and
portal triads, which were, however, occasionally displaced.
Vascular spaces were indistinct.
Adenomatous Nodules. These measured more than 3
mm in diameter, encompassing
a number of liver lobules
and even entire liver lobes. Their color ranged from tan to
red-brown. Microscopically,
this type of nodule was com
posed of well-differentiated,
normal to large polygonal
hepatic cells. Tinctorially, the cells were clear, eosinophilic,
and/or basophilic (Fig. 2), showing occasionally
more hyperchromasia than did cells found in the hyperplastic nod
ules. Cytoplasmic inclusion bodies were sometimes pres
ent, occasionally
in many cells. Adenomatous
nodules
showed an expansive type of growth, compressing but not
invading surrounding tissues, as indicated by sharp demar
cation lines (Fig. 3). Hepatic cells were usually arranged in
single but distorted cords (Fig. 4). Vascular spaces were
distinct only occasionally
in certain areas of the nodule.
Portal triads and central veins were generally absent, espe
cially when nodular structure occupied the major portion of
a liver lobe (Fig. 5). Mitotic activity was usually slight, and
the appearance of the mitotic figures was normal. Necrotic
changes were rarely seen. Occasionally, nuclei were vesicu
lar and thus showed prominent nucleoli (Fig. 6).
Trabecular Nodules. Trabecular nodules (Figs. 7 to 16,
including pulmonary métastases) were similar in size to
large adenomatous nodes, frequently involving several liver
lobes. Upon gross inspection they were brown-gray, the
gray appearance being more prominent on the cut surface.
Microscopically,
cellular morphology ranged from uniform
to pleomorphic
and from well differentiated
to anaplastic.
The hepatocyte size was the same, smaller (microcellular)
or larger (macrocellular)
than normal. The neoplastic cells
tended to grow in broad trabeculae (macrotrabecular)
(Fig.
11), in moderately wide plates (Figs. 7 and 13), or in narrow
trabecular
(microtrabecular)
formations
(Fig. 16). Occa
sionally, cellular borders were indistinct, giving a syncytiallike appearance to these lesions (Fig. 15). Trabeculae, re
gardless of the diameter, alternated with vascular spaces of
varied width. Intravascular invasion and free-floating single
or multiple tumor cells were frequent findings.
Tumor
growth was invasive and destructive, infiltrating surround
ing hepatic
parenchyma.
Trabecular
structures
were
2004
ofmice
at
of
at
treatment
mice ex
termina
(days)1,15,42 amined225 tion
(wk)110
236
90
325478Age 9090
aligned either parallel or haphazardly (Figs. 7 and 15). The
anatomic liver landmarks were not seen within the nodules.
Depending upon the width of trabeculae or sinusoids, the
cell morphology and their size, and the section plane, the
trabecular growth assumed a tubular, acinar (Fig. 9), glan
dular, organoid (Fig. 16), or even hemangiomatous
appear
ance. The highly anaplastic cells usually grew in sheets.
Mitotic activity ranged from slight to abundant, the figures
frequently being bizarre. Broad trabeculae
had the ten
dency to undergo central necrosis with formation of cystic
spaces. Hemorrhage with formation of blood lakes or blood
cysts was usually found in connection with necrosis. Occa
sionally, the tumor grew in apparent sheets that under high
magnification
revealed abortive organoid formations (Fig.
16) composed of basophilic cells with an increased nucleocytoplasmic ratio. The sheet type of growth was sometimes
composed of alternating and intercepting areas composed
of abnormal micro- and macrohepatocytes.
All of the above
architectural
arrangements were viewed as variants of the
most commonly seen abnormal trabecular pattern.
Pulmonary métastases (Figs. 8, 10, 12, and 14) were
either single (Fig. 10) or multifocal (Fig. 12), composed of
small groups of cells or of large tumor masses showing
frequently a close architectural
resemblance to the primary
trabecular nodules (Figs. 8 and 14). On occasion metastatic
cells did not resemble those of the primary lesion. Meta
static foci occupied mainly the alveolar capillaries and the
small and medium branches of the pulmonary artery adja
cent to the small bronchi and bronchioles.
Primary tumors
metastasized mainly by the hematogenous route. They grew
concentrically,
distorting
bronchiolar
lumen (Fig. 12) or
replacing lung parenchyma.
Most of the metastatic foci,
however, were small and discrete, which explains their rare
detection on gross examination.
Structural
morphology
varied from abortive to reproduction
of normal hepatic
architecture to the formation of acini (Fig. 8) and trabeculae
(Fig. 14).
Correlation
Metastasis
between Nodular Morphology and Pulmonary
Table 2 lists the number of mice with specific hepatic
nodular lesions and their metastatic rates. Of 1264 mice the
livers of 114 (9%) were free of any nodular lesions. Hyperplastic nodules were seen in 126 (10%) animals. None of
CANCER
RESEARCH
VOL. 38
Morphology of Hepatocellular Carcinoma in Mice
Table 2
Number and percentage of mice with nodular hepatic lesions and pulmonary métastases
Nodular hepatic lesions
Hyperplastic
tastases"No.19
of
free of
mice ex
nodular
CarcinogenBenzo(a)pyreneamined225 lesions49
Adenomatous
mé
métastases"-*No.0
Trabecular
Métastases"'*"No.47
Ethylnitrosourea
34.3
236
1.5
143
49
10
16
0.00.0 67
0
1
Benzidine-2HCI
39.8
325
4843No.000%0.0
161
64106%49.5
4213Pulmonary
7490Pulmonary
12%0.01.4
DiethylnitrosamineNo.
478mice
0.0No.60
2.2No.97
332Pulmonary 31.9
" Pulmonary métastases,totals: hyperplastic (0 of 126) versus adenomatous (4 of 291), p > 0.99; adenomatous (4 of
291) versus trabecular (266 of 733), p < 0.001.
6 Average age of mice with pulmonary métastases,76 weeks.
c Average age of mice with pulmonary métastases,78 weeks.
Table 3
Cumulative metastatic rates of hepatocellular carcinomas at specified age periods
age:660
with pulmonary métastasesat
no.with
tra
100wkNo.354964106%
wk
wkNo.002317%00145570
wkNo.3113236%3820111180
wkNo.21294263%222026192190
becularnodules"97143161332Mice
CarcinogenBenzo(a
%36 No.
(pyreneEthylnitrosoureaBenzidine-2HCIDiethylnitrosamineCumulative
4734403235
46
%47
4836
weighted av. (%)Total
36110wkNo.
" Total number of mice that showed trabecular nodules at the time of autopsy regardless of age at death.
6 Each column lists number and percentage of mice that showed pulmonary métastasesby the specified age
(cumulative incidences). Percentages were calculated on the basis of the total number of mice showing trabecular
nodules at autopsy for each carcinogen series.
these animals showed pulmonary métastases.Adenoma
tous nodules were observed in 291 (23%) animals surviving
on the average 76 weeks. Four of these animals (1.4%)
showed métastases.Fifty-eight % of examined animals (733
of 1264) showed the trabecular type of liver nodules. Thirtysix % of these mice (266 of 733) had pulmonary métastases
by an average age of 78 weeks.
For evaluation of the possible dependence of pulmonary
métastasesupon the age at the time of death of the animal,
the available data were arranged to present cumulative
pulmonary metastatic rates for mice that died by 60, 70, 80,
90, 100, and 110 weeks of life (Table 3). The animals dying
by 60 weeks of age showed a weighted metastatic average
of 5%. The pulmonary metastatic rate increased as a func
tion of time with each carcinogen. The cumulative weighted
averages were 11, 21, 35, and 36%. This increase in the
incidence of métastasesbecame even more manifest when
the data were assessed for cohorts of animals dying be
tween 61 and 70, 71 and 80, and 81 and 90 weeks. The
observed incidences were: 21% (42 of 201), 40% (73 of 181),
and 51% (99 of 196) for these 3 age periods, respectively.
DISCUSSION
This study provides additional information concerning
the biological behavior of 3 types of chemically induced
nodular liver lesions in the mouse. Thus, regardless of the
carcinogen used, mice surviving 81 to 90 weeks of age and
JULY 1978
bearing the trabecular type of growth and its variants
showed pulmonary métastasesin 51% of cases. In the
absence of these trabecular nodular lesions, the occur
rence of pulmonary métastases became insignificant
(1.4%). The high association between the presence of
trabecular nodular lesions and pulmonary métastasesdem
onstrates unquestionably the malignant character of tra
becular nodular lesions. This is further substantiated by
high transplantability rates of the trabecular type of nodules
induced by the 4 carcinogens. The observed incidences
were 70% for benzo(a)pyrene, 75% for ethylnitrosourea,
38% for benzidine-2HCI, and 80% for diethylnitrosamine
(unpublished data). Such transplants grew rapidly, invaded
locally, metastasized broadly, and killed the hosts within
6 weeks. Some of these transplantable tumors were
carried for over 100 generations. This additional character
istic further confirms their malignant nature.
The finding of 1.4% (4 cases) pulmonary métastasesin
animals showing only the adenomatous type of nodule is
worthy of discussion regarding the origin and biological
significance of these métastases.Thus, because additional
samples of hepatic tissues were not available in these 4
cases for histological réévaluation,
it was not possible to
resolve whether these pulmonary métastasesarose from
the identified adenomatous nodules or from undiscovered
trabecular lesions. The latter possibility has been raised
because, in another group of mice showing in original
slides adenomatous nodules, we observed the trabecular
2005
S. D. Vesselinovitch et al.
nodular pattern upon examination of the additional speci
mens. Also, in a recently terminated study with benzidine
in which 560 mice were examined at 80 weeks for the
presence of various hepatic nodular lesions, 202 animals
showed the presence of adenomatous lesions and none of
these animals had pulmonary métastases.In contrast, only
mice showing moderately to poorly differentiated trabecular
hepatocellular carcinomas showed pulmonary métastases
(39 of 128; 30%) (unpublished data).3
The proposition that some of the pulmonary métastases
observed in animals with both types of lesions could have
originated from the adenomatous rather than from the more
advanced trabecular type of nodule does not seem to be
biologically tenable because (a) in the factual absence of
the trabecular nodules in the liver, as documented by the
recently terminated study, pulmonary métastaseswere not
present; (D) the adenomatous nodules never showed an
invasive growth, prominent vascular channels, and marked
cellular atypia, morphological attributes usually associated
with metastatic behavior; and (c) the trabecular rather than
adenomatous nodules showed the invasive and metastatic
type of growth upon isogeneic transplantation (8). There
fore the questionable association between adenomatous
nodules and metastasis and their noninvasive growth char
acteristics leaves the malignant nature of these lesions
unsubstantiated.
The detection of a high metastatic rate in this study was
related to the high incidence of hepatocellular carcinomas
occurring relatively early in life, the good survival of tumorbearing animals, and proper sampling and histological
evaluation of lung tissues (7). Pulmonary métastasesof liver
tumors were observed by other investigators, although in
most instances with significantly lower rates. Thus Stewart
(12) reported a 1 to 2% rate for spontaneously developing
liver tumors. Tomatis ef al. (19) and Turusov ef al. (20)
observed an average of 1.4% métastasesin CF-1 mice
exposed to dichlorodiphenyltrichloroethane.
Gellatly (2),
using 4-dimethylaminoazobenzene, found pulmonary mé
tastases in 13% of C57BL mice bearing trabecular hepato
cellular carcinomas. Gorer (3) observed 7% métastasesin
CBA mice living beyond 56 weeks. Thorpe and Walker (17)
found 13% and 61% pulmonary métastasesof type B liver
tumors in dieldrin-treated male and female CF-1 mice,
respectively. Takayama ef al. observed in mice malignant
liver tumors that were induced by /V,A/'-2,7-fluorenylenebisacetamide (13, 15) and nitroso compounds (16). Histologically, the malignant liver tumors observed by Takayama
ef al. were similar in both morphology and behavior to those
reported here; they were trabecular hepatocellular carcino
mas that metastasized to the lungs in 13 to 31%. Such
lesions were also readily transplantable (13). Also in accord
ance with the current report is the recent study by Reddy ef
al. (9), who observed 24% pulmonary métastasesof malig
nant hepatic cell tumors in nefenopin-treated mice.
This study showed that the chemical carcinogens in
duced 3 distinct nodular liver lesions. Of these, only nod
ules showing the trabecular as well as the more anaplastic
pattern could be classified as hepatocellular carcinomas.
Thus it could be concluded that the mouse liver system may
3 From studies supported
2006
in part by NCTR Contract:
222-76-2004 (C).
serve its purpose. However, the observed nodular lesions
should be appropriately classified, the experimental condi
tions must be fully assessed, and the factors modifying
hepatocarcinogenesis should be duly considered before
bioassay data are interpreted.
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CANCER
RESEARCH
VOL. 38
Morphology of Hepatocellular Carcinoma in Mice
•OBBÉMOMT^-1 -•.•&*<£
Fig. 1. Liver; discrete hyperplastic nodule. Note slight compression of surrounding parenchyma and slightly shaded (eosinophilic) cytoplasm of bordering
cells. Benzo(a)pyrene, 38-week-old male. H & E, x 100.
Fig. 2. Liver; a small (3-mm) adenomatous nodule (upper right) and a dark-shaded (basophilic) focus (bottom). The nodule is composed of cells
resembling the normal hepatocytes. Cytoplasm gives clear to slightly shaded (acidophilic) appearance. Vascular spaces are indistinct. Diethylnitrosamine,
81-week-old male. H & E, x 50.
Fig. 3. Liver; 2 adenomatous nodules, 5mm (upper) and 8mm (lower) in diameter, compressing normal liver tissue. Nodular areas show lighter tinctorial
characteristics when compared with normal liver tissues. Ethylnitrosourea, 51-week-old male. H & E, x 50.
Fig. 4. Liver; adenomatous node replacing the main portion of a liver lobe. Characteristic liver structure is absent. Hepatocytes are arranged in 1-cellthick convoluted cords. Benzo(a)pyrene, 72-week-old male. H & E, x 100.
JULY 1978
2007
S. D. Vesselinovitch et al.
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Fig. 5. Liver; adenomatous nodule occupying the major portion of a liver lobe. Note round vacuoles of varying size, mostly multiple, within hepatic cells.
Lobular structure is not preserved. Ethylnitrosourea, 43-week-old male. H & E, x 50.
Fig. 6. Liver; adenomatous nodule, portion of a nodular mass that replaced the entire liver lobe. Note tinctorial cytoplasmic variation and distorted single
plates. The majority of the nuclei are vesicular and show prominent nucleoli. Ethylnitrosourea, 74-week-old male. H & E. x 250.
Fig. 7. Liver; trabecular nodule. Note the pleomorphic appearance of the cells and the discrete endothelial lining of trabecular formations. Vascular
spaces contain blood. Diethylnitrosamine, 65-week-old male. H & E, x 250.
Fig. 8. Lung; pulmonary metastasis from the animal bearing the primary trabecular nodule illustrated in Fig. 7. Note acinar formations within the
metastasis and 2 capillary emboli. H & E, x 250.
2008
CANCER
RESEARCH
VOL. 38
Morphology of Hepatocellular Carcinoma in Mice
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Fig. 9. Liver; trabecular microcellular basophilic tumor. Note trabecular and acinar structures, cellular pleomorphism, variation in nuclear size, and dark
(basophilic) cytoplasm. Diethylnitrosamine, 80-week-old female. H & E. x 250.
Fig. 10. Lung; pulmonary metastasis from the animal bearing the lesion illustrated in Fig. 9. Note central vascular core and marked cellular pleomorphism.
H&E, x 250.
Fig. 11. Liver; trabecular nodule composed of macrotrabecular (broad) structures. Neoplastic cells appear to be relatively uniform, showing vesicular
nuclei and prominent nucleoli. Diethylnitrosamine. 58-week-old male. H & E, x 250.
Fig. 12. Lung; multiple pulmonary métastases(vascular and lymphatic) from the mouse bearing the trabecular nodule illustrated in Fig. 11. Note varied
size of metastatic foci and intra- and perivascular metastasis in the lower right. H & E, x 50.
JULY
1978
2009
S. D. Vesselinovitch et al.
Fig. 13. Liver; trabecular nodule composed of moderately wide plates and distinct vascular spaces. Benzo(a)pyrene, 91-week-old male. H & E, x 100.
Fig. 14. Lung; pulmonary metastasis from the animal with the trabecular nodule illustrated in Fig. 13. Note moderately wide trabecular formations and
compression of lung parenchyma (extreme lower left). H & E, x 250.
Fig. 15. Liver; trabecular nodule. Note haphazard branching of broad trabeculae. In general, cell borders are indistinct, giving a syncytial-like appearance
to this tumor. Benzo(a)pyrene, 73-week-old male. H & E. x 100.
Fig. 16. Liver; trabecular nodule composed of dark (basophilic) cells arranged in an organoid pattern. Nucleocytoplasmic ratio is markedly increased.
Nuclear chromatin varies in density. Diethylnitrosamine, 62-week-old male. H & E, x 250.
2010
CANCER
RESEARCH
VOL. 38