[CANCER RESEARCH 48, 148-160, January 1. 1988)
Mouse Papillary Lung Tumors Transplacentally
Induced by A^-Nitrosoethylurea:
Evidence for Alveolar Type II Cell Origin by Comparative Light Microscopic,
Ultrastructural, and Immunohistochemical Studies1
Sabine Rehm,2 Jerrold M. Ward, Ank A. W. Ten Have-Opbroek,
Lucy M. Anderson, Gurmukh Singh,
Sikandar L. Katyal, and Jerry M. Rice
Tumor Pathology and Pathogenesis Section and Perinatal Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National
Cancer Institute, Frederick, Maryland 21701 [S. R., J. M. W., L. M. A., J. M. R.J; Department of Anatomy and Embryology, University of Leiden, Leiden, The Netherlands
[A. A. W. T. H.-OJ; and Department of Pathology, University of Pittsburgh, Veterans Administration Medical Center, School of Medicine, Pittsburgh, Pennsylvania 15261
[G. S., S. L. K.J
ABSTRACT
A histogenetic study was designed to evaluate controversial findings
on the cell of origin of tubular/papillary lung tumors in mice, i.e.,
bronchiolar Clara cell versus alveolar type II cell. A/-Nitrosoethylurea
(0.5 mmol or 0.74 mmol/kg) was given to pregnant C3H (C3H/HeNCr
MTV) and Swiss Webster |Tac:(S\V)fBR| mice as a single i.p. injection
on Day 14, IS, 16, or 18 of gestation. The offspring were studied at
various ages ranging from 7 days to 52 wk.
Serial sections of the whole lung (100 to 200 sections per mouse)
showed that solid/alveolar and papillary tumors arose from the pulmonary
acinus, invading the bronchioles only as the tumors grew. Furthermore,
a mixture of solid and papillary patterns within a single nodule did not
represent a merging of two tumors but a progression from the solid to
the papillary form. By use of two rabbit antisera against mouse lung
surfactant apoproteins found in normal alveolar type II cells, it was
shown by the avidin-biotin peroxidase complex procedure, by the peroxidase-antiperoxidase technique, and by indirect immunofluorescence that
both solid and papillary tumors contained these proteins that are specific
markers for alveolar type II cells. With a rabbit anti-rat Clara cell
antiserum, none of the tumors studied was ¡mmunoreactive while normal
Clara cells were reactive. The nitroblue tetrazolium formazan stain for
dehydrogenase enzymes, found particularly in Chira cells, did not reveal
these enzymes in any lung tumors from either strain.
Ultrastructurally, no typical features of the mature Clara cell were
detected in papillary or other pulmonary neoplasms. However, all tumors
showed characteristic alveolar type II cell structures such as various
stages of lamellar body formation, although these features were less well
differentiated in the papillary tumors. Argentaffin dense bodies, repre
senting lysosomes and immature forms of lamellar bodies, were commonly
observed in pupillary rumors. Some features of the papillary tumors such
as cell shape, high glycogen content, and primary cilia were equivalent
to those seen in pulmonary epithelial precursor cells during fetal devel
opment. With age, the papillary tumors became invasive, accumulated
neutral lipids, and developed bizarre cleaved nuclei and lamellated nuclear
pseudoinclusions.
In conclusion, the papillary lung tumors of the mouse, at least those
induced transplacentally by yV-nitrosoethylurea, constitute less well-dif
ferentiated or poorly differentiated
carcinomas with fetal morphological
alveolar type II cell adenomas
and biochemical properties.
this consensus developed involved spontaneous and induced
tumors, light microscopy of serial sections from single tumors
or lung lobes (1, 5), and ultrastructural demonstration of type
II cell features (6, 7). Larger papillary tumors, however, fre
quently grew invasively into bronchioles, and since their colum
nar cell shape superficially resembled that of the bronchiolar
cells, such origin was occasionally suggested (8-10).
In studies on N FU '-induced papillary lung tumors of Swiss
Webster mice, ultrastructural proof was presented for a histo
genetic origin of the papillary mouse lung tumor from the
bronchiolar Clara cell (11-15). Main criteria were the presence
of dense secretory granules and membrane-enclosed crystals,
nuclear indentations, a cuboidal to columnar cell shape, nu
merous microvilli at the luminal surface, and interdigitating
membranes between adjacent cells. The Clara cell origin was,
however, questioned since it later proved impossible to dem
onstrate a Clara cell antigen in papillary neoplasms of C57BL/
6 x C3H F, (B6C3F,) and BALB/c mice, while many of these
tumors were immunoreactive for surfactant apoproteins of nor
mal alveolar type II cells (16). Since then a number of studies
have been published referring to the mouse Clara cell tumors
(17-23), often without acknowledging that uncertainty still
exists regarding the cell of origin. Resolution of this question
is important, since the mouse lung tumor is a common end
point in both mechanistic and bioassay Carcinogenesis experi
ments (2).
The present study with C3H and Swiss Webster mice was
undertaken to help clarify the histogenetic origin of the mouse
papillary lung tumor, combining various light microscopic and
ultrastructural techniques and using two different rabbit antisera specific for antigens found in mouse alveolar type II cells
and one antiserum specific for Clara cells.
MATERIALS AND METHODS
or
INTRODUCTION
Until 1979, there was general agreement that both solid/
alveolar and tubular/papillary lung tumors of the mouse origi
nated from the alveolar type II cell (1-3). Morphological vari
ations among tumors were considered to represent different
stages of progression of the neoplasms (4). Studies from which
Received 7/2/87; revised 9/29/87; accepted 10/5/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by USPHS Contracts N01-CO-23910and N10-CP-41014
to Program Resources, Inc., and Microbiological Associates, Inc., respectively.
2To whom requests for reprints should be addressed, at National Cancer
Institute, FCRF Bldg. 538, Room 220, Frederick, MD 21701-1013.
Animals and Chemicals. Male and female C3H/HeNCr
MTV mice
were obtained from the Animal Production Area, National Cancer
Institute-Frederick Cancer Research Facility, Frederick, MD. All ani
mals were housed in an environment maintained at 22-26T
with SO
±10% relative humidity and a 12-h light/dark (6 a.m. to 6 p.m.) cycle.
The mice were free of serum antibodies to Mycoplasma sp. and to all
known murine viruses, including pneumotropic agents such as Sendai
virus and pneumonia virus of mice. At the age of 8 wk the mice were
mated, and the females were checked every morning for vaginal plugs
to determine the first day of gestation. Lung tumors were induced
transplacentally in fetal mice (24) on gestational Days 14, 16, and 18
'The abbreviations used are: NEU, W-nitrosoethylurea; MTV, mammary
tumor virus negative; PAS, periodic acid-Schiff; NBT, nitroblue tetrazolium; unti
CCA, antiserum to rat Clara cell antigen; CCA, Clara cell antigen; anti-SAP-M,
antiserum to surfactant apoproteins of the mouse; SAP-M, surfactant apoproteins
of the mouse; SAALS, specific anti-adult lung serum; ABC, avidin-biotin perox
idase complex; FITC, fluorescein isothiocyanate; PAP, peroxidase-antiperoxidase; H & E, hematoxylin and eosin.
148
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS OF MOUSE LUNG TUMORS
by i.p. injection of 0.5 mmol of NEU per kg (Sigma, St. Louis, MO)
dissolved in trioctanoin (Eastman Kodak, Rochester, NY). Two male
progeny from each gestational treatment day were sacrificed for serial
lung sections at the age of 1, 2, 4, 8, 16, and 32 wk. Additional mice
were sacrificed for ultrastructural, biochemical, and immunocytochemical studies at ages of 4 to 52 wk. Control lung tissues from untreated
mice or from mice that received trioctanoin only were obtained from
fetuses on gestational Days 14, 16, and 18, or from neonates on the
first postnatal day, and 1, 2, 4, and 52 wk after birth.
Timed pregnant female Swiss Webster mice [Tac:(SW)fBR] were
purchased from Taconic Farms, Germantown, NY. They received 0.74
mmol of NEU/kg in trioctanoin by i.p. injection on Day 15 of gestation.
Offspring were killed for light microscopic studies comparable to the
C3H mice at ages ranging from 4 wk to 6 mo.
Necropsy. At necropsy all grossly visible lung tumors were recorded.
For studies on paraffin-embedded sections by light microscopy, lungs
were inflated and fixed with Bouin's solution for 4 to 5 h and placed
teins and a M, 12,000 protein, presumably also a surfactant protein
(34).
ABC Immunohistochemistry. The ABC immunohistochemical pro
cedure was followed as previously described (35). Anti-CCA was used
diluted 1:800 with phosphate-buffered saline, anti-SAP-M was diluted
1:300, and normal rabbit sera diluted likewise were used as controls at
similar concentrations for an incubation period of 30 min. The rabbit
Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used
for the localization of CCA and SAP-M in Bouin's fixed sections of
mouse lung tumors from C3H and Swiss mice. 3,3-Diaminobenzidine
was the chromogen (16), and sections were counterstained with hematoxylin.
Fluorescence Microscopy. The indirect immunofluorescence tech
nique was applied as described previously in detail (32, 36) to sections
of alcohol-fixed or fresh-frozen lung tumors from C3H or Swiss Web
ster mice using the rabbit antiserum SAALS at a dilution of 1:40 or
1:100. Control sections were treated with pn-i ninnine rabbit serum and
FITC conjugate, or FITC conjugate only.
PAP Technique. The PAP technique was applied to sections of lung
tumors from C3H and Swiss mice fixed in Bouin's solution, formalin,
overnight in 70% alcohol. In selected cases neutral buffered formalin
or 70% alcohol alone was used as fixative. After fixation all five lung
lobes were separated from each other but embedded together in one
block. For enzyme and lipid stains on fresh frozen sections, tissues
were embedded in O.C.T. compound (Miles Laboratories, Naperville,
IL) on dry ice.
Morphological Classification. Lesions were classified as previously
described (16) into three categories, i.e., alveolar/solid, tubular/papil
lary, and mixed showing both solid and papillary growth patterns.
Alveolar and solid lesions were evaluated together, since a mesh-like
alveolar pattern was often seen early before the development of a more
solid appearance or, as evident from serial sections, a compact center
was often surrounded by an alveolar area. Likewise, no distinction
between initial, small tubular lesions and larger papillary structures
seen in older mice was made.
Serial Sections of Lung. Hematoxylin:eosin-stained serial sections
(100 to 200 sections per mouse), 5 pm thick, were cut from the entire
lung of two C3H mice from each gestational treatment day at the ages
of 1, 2,4, 8,16, and 32 wk to localize developing tumor sites in relation
to lung structures. A Zeiss Videoplan image analysis system (Zeiss,
New York, NY) was used to measure and outline tumor diameters. For
comparison, 4 lungs from NEU-treated Swiss mice aged 4 to 5 wk were
also cut serially.
Special Stains. Special histochemical and immunological studies
were carried out on control tissue and tumors from C3H and Swiss
mice at different ages. Sections were stained with alcian blue, and
subjected to the PAS reaction with or without prior diastase treatment
to demonstrate acid or neutral mucopolysaccharides. Frozen sections
prepared from formalin-fixed or fresh tissues were stained with Oil Red
0 for neutral lipids or tested for dehydrogenase enzymes (3-hydroxybutyrate dehydrogenase) by the NBT reducÃ-asemethod (17, 25, 26).
Antigens and Antisera. Rabbit anti-CCA was prepared as described
before (27). Rabbit anti-SAP-M was obtained and characterized as done
previously for antiserum to rat lung surfactant apoproteins (28-31).
Briefly, mouse lung surfactant was isolated by salt gradient centrifugation and used as an immunogen in rabbits. The resulting antiserum was
absorbed with mouse serum, and the rabbit serum proteins not binding
to mouse serum were collected. A -, globulin fraction of rabbit antiserum to mouse surfactant was prepared by ammonium sulfate precip
itation, and the precipitate was dialysed in saline. In addition to the
tissue reactivity of the antiserum, the preparation was tested by immunoblotting. Briefly, mouse surfactant was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, the electrophoresed proteins
were transferred to nitrocellulose paper, and the paper was stained by
the immunoperoxidase method using rabbit antisenim to mouse sur
factant (30).
SAALS was prepared by immunizing rabbits with adult mouse lung
homogenate (supernatant fraction), and its specificity for alveolar type
II cell-specific antigen was obtained by absorption with cross-reacting
organs and normal mouse serum (32). In fetal lung tissue, antigens
reactive with SAALS can be detected in prospective cells of the pul
monary acinus from Day 14.2 of gestation onward (33). Histochemically, SAALS was characterized as being specific for surfactant apopro
or alcohol to test for SAALS reactivity at dilutions of 1:40 or 1:100.
The rabbit PAP technique (Nordic Immunological Laboratories, Tilburg, The Netherlands) was used according to the double bridge method
in combination with CuSO4 enhancement (37). Control sections were
treated with preimmunization serum and PAP according to the double
bridge method, PAP, or diaminobenzidine only.
Ultrastructnre. Control and tumor tissues from C3H mice were fixed
in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4"C and
postfixed with 1% osmium tetroxide for 1 h. Except for tumors from
6-mo-old mice, staining with 0.5% uranyl acetate was performed prior
to embedding in epoxy resin.
Two solid and 4 papillary neoplasms from 4-wk-old mice and 6
papillary tumors from 6-mo-old mice were studied. Semithin sections
B
Fig. 1. Mouse surfactant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting using rabbit anti-SAP-M.
Band A, dimeric form of the M, 35,000 surfactant apoprotein; the two Bands B,
the M, 35,000 apoprotein.
149
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS OF MOUSE LUNG TUMORS
Table 1 Age-dependent location of lung tumors induced by NEU in two strains of mice
Location"No.
of lung tumorsBronchiolar
lumenPleural
contactNo
connection
to bronchiolesor
pleuraStrainC3H/HeNCr
ofmice6'666664'Solid*(»)18422232218Papillary(")2251840373634Solid*M0(0)"1(13)1(25)9(41)12(52)1
MTV-Swiss
(64)9(50)22
(73)15
(55)24
(54)26
(65)28
(65)17(77)12(67)Papillary(«)2(100)16
(50)4(22)Papillary(«)0(0)3(12)6(33)19(48)20
1
(72)5(15)Solid*(»)0(0)4(50)3(75)16
(78)17(50)Solid*(«)0(0)4(50)1(25)4(18)4(17)1(5)6(33)Pa
Webster [Tac: (SW)fBR]Age(wk)124816324-5No.
" Some tumors are both in contact with pleura and invade bronchioles.
* Includes mixed tumors from age 8 wk.
' Each of two mice treated on gestation Days 14. 16, and 18 with 0.5 mmol NEU/kg. i.p.
* Numbers in parentheses, percentage.
' Treated on gestation Day 15 with 0.74 mmol NEU/kg, i.p.
were stained with toluidine blue or with a silver nitrate solution for the
demonstration of glycogen and other argentaffm cell structures (38,
39). The argentaffin reaction is an osmium-dependent phenomenon
(39). Thin sections were mounted on film-coated nickel grids and
stained with uranyl acetate/lead citrate, or mounted on uncoated gold
grids and stained with silver nitrate (39) and examined with an electron
microscope (Phillips 300).
RESULTS
Immunoblotting of Rabbit Anti-Mouse Surfactant. Anti-SAPM stained two bands in the M, 35,000 region representing
surfactant apoprotein A (Fig. 1). A stained M, ~70,000 band
represents dimers of the apoprotein A. The antiscrum did not
react with any serum proteins.
Histogenesis of Lung Tumors from Study of Serial Sections.
From serial sections of the entire lung the localization of lung
tumors in relation to surrounding structures was studied as a
function of age (Table 1). No differences with respect to tumor
location were observed among the various days of treatment.
Lung tumors rapidly increased in size, and therefore, only very
early stages ( 1 to 2 wk) allowed an exact observation on the site
Fig. 2. Mouse lung tumors induced transplacentally by NEU. I. solid lung
of origin. No papillary tumors were found arising directly from
tumor from 4-wk-old Swiss Webster mouse growing into bronchiolar lumen. H
& E, x 75. B, papillary lung tumor from 2-wk old C3H mouse associated with
the bronchioles in either C3H or Swiss Webster mice. Both
pleura and separate from bronchiolar lumen by alveolar walls. H & E, x 100.
solid and papillary tumors were very similarly distributed (Table
1). The incidence of tumors possessing an open contact to
bronchioles, i.e., not interrupted by alveolar walls as shown in
Fig. 2A for a solid lung tumor, was very low early in the study
but increased as the mice aged and the tumors grew in size or
became invasive. However, an intimate pleural contact as shown
in Fig. 2B for a papillary tumor was observed in at least 50%
of both tumor types at all age periods investigated in both
strains.
Both pure solid and pure papillary tumors were seen at the
earliest time points of sacrifice. Mixed tumors were observed
for the first time in C3H mice aged 4 wk that had been exposed
to NEU on Day 14 of gestation. Serial sections of mixed
tumors, particularly of early ones, indicated that papillary struc
6070 — pleura
tures arose within originally pure solid tumors as illustrated
graphically in Fig. 3. Fig. 4B gives an example of a mixed
tumor with a single central papillary focus. More frequently,
however, neoplasms showed multiple separate foci of columnar
cells that formed small rosettes or a papillary pattern within
10— 20— 30— 40— 50.
the solid tumors, lacking contact with a papillary tumor beyond
Fig. 3. Enhanced computerized image analysis printout. Histological sections
the solid area. In late stages of large mixed tumors, in which
of two early mixed (solid/papillary) lung tumors (a and ft) from C3H mice shown
the papillary growth exceeded that of the solid area, this devel
in steps of 10 sections. Outlined area, solid tumor growth; Mack area, papillary
tumor growth.
opment could no longer be recognized.
150
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
-
* -T
i#*
«fcj%
OF MOUSE LUNG TUMORS
i.-
•-'..'-
;-££*££
- •;•§**
Fig. 4. Mouse lung tumors induced transplacentally by NEU. A, mixed mouse lung tumor from C3H mouse. Early central papillary' growth of columnar cells
within solid tumor; compare with Fig. 3. H & E, x 100. B, frozen section of two papillary lung tumors (p) from C3H mouse lacking positive NBT formazan staining
as evident in normal bronchiolar cells (arrows), a, alveolar cells, x Ino. C, cystic papillary lung tumor from Swiss Webster mouse staining negative for CCA. Note
immunoreactive bronchiolar cells. ABC immunohistochemistry. Hematoxylin. x lOO. D. papillary lung tumor from Swiss Webster mouse with positive cytoplasmic
immunostaining for SAP-M of tumor cells lining the papillary folds, free macrophages (arrows), and normal alveolar type II cells at tumor border. ABC
immunohistochemistry. Hematoxylin, x 250. E and F, indirect immunofluorescence of SAALS in paraffin sections from lungs of C3H mice fixed in alcohol. E,
hyperplastic alveolar walls at edge of solid lung tumor with positive cytoplasmic immunofluorescence of SAALS specific for alveolar type II cells, x 270. F, papillary
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
Table 2 Comparison of different features of normal mouse pulmonary epithelium
and lung tumors
lungFeatureAlveolar
Normal adult
Fetal lung
tumorsSolid
type II
Clara cellLung
tumors
cellBronchiolar
Papillary
tumors
Columnar shape
Clara cell antigen"
Surfactant apoproteins
SAP-M"
SAALS*
NBT reducÃ-aseactivity'
Neutral lipids
Glycogen fields
Lamellar bodies
Osmiophilic multivesicular bodies
Osmiophilic dense
bodies
Nonosmiophilic secre
tory granules of
Clara cells
Primary cilia
" As observed by the ABC technique on Bouin's fixed tissue.
* As observed by the PAP technique or immunofluorescence.
' With 30-s formalin pretreatment.
d Undetermined.
' Refs. 55 and 56.
1 Lysosomes only.
Nitroblue Tetrazolium ReducÃ-ase Activity. From 4-wk-oId
C3H mice, unfixed frozen sections of 21 papillary tumors were
studied for the staining patterns of NBT forma/an in an effort
to identify Clara cell dehydrogenases. None of the tumors
exhibited a staining intensity exceeding that of the surrounding
alveoli after formalin pretreatment (Table 2). This contrasted
strongly with the dark staining of bronchiolar cells (Fig. 4H).
Likewise, no significant staining was observed in 6 solid and
31 papillary tumors obtained from 6-mo-old Swiss Webster
mice. The formalin pretreatment of 30 s abolishes a low level
of NBT reducÃ-aseactivity in cells other than Clara cells (25,
26). When this pretreatment was omitted, 2 of the alveolar and
7 of the papillary tumors exhibited noticeably higher enzyme
activity than the surrounding normal type II cells.
Alcian Blue. None of the tumors studied produced secretions
of acid mucopolysaccharides.
Periodic Acid-Schiff Reaction. In fresh frozen sections of
normal fetal lungs, glycogen could be detected only from Day
16 onward by the PAS reaction. In tumors, glycogen was found
particularly in early papillary and mixed neoplasms. Diastase-
resistant intranuclear inclusions were seen in some of the pap
illary tumors in mice over 2 mo of age but not in solid tumors.
Oil Red 0. In normal fetal lungs, single tiny droplets were
first observed on Day 18. In mice 1 wk of age, the pulmonary
acini contained considerable amounts of lipids. Neutral lipids
were frequently present in papillary tumors of both strains, the
amounts increasing with age. Some of the larger tumors con
tained considerable quantities of lipids (Table 2).
Immunocytochemistry. By the ABC technique, CCA was first
detected in normal fetal lung on Day 18 of gestation within the
apical cytoplasm of normal bronchiolar cells. Of all papillary
and mixed tumors examined for CCA, none exhibited a positive
reaction. This contrasted sharply with the intense intracytoplasmic staining of normal bronchiolar Clara cells (Fig. 4C) as
was shown previously (16). The antiserum used proved to be
highly specific for CCA, since in all cases studied, no pulmonary
cells other than Clara cells reacted positively. The numbers of
tumors tested for CCA were generally comparable to those
examined for SAP-M (Table 3).
SAP-M was observable for the first time in developing mouse
lung parenchyma only after birth. SAP-M was localized within
type II cells and also formed a superficial lining of the bron
chiolar epithelial surface, but with anti SAP-M there was no
staining of the cytoplasm of bronchiolar cells. All solid areas
from pure or mixed tumors tested for the presence of cytoplasmic SAP-M (Table 3) were moderately to strongly positive
focally or diffusely. Of the papillary tumors, 75% were immunoreactive for SAP-M in the C3H and 80% in the Swiss mice.
A tumor was considered reactive if at least one-third of the cells
showed a yellow-brown cytoplasmic staining that was often
granular and similar to that observed in the normal type II
cells. This staining pattern was distinctly different from a light
gray-brown diffuse background staining. Most papillary neo
plasms possessed a mild-to-moderate staining reaction for SAPM with regard to the degree of staining intensity and size of
the positive tumor area. Only a few papillary tumors contained
cells that stained as intensely dark-brown as many of those in
solid tumors (Fig. 4D). The degree of positive staining was
similar in all age groups of both strains. However, the occur
rence of intranuclear inclusions described previously (16) was
observed in C3H mice only after the age of 2 mo. Characteris
tically, the cytoplasm of cells with intranuclear inclusions was
negative for SAP-M. Many tumors also possessed intraluminal
black-brown deposits, presumably representing secreted surfac-
Table 3 Immunohistochemical localization of SAP-M in mouse lung tumors by the ABC technique
Morphological tumor type (no. positive for SAP-M/no. tested)
MixedAge
of
mice5
(wk)C3H°
Strain
staining17/17
staining0/17
2
4
8
16
32
52No.
Total
4
3
34
2/2
8/8
5/5
6/6
3/3Solid0/0
8Solid1/1
0/0
0/00/0
10/10
18/18Papillary0/0
12/15
8/14
12/18
5/10
23/30
13/18PapillaryCytoplasmic
32/44Intranuclear
0/15
0/14
4/18
4/30
17/44
28/28(100)
18/28(64)
104/138(75)
25/138(18)
0/1
5/85/9
5/10
20/2233/41
2/10
0/222/41
27
25/25(100)*
5
3101/13/3
18/1822/22(100)0/01/1 8/89/9(100)0/0
0/0
0/00/0
Webster*Total4
Swiss
12
242
(56)8/9
(80)0/9
(5)
* Treated on gestation Day 16 with 0.5 mmol NEU/kg, i.p.
* Numbers in parentheses, percentage.
'' Treated on gestation Day 15 with 0.74 mmol NEU/kg, i.p
152
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
.
OF MOUSE LUNG TUMORS
.
Table 4 Localization of reactivity to SAALS in mouse lung tumors by PAP
technique and immunofluorescence
tumor
type (no. positive for
tested)Solid6/60/03/33/30/01/1Mixed0/04/42/20/01
SAALS/no.
ofmice531222Morphological
TechniquePAPifStrainC3H°C3HSwiss
•A
V
WebsterAge(wk)4421242424No.
" Treated on gestation Day 16 with 0.5 mmol NEU/kg, i.p.
* Treated on gestation Day 15 with 0.74 mmol NEU/kg, i.p.
' IF, immunofluorescence.
ser
s
m
m
m
ser
m
m
s
ser
Fig. 5. Bronchiolar cells of normal adult C3H mice. I. semithin epoxy section
of normal mouse (C3H) bronchiolar cells stained with toluidine blue. Most of the
dense granules located beneath the apical plasma membrane of the nonciliated
cells represent secretory granules of Clara cells. Similar staining of lysosomes in
alveolar macrophage (arrow), x 630. B, electron micrograph of normal mouse
Webster*C3HC3HSwiss
tant. The cytoplasm of macrophages that were regularly present
often also stained positively (Fig. 4D), probably due to phagocytized surfactant. Several papillary tumors in animals aged 52
mo showed central necrosis and possessed cholesterol clefts;
the necrotic material in such tumors stained positive for SAPM.
By the PAP technique, all solid and papillary tumors stained
with SAALS (Table 4) showed a positive staining reaction. A
fine granular yellow-brown pattern was present in most tumor
cells that was similar to the staining of normal alveolar type II
cells. The staining intensity varied among tumor cells and was
generally weaker than in normal type II cells. In solid tumors
the staining occurred throughout the cytoplasm, whereas in
papillary tumors it was generally restricted to the apical por
tions of the cells. Solid tumors stained more intensely than
papillary tumors. Occasionally, intranuclear positive staining
was seen. No strain differences were observed (Table 4). A
superficial, extracellular lining layer reactive with SAALS was
observed along all air spaces, i.e.. lining alveolar septa as well
as the bronchioles. By CuSO4 enhancement the intensity of the
staining was improved considerably.
Fluorescence Microscopy. All solid tumors tested for SAALS
reactivity were positive using immunofluorescence (Fig. 4E).
Similarly, all papillary neoplasms (Fig. 4F) were stained by
SAALS (Table 4). An exceptionally intense staining was found
in the lumina of papillary tumors by immunofluorescence (Fig.
4F). Localization and intensity of the fluorescence in lung
tumors and normal pulmonary structures were comparable to
the results obtained by the PAP technique.
Ultrastructure of Fetal Lungs, Mature Bronchiolar Clara
Cells, and Lung Tumors. On Day 14 of gestation the yet undifferentiated developing lung consisted of tubules lined by high
columnar epithelial cells. Some cells had cytoplasmic glycogen
deposits, and occasionally a single apical primary cilium was
present. On Day 16 of gestation, the prospective bronchial
system consisted of tubules lined by very tall and slender
columnar epithelium lacking glycogen and characteristic cyto
plasmic organdÃ-es. Precursor cells of alveolar type II cells lining
the prospective respiratory system were cuboidal and possessed
glycogen deposits as well as multivesicular and dense bodies.
On Day 18, cells from both systems of the respiratory tract had
(C3H) bronchiolar Clara cell stained with uranyl acetate and lead citrate. Dense
secretory granules (,v)located beneath apical plasma membrane; m. mitochondria;
ser. smooth endoplasmic reticulum among and circling mitochondria, x 28,945.
C, somit hi»epoxy section of normal mouse (C3H) bronchiolar cells stained with
ammonium silver nitrate. Scattered osmiophilic dense granules representing
lysosomes in various cell areas of ciliated and nonciliated cells. Granular appear
ance of Clara cell cytoplasm due to presence of mitochondria, x 630. D, electron
micrograph of normal mouse (C3H) bronchiolar Clara cell stained with ammo
nium silver nitrate. Osmiophilic lysosome (/) can easily be distinguished from
nonosmiophilic secretory granules (s); m, mitochondria; ser, smooth endoplasmic
reticulum. X 25,200.
153
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
attained more mature features. Bronchial low columnar nonciliated cells, some with large amounts of glycogen, were distin
guishable from developing ciliated cells. Alveolar cells showed
a pyramidal shape, some were without glycogen, and mature
lamellar bodies were present and released into the fetal alveolar
spaces. At all stages of development single primary cilia were
seen in all epithelial cell types.
Characteristic features of the mouse bronchiolar Clara cell
from 24-wk-old C3H mice are shown in Fig. 5. Thick sections
stained with toluidine blue (Fig. 5A) showed dense foci beneath
the luminal cell membrane of Clara cells and distributed
throughout the cytoplasm. Some of these foci were also seen in
ciliated cells. As observed with conventional staining proce
dures for electron microscopy, secretory granules often ap
peared dense (Fig. 5B) and could not be distinguished from
lysosomes. However, using only a silver stain, a clear distinction
between osmiophilic lysosomes and nonosmiophilic secretory
granules was possible (Fig. 5, A and D) due to a reduction of
the silver by osmium (Table 2). Furthermore, Fig. 5, B and D,
shows a smooth cell surface, the typical large round or ovoid
mitochondria of the mouse Clara cell with very short cristae,
and an abundance of smooth endoplasmic reticulum often
encircling the mitochondria.
In solid lung tumors, the cells were typically packed with
large lamellar bodies (Fig. 6, A and B). Various osmiophilic
stages of lamellar body formation (Fig. 6B) were observed as
described for the normal type II cell (40). The cross-sectional
profile of cells of solid tumors and their nuclei were generally
round, and the free surfaces showed several irregular microvilli
(Fig. 6A). Cells were connected by interdigitating membranes
(Fig. dB) and tight junctions. Glycogen granules were scattered
throughout the cytoplasm.
An early papillary tumor with apparent transitional features
is shown in Fig. 7. All cells contained irregularly shaped dense
bodies, multivesicular bodies, and occasionally mature lamellar
bodies (Fig. 7, inset). The cell shape was elongated, resembling
fetal type II cells (Fig. 8), and the nuclei were mostly round,
located basally. The surface of the tumor cells possessed nu
merous microvilli. Mitochondria varied largely in number, size,
and shape, but always showed well-developed cristae. Cytoplasmic glycogen was distributed either randomly as single
granules or as larger glycogen deposits similar to those found
in cells of fetal lung (Fig. 8). Further similarities between the
fetal lung and features of papillary tumors are shown in Fig. 9
with regard to cell shape, glycogen deposits, and the occurrence
of primary cilia (41).
In older papillary tumors a further morphological dedifferentiation could be observed. The number of cytoplasmic dense
bodies varied, and mature lamellar bodies were seen less fre
quently, whereas the number of multivesicular bodies was in
creased. Occasionally, dense paracrystalline structures were
seen, appearing rectangular or as a square (Fig. 10.-I) and
surrounded by a membrane similar to that of the dense bodies.
Papillary tumors of 6-mo-old C3H mice shown in Fig. 10, A
and B, were subjected to the silver stain in the same fashion as
the Clara cell specimen in Fig. 5D. The dense and multivesicular
bodies as well as the paracrystalline structures were argentaffin
which proved that they were osmiophilic. Most of the dense
bodies in the papillary tumors did not possess a homogeneous
matrix. Frequently, electron-lucent areas were present, shaped
as crescents or vacuoles, and often located eccentrically (Fig.
10Ä). All older papillary tumors possessed cells with lipid
vacuoles or droplets that were frequently surrounded by glyco
gen deposits (Figs. 9F and 10, A and C). The cell shape was
usually tall columnar, and the nuclei were regularly cleaved and
occasionally contained nuclear pseudoinclusions of mature la-
mvb 'i.
m
v'^'Vf-
-•
-li
Fig. 6. Electron micrographs of solid lung tumor from C3H mouse. In A, cells are round or pyramidal with microvilli on their free surface and numerous large,
cytoplasmic lamellar bodies (Ih): m, mitochondria. I nun I acetate/ammonium silver nitrate, x 5,250. B, formation of lamellar bodies (Ib) from multivesicular bodies
(mvb); large, dense incomplete lamellar body in upper-right corner, whorled round cytoplasmic sequestrations (sq) with glycogen granules. I ram 1acetate/ammonium
silver nitrate, x 32,400.
154
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
In the periphery of all older papillary tumors, cells were
present with cytoplasmic accumulations of osmiophilic, lami
nated material that was only preserved in epoxy resin-embedded
material (Fig. 9G). Fig. 9F shows a semithin section of a
papillary tumor with many cells possessing glycogen and lipid
vacuoles as well as macrophages with crystal-like inclusions of
unknown origin. Ultrastructurally, these configurations were
membrane bound and electron lucent.
DISCUSSION
The two morphological variants of mouse lung tumors have
commonly been considered benign (solid/alveolar adenoma) or
malignant (papillary carcinoma) growths arising from alveolar
type II cells (2). The papillary pattern of growth has been found
both as a uniform feature (1) and in foci within solid tumors
(4, 42). To our knowledge, no malignant solid neoplasms have
been described in detail, although such tumors are transplantable (4).
The present study on solid mouse lung tumors induced by
transplacental exposure to NEU confirms their origin from the
alveolar type II cell. Tumor cells in tissue sections were con
sistently large, with round profiles and round nuclei, abundant
pleomorphic lamellar bodies, and a strongly positive immunohistochemical reaction for intracytoplasmic surfactant apoproteins using two different antibodies.
No biochemical, immunocytochemical, or morphological evi
Fig. 7. Electron micrograph of early papillary (tubular) lung tumor in 4-wkdence
could be found for the proposed Clara cell origin (11) of
old C3H mouse. Cells are pyramidal or cuboidal in cell shape, nuclei located
the papillary lung tumor in either C3H or Swiss Webster mice.
basally are mostly round, one cleaved nucleus. Free surface with numerous
microvilli, and cytoplasm contains many dense secretory granules, p, glycogen; *,
Therefore, suspected strain differences (43) do not seem to be
area enlarged in insel showing lamellar and dense bodies. Uranyl acetate/ammo
justified.
As previously shown by others (1,5,44), serial sections
nium silver nitrate, x 5,250. Inset, x 13,920.
failed to reveal original growth from the bronchioles, the site
of the Clara cell. According to recent understanding of lung
differentiation (32, 33, 45-47), an early, strictly separate devel
opment of the alveolar and the bronchiolar systems is recogniz
able in the mouse from the 14th day of gestation onward.
Therefore, it appears unlikely that Clara cells are present in
alveolar walls (15) or that type II cells are transformed into
Clara cells. Serial sections of larger, grossly visible, invading
tumors can give no evidence as to their site of origin (8, 9). Few
of the early tumors seen in serial sections in the present study
were not separated by alveolar walls from bronchiolar lumina.
These probably arose from the alveolar tubules (46) (formerly
termed the distal part of the respiratory bronchiole). As the
tumors increased in size and invasive growth with age, more
neoplasms of both morphological types were found within
bronchioles. This may account for different rates of airway
invasion noted by others (48). However, 50% of both tumor
types originated from alveolar walls adjacent to the pleura,
where mouse lung tumors have always been well recognizable
at necropsy (2, 49).
The present study of serial sections from mixed tumors
containing both solid and papillary structures gave no evidence
for invasion of one neoplasm by the other (11, 13). As suggested
previously (4, 42, 50, 51), the mixed lung tumors probably
represent a stage of tumor progression from a benign solid,
alveolar type II cell adenoma to a less well-differentiated, ma
Fig. 8. Electron micrograph of normal alveolar type II precursor cells on
gestation Day 18 from C3H mouse; mv, microvilli; m, mitochondria; /ft, lamellar
lignant papillary tumor. In lung tumors arising naturally in
bodies; g, glycogen deposits. Uranyl acetate/ammonium silver nitrate, x 10,920.
mice not given a carcinogen, tumor appearance and dedifferentiation start relatively late and can extend over almost the
entire life span of the mouse (50). Development is accelerated
mellar bodies (Fig. IOC). In all types of mouse lung tumors,
in chemically induced tumors and varies with the strain and
cytoplasmic membranes were often present, forming seques
tered circles or rod-like structures (Figs. 6 and \OA and C) age of the mouse at treatment (4), type of compound, and
dosage (52). In transplacental carcinogenesis, the target tissue
often in connection with glycogen.
155
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
Tf*¿v•'
i "TF
V*,
•
-V. ^p^
r* ^*^T
Fig. 9. Comparison of features from C3H mouse lung tumors with normal fetal C3H mouse lung.. I. electron micrograph of cells from papillary lung tumor from
C3H mouse. Cuboidal cell shape; indented nuclei; variably shaped, homogenous, or vesiculated dense bodies; and lamellar bodies. Uranyl acetate/ammonium silver
nitrate, x 7,000. B, electron micrograph of normal alveolar type II precursor cells on gestation Day 16 from C3H mouse; note cuboidal shape, basally oriented nuclei,
glycogen deposits, dense bodies (arrows), and primary cilium (arrow head). Uranyl acetate/ammonium silver nitrate, x 5,250. C, electron micrograph of cell from
papillary lung tumor from C3H mouse. Primary cilium, glycogen deposits, multivesicular and dense bodies. Uranyl acetate/ammonium silver nitrate, x 21,600. D,
electron micrograph from normal fetal lung epithelium on gestation Day 16 from C3H mouse with primary cilium. Uranyl acetate, x 21,600. E, semithin section of
normal C3H mouse alveolar type II precursor cell on gestation Day 16; note cuboidal cell shape, basally oriented nuclei, and glycogen deposits (arrows). Ammonium
silver nitrate, x 630. /•',
semithin section of papillary lung tumor from C3H mouse aged 6 mo. Columnar cells with glycogen deposits and lipid vacuoles. Alveolar
macrophages with crystals in lumen. Ammonium silver nitrate, x 630. (i. semithin section from papillary lung tumor of C3H mouse aged 6 mo with cells containing
congested osmiophilic, lamellar material. Ammonium silver nitrate, x 630.
is different from that of postnatal lung, and the induction period
of lung tumors is reduced even further (24, 44). Therefore,
direct comparisons of lung tumor latency periods according to
tumor type (23) should only be done if the conditions of tumor
induction are considered accordingly. It seems very unlikely
that papillary mouse lung tumors should have a different origin
depending on whether they arise de novo or from within solid
neoplasms.
Dehydrogenase enzymes (mainly 3-hydroxybutyrate dehydrogenase) are present in large quantities in Clara cells of
various species including the mouse and can be demonstrated
histochemically by NBT formazan staining (25). Not a single
lung tumor of C3H or Swiss mice studied for NBT reducÃ-ase
activity revealed a staining intensity exceeding that of the
surrounding normal alveolar septa. In contrast, one conflicting
report described 13 of 19 papillary tumors induced transplacentally in Swiss mice by NEU as possessing significant
activity (17). The reason for this difference is not immediately
clear, since mouse strain, NEU dose, and the day of treatment
were the same as ours. However, it must be remembered that
156
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
•W
A
\
sq
B
Fig. 10. Electron micrographs of papillary lung tumors from C3H mice aged 6 mo. I. high columnar cells with microvilli, interdigitating membranes, bizarre
cleaved nuclei, lipid vacuoles, osmiophilic dense bodies, membrane-bound paracrystalline structure (arrow), longitudinal sequestrations (sq), and granular glycogen
(A')-Ammonium silver nitrate, x 6,562. B, assortment of multivesicular, dense, and partially lamellated bodies in cytoplasm. Ammonium silver nitrate, x 29.400. C,
lamellar nuclear inclusion. Cytoplasm with lipid vacuoles and sequestrations (SQ).Uranyl acetate/lead citrate, x 16,380.
were based on the presence of dense secretory granules, mem
brane-bound crystalline structures, numerous microvilli, and
expected to be present in some tumors of alveolar type II cell interdigitating membranes between adjacent cells, as well as the
origin as observed in the present study, especially if the inhibi- cuboidal-to-columnar cell shape and deeply cleaved nuclei (11,
tive pretreatment with formalin is omitted (26).
53). The shape of papillary tumor cells bears a resemblance to
Major arguments for the Clara cell origin of papillary tumors
Clara cells, but also resembles epithelial cells in the fetal mouse
157
type II cells also contain the enzyme but in smaller quantities
(25). It is thus not a specific1marker for Clara cells and can be
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HIST(X7ENESIS OF MOUSE LUNG TUMORS
lung. Likewise, the reappearance of primary cilia and the high
glycogen content in some cells of the papillary tumors signal a
regressive development resembling a fetal stage. Although gly
cogen is also present in developing Clara cells, this occurs only
relatively late in gestation, whereas glycogen is consistently
present in alveolar type II precursor cells. Previous biochemical
studies also noted an enzyme expression in mouse lung tumors
comparable to the fetal lung that was absent in mature alveolar
type II cells (2). Parallel reexpressions of fetal properties are
known for other organs and their tumors (54).
Increasing quantities of neutral lipids, found with age in the
papillary tumors in the present study, could result from a
biochemical transformation of the glycogen. Frequently, both
were seen together within the same cell. Lipid droplets were
found in mouse alveolar type II cells during early postnatal
development (55) and in tissue cultures of normal alveolar type
II cells from rats (56).
The use of antigenic markers utilizing immunological tech
niques for cell identifications has become widely acknowledged.
Also as shown previously (16), not a single papillary neoplasm
of more than 150 tumors in the present study was immunoreactive for Clara cell antigens. However, if tested for mouse
surfactant apoproteins with two different antibodies using the
ABC and PAP technique and indirect immunofluorescence, all
solid tumors and the majority of papillary tumors reacted
positively. Staining intensity was lower in the papillary tumors
than in normal type II cells, probably as a result of dedifferentiation with partial loss of mature antigenic determinants. These
results clearly confirm those obtained with an anti-rat surfactant
antibody showing mouse lung tumors to be of type II cell origin
(16). SAALS is able to detect type II cell antigens at a very
early stage of lung development in the mouse (33; Day 14 of
gestation versus first day of life for anti-SAP-M) and in the rat
(36; Day 16 of gestation). Therefore it appears that SAALS is
specific for a different range of surfactant antigens. Variations
in antibody specificity could in part account for different ultrastructural localizations of surfactant (57-60), since the antisera
were prepared at different locations, at different times, or
according to different methods.
To clarify discrepancies between previous electron micro
scopic (11) and immunocytochemical findings (16), an addi
tional ultrastructural study was performed. Other ultrastruc
tural investigations, presenting the alveolar type II cell as origin
for mouse lung tumors, sometimes did not strictly separate
solid and papillary tumor types. Nevertheless, it is obvious from
descriptions and micrographs that the authors dealt with pap
illary tumors (6, 7, 61-63), and we can confirm most of their
observations. As evidence for the Clara cell origin of papillary
tumors, osmiophilic bodies and dense secretory granules were
shown in specimens stained conventionally with toluidine blue
or uranyl acetate (11, 15). Therefore, no distinction was possible
between nonosmiophilic electron-dense secretory granules of
Clara cells (64-66) and other osmiophilic dense bodies. Crys
tals, quoted as Clara cell markers (11, 15), have never been
described in mouse Clara cells (64-73) and cannot, therefore,
be considered as morphological evidence that mouse papillary
tumors originate from Clara cells. Membrane-bound crystallike formations have previously (7, 74), however, been noted in
lung tumors of mice, and we found them to be osmiophilic.
Multiple microvilli, characteristic of papillary tumors, con
trasted with the relatively smooth surface of Clara cells, and
the tumors usually possessed more rough endoplasmic reticulum (75) as opposed to the abundance of smooth endoplasmic
reticulum in Clara cells (67, 72, 73). Cleaved nuclei, interdigi-
tating membranes, and tight junctions represent nonspecific
features also found in normal alveolar type II cells and solid
tumors. Characteristic mouse Clara cell mitochondria (65, 73),
however, were not present in the papillary tumors.
Significant structures found in papillary tumors were osmio
philic multivesicular bodies (Figs. 9Cand IOB). M ulti vesicular
bodies have been noted as a source of lamellar body formation
(40) and a site of localization of surfactant antigens in alveolar
type II cells (57-60). Clara cells may also possess multivesicular
bodies (68). However, these are infrequent, lack a dense matrix,
and show no lamellar formations. Some of the dense cell
organelles seen in the tumors are probably lysosomes, but many
of them are most likely involved in the production of surfactant,
explaining the high incidence of tumors reacting positively for
SAP-M and with SAALS. The formation of mature or imma
ture lamellar bodies depends on surrounding growth conditions
(76, 77), and cell organelles can even be lost (77), as in the
normal alveolar type I cell derived from the alveolar type II
cell (78). Earliest prenatal alveolar cells possess membranebound round granules with a dense matrix which have been
suggested to represent precursors of lamellar bodies (47, 79).
Some lamellar configurations are known to occur as artifacts
(80). Such structures can be of lysosomal origin and are found
in all cell types; they are inducible by drugs (81). Myelin figures
similar to lamellar bodies have not been described to occur in
normal mouse Clara cells (64-73). One could argue against the
specificity of lamellar bodies found in papillary tumors as
evidence for alveolar type II cell function if the identification is
by morphology alone. However, the same should then be ap
plied to their presence in solid tumors. Immunoultrastructural
studies on the localization of surfactant apoproteins in papillary
mouse lung tumors should help clarify these questions.
Tubular myelin constitutes the hypophase of the surfaceactive alveolar surfactant layer (82, 83) and is phagocytized by
macrophages (84, 85). Tubular myelin was observed in the
lumina of papillary mouse lung tumors, and myelin figures were
also present in some of 18 putative Clara cell tumors (11). Its
origin within complex papillary tumors could stem from some
remaining normal alveolar type II cells or from tumor cells.
Osmiophilic lamellar accumulations observed in alveolar type
II cells surrounding or within papillary and mixed tumors (17)
probably represent a disturbed host response that can also be
induced by radiation (86) or bleomycin (87). The significance
of membranes in the cytoplasm of tumor cells (Figs. 6 and 10,
A and C) is unknown (6, 88, 89). They might be comparable to
the bar-like structures seen in rat alveolar type II cells (90) and
have been suggested to be involved in surfactant production
(47).
Human lung tumors with immunological markers of alveolar
type II cells have been described to consist of cuboidal or high
columnar cells and to grow in a papillary pattern (91-94).
These tumors showed intranuclear staining for surfactant that
was attributed to ultrastructural tubular configurations and may
represent nuclear membranes with surfactant apoprotein (93).
Only recently have the tubular intranuclear inclusions been
suggested as a marker for human alveolar cell carcinoma (95).
In the present study and in others on mouse lung tumors (6,
61, 89, 96), nuclear pseudoinclusions consisting of membrane
invaginations with cytoplasmic contents, mainly lamellar struc
tures, were a consistent finding in older papillary tumors and
are probably responsible for the positive intranuclear surfactant
staining (16).
In summary, we could not find specific enzymatic, antigenic,
morphological features in the papillary tumors of the mouse
158
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS OF MOUSE LUNG TUMORS
71-83. Lyon: International Agency for Research on Cancer, 1973.
25. Etherton, J. E., and Conning, D. M. Enzyme histochemistry of the lung. In:
Y.S. Bakhle and J. R. Vane (eds.). Metabolic Functions of the Lung, pp.
233-259. New York: Marceli Dekker, Inc., 1978.
26. Devereux, T. R., and Fouts, J. R. Isolation and identification of Clara cells
from rabbit lung. In Vitro, 16:958-968. 1980.
27. Singh, G., and Katayal, S. L. An immunolgic study of the secretor)- products
of rat Clara cells. J. Histochem. Cytochem., 32:49-54. 1984.
28. Katyal, S. L.. and Singh, G. An immunologie study of the apoproteins of rat
lung surfactant. Lab. Invest., 40: 562-567, 1979.
29. Katyal, S. L., and Singh, G. Structural and ontogenetic relationships of rat
lung surfactant apoproteins. Exp. Lung Res., 6: 175-189, 1984.
30. Singh. G., Katyal, S. L., Ward, J. M., Gottron, S. A., Wong-Chong, M.-L.,
and Riley, E. J. Secretory proteins of the lung in rodents: immunocytochemistry. J. Histochem. Cytochem., 33: 564-568, 1985.
31. McMahon, J. B., Smith, A. C., del Campo. A., Singh, G., Katyal, S. L., and
Schuller, H. M. Characterization of rat alveolar type II cells in vitro by
immunological, biochemical, and morphological criteria. Exp. Lung Res.,
77:263-275,1986.
32. Ten Have-Opbroek, A. A. W. Immunological study of lung development in
the mouse embryo. Appearance of a lung-specific antigen, localized in the
great alveolar cell. Dev. Biol., 46: 390-403, 1975.
33. Ten Have-Opbroek, A. A. W. Immunological study of lung development in
the mouse embryo. II. First appearance of the great alveolar cell, as shown
by immunofluorescence microscopy. Dev. Biol., 69: 408-423, 1979.
34. Van Hemert, F. J., Ten Have-Opbroek, A. A. W., and Otto-Verberne, C. J.
M. Histochemical characterization of an antigen specific for the great alveolar
cell in the mouse lung. Histochemistry, 85: 497-504, 1986.
35. Ward, J. M., Reynolds, C. W., and Argilan, F. Immunoperoxidase localiza
tion of large granular lymphocytes in normal tissues and lesions of athymic
nude rats. J. Immuni,!.. 131: 132-139. 1983.
36. Otto-Verberne, C. J. M., and Ten Have-Opbroek, A. A. W. Development of
the pulmonary acinus in fetal rat lung: a study based on an antiserum
recognizing surfactant-associated proteins. Anat. Embryol., 175: 365-373,
1987.
37. De Jong, A. S. H., Van Kessel-Van Vark, M., and Raap. A. K. Sensitivity of
various visualization methods for peroxidase and alkaline phosphatase activ
ity in immunoenzyme histochemistry. Histochem. J., 17: 1119-1130, 1985.
38. Hayat, M. A. Principles and Techniques of Electron Microscopy, Vol., 1, pp.
370-383. Baltimore: University Park Press, 1981.
39. Klein, W., and Bock, P. Argentaffin reaction of glycogen in semithin and
thin sections. Mikroskopie, 41: 318-325, 1984.
40. Sorokin, S. P. A. morphologic and cytochemical study on the great alveolar
cell. J. Histochem. Cytochem., 14: 884-897, 1967.
41. Sorokin, S. P. Reconstructions of ceninoli- formation and ciliogenesis in
mammalian lungs. J. Cell Sci., 3: 207-230, 1968.
42. Mori, K., and Hirafuku, I. Histogenesis of lung carcinoma in mice induced
by 4-nitroquinoline 1-oxide: carcinoma arising from areas of adenoma. Gann,
55:205-209, 1964.
43. Witschi, H., and Haschek, W. M. Letters to the editor. Cells of origin of
lung tumors in mice. J. Nati. Cancer Inst., 70: 991, 1983.
44. Smith, W. E., and Rous, P. The neoplastic potentialities of mouse embryo
tissues. IV. Lung adenomas in baby mice as result of prenatal exposure to
urethane. J. Exp. Med., 88: 529-563, 1949.
45. Ten Have-Opbroek, A. A. W. The development of the lung in mammals: an
analysis of concepts and findings. Am. J. Anat., 162: 201-219, 1981.
46. Ten Have-Opbroek, A. A. W. The structural composition of the pulmonary
acinus in the mouse. A scanning electron microscopical and developmentalbiological analysis. Anat. Embryol., 774:49-57, 1986.
47. Ten Have-Opbroek, A. A. W., Dubbeldam, J. A., and Otto-Verberne, C. J.
M. Ultrastructural features of type-II alveolar epithelial cells in early embry
onic mouse lung. Anat. Ree., in press, 1988.
48. Anderson, L. M., and Budinger. J. M. Qualitative histological differences
between transplacentally-induced lung tumors in young and aging mice.
Cancer Lett., //: 285-293. 1981.
49. Stewart, H. L., Dunn, T. B., and Snell, K. C. Pathology of tumors and
nonneoplastic proliferative lesions of the lungs of mice. In: P. Nettesheim,
M. G. Hanna, and J. W. Deatherage (eds.). Morphology of Experimental
Carcinogenesis, No. 21 AEC Symp. Ser., pp. 161-181. Oak Ridge: ESAEC
Technical Information Center, 1970.
50. Deerberg, F., Pittermann, W., and Rapp, K. G. Der Lungentumor der Maus:
ein progressiver Tumor. Vet. Pathol., 77:430-441, 1974.
51. Matsuzaki, O. Histogenesis and growing patterns of lung tumors induced
by potassium
1-methyl-l,4-dihydro-7-[2-(5-nitrofuryl)vinyl]-4-oxo1,8-na
phthyridine-3-carboxylate in ICR mice. Gann. 66: 259-267, 1975.
52. Shimkin, M. B. Pulmonary tumors in experimental mice. Adv. Cancer Res.,
3: 223-267, 1955.
53. Fry, R. J. M., and Witschi, J. P. Lung tumors in mice. In: J. F. Douglas
(ed.), Carcinogenesis and Mutagenesis Testing, pp. 63-78. Clifton: Humana
Press, 1984.
54. Escribano, M. J., Cordier, J.. Nap, M.. Ten Kate, F. J. W., and Burtin, P.
Differentiation antigens in fetal human pancreas. Reexpression in cancer.
Int. J. Cancer. 38: 155-160. 1986.
55. Blanchette-Mackie, E. J., Wetzel, M. G., Chernick, S. S., Paternitis, J. R.,
Brown, W. V., and Scow, R. O. Effect of the combined lipase deficiency
mutation (cld/cld) on ultrastructure of tissues in mice. Lab. Invest., 55: 347362, 1986.
lung that were unequivocally specific for Clara cells and that
would clearly support an origin of these tumors from Clara
cells. Substantial evidence is presented, however, for the alveo
lar type II cell as the origin of the papillary tumor. These
features include origin from the pulmonary acinus, the high
incidence of tumors immunoreactive to the different surfactant
apoproteins, and the ultrastructural presence of cell organelles
specific for alveolar type II cells.
ACKNOWLEDGMENTS
The authors thank John Henneman for excellent photographical
assistance and Deborah Devor, Shirley Hale, Dr. Ursula Heine, Susan
Kenney, luana Muñoz,Kunio Nagashima, Eleonore Stanfield, Cindy
Thompson, and Dr. Martin Wenk for advice and technical support.
REFERENCES
1. Grady, H. G., and Stewart, H. I Histogenesis of induced pulmonary tumors
in strain A mice. Am. J. Pathol., 16: 417-432, 1940.
2. Shimkin, M. H.. and Stoner, G. D. Lung tumors in mice: application to
carcinogenesis bioassay. Adv. Cancer Res., 21: 1-58, 1975.
3. Stewart, H. L., Dunn, T. B., Snell, K. C, and Deringer, M. K. Tumors of
the respiratory tract. In: V. S. Turusov (ed.). Pathology of Tumours in
Laboratory Animals, Vol. 2, pp. 251-287. Lyon: International Agency for
Research on Cancer, 1979.
4. Riunirà . I. Progression of pulmonary tumor in mice. 1. Histológica! studies
of primary and transplanted pulmonary tumors. Acta Pathol. Jpn., 21: 1356, 1971.
5. Mostofi, F. K., and Larsen, C. D. The histogenesis of pulmonary tumors
induced in strain A mice by urethan. J. Nati. Cancer lust.. //. 1187-1221,
1951.
6. Svoboda, D. J. Ultrastructure of pulmonary adenomas in mice. Cancer Res.,
22: 1192-1201, 1962.
7. Brooks, R. E. Pulmonary adenoma of strain A mice: an electron microscopic
study. J. Nati. Cancer Inst., 41: 719-742, 1968.
8. Mori, K. Further studies on the histogenesis of pulmonary tumors in mice
induced by 4-nitroquinoline 1-oxide. Gann, 55: 315-323, 1964.
9. Amaral-Mendes, J. J. Histopathology of primary lung tumours in the mouse.
J. Pathol., 97: 415-427, 1969.
10. Kanisawa, M. Induction of pulmonary tumors in mice by oral administration
of a 5-nitrofuran derivative. In: E. Karbe and J. F. Park (ed.). Experimental
Lung Cancer, pp. 246-256. New York: Springer-Verlag, I974.
11. Kauffman, S. L., Alexander, L., and Sass, L. Histologie and ultrastructural
features of the Clara cell adenoma of the mouse lung. Lab. Invest., 40: 708716, 1979.
12. Sato, T., and Kauffman, S. L. A scanning electron microscopic study of the
type II and Clara cell adenoma of the mouse lung. Lab. Invest., 43: 28-36,
1980.
13. Kauffman, S. L. Histogenesis of the papillary Clara cell adenoma. Am. J.
Pathol.. 103: 174-180. 1981.
14. Parsa, I., and Kauffman, S. L. Malignant Clara cell line derived from ethylnitrosourea-induced murine lung adenomas. Cancer Lett., IK: 311 -316,1983.
15. Kauffman, S. L., and Sato, T. Bronchiolar adenoma, lung, mouse. In: T. C.
Jones, U. Mohr, and R. D. Hunt (eds.). Respiratory System, Monographs
on Pathology of Laboratory Animals, pp. 107-111. Berlin: Springer-Verlag,
1985.
16. Ward, J. M., Singh, G., Katyal, S. L., Anderson. L. M., and Kovatch, R. M.
Immunocytochemical localization of the surfactant apoprotein and Clara cell
antigen in chemically induced and naturally occurring pulmonary neoplasms
of mice. Am. J. Pathol., 118:493-499, 1985.
17. Palmer, K. C. Clara cell adenomas of the mouse lung. Interaction with
alveolar type II cells. Am. J. Pathol., 120:455-463, 1985.
18. Palmer, K. C., and Grammas, P. /3-Adrenergic regulation of secretion from
Clara cell adenomas of the mouse lung. Lab. Invest., 56: 329-334, 1987.
19. Beer, D. G., and Malkinson, A. M. Genetic influence on type II or Clara cell
origin of pulmonary adenomas in urethan-treated mice. J. Nati. Cancer Inst.,
75; 963-969, 1985.
20. Malkinson, A. M., and Thaete, L. G. Effects of strain and age on prophylaxis
and co-carcinogenesis of urethan-induced mouse lung adenomas by butylated
hydroxytoluene. Cancer Res., 46: 1694-1697, 1986.
21. Lindenschmidt, R. C., Tryka, A. F., and Witschi, H. P. Inhibition of mouse
lung tumor development by hyperoxia. Cancer Res., 46: 1994-2000, 1986.
22. Last, J. A., Warren, D. L., Pecquet-Goad, E., and Witschi, H. Modification
by ozone of lung tumor development in mice. J. Nati. Cancer Inst., 78: 149154, 1987.
23. Thaete, L. G., Gunning, W. T., Stoner, G. D., and Malkinson, A. M. Cellular
derivation of lung tumors in sensitive and resistant strains of mice: results at
28 and 56 weeks after urethan treatment. J. Nati. Cancer Inst., 78:743-749,
1987.
24. Rice, J. M. The biological behaviour of transplacentally induced tumors in
mice. In: L. Tomatis and U. Mohr (eds.), Transplacental Carcinogenesis, pp.
159
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
HISTOGENESIS
OF MOUSE LUNG TUMORS
56. Mason, R. J., and Williams. M. C. Phospholipid composition and ultrastruc
ture of A549 cells and other cultured pulmonary epithelial cells of presumed
type II cell origin. Biochim. Biophys. Acta, 617: 36-50, 1980.
57. Williams, M. C., and Benson, B. J. Immunocytochemical localization and
identification of the major surfactant protein in adult rat lung. J. Histochem.
Cytochem., 29:291-305, 1981.
58. Balis, J. U., Paterson, J. F., Paciga, J. E., Haller, E. M., and Shelley, S. A.
Distribution and subcellular localization of surfactant associated glycoproteins in human lung. Lab. Invest., 52: 657-669, 1985.
59. Walker, S. R., Williams, M. C., and Benson, B. Immunocytochemical local
ization of the major surfactant apoproteins in type II cells, Clara cells, and
alveolar macrophages of rat lung. J. Histochem. Cytochem., 34:1137-1148,
1986.
60. Coalson, J. J., Winter, V. T., Martin, H. M., and King, R. J. Colloidal gold
immunoultrastructural localization of rat surfactant. Am. Rev. Respir. Dis.,
133: 230-237, 1986.
61. Flaks, B., and Flaks, A. Fine structure of murine pulmonary adenomata
induced by carcinogen treatment in organ culture. Cancer Res., 29: 17811789, 1969.
62. Toth, B., and Shimizu, H. l-Carbamyl-2-phenylhydrazine tumorigenesis in
Swiss mice. Morphology of lung adenomas. J. Nati. Cancer Inst.. 52: 241251, 1974.
63. Franks, L. M., Carbonell, A. W., Hemmings, V. J., and Riddle, P. N.
Metastasizing tumors from serum-supplemented and serum-free cell lines
from a C57BL mouse lung tumor. Cancer Res., 36: 1049-1055, 1976.
64. Stinson, S. F., and Loosli, C. G. Ultrastructural evidence concerning the
mode of secretion of electron-dense granules by Clara cells. J. Anal.. 727:
291-298, 1978.
65. Plopper, C. G., Mariassy, A. T., and Hill, L. H. Ultrastructure of the
nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. I. A
comparison of rabbit, guinea pig, rat, hamster, and mouse. Exp. Lung Res.,
/: 139-154, 1980.
66. Widdicombe, J. G., and Pack, R. J. The Clara cell. Eur. J. Respir. Dis., 63:
202-220, 1982.
67. Karrer, H. E. Electron microscopie study of bronchiolar epithelium of normal
mouse lung. Exp. Cell Res., 10: 237-241, 1956.
68. I lutiseli, M. M., and Moretti, R. L. Ultrastructure of the mouse trachéal
epithelium. J. Morphol., 128: 159-170, 1969.
69. Petrik, P., and Collet, A. J. Infrastructure des cellules bronchiolaires non
ciliées
chez la souris. Rev. Cancer Biol., 29: 141-152, 1970.
70. Etherton, J. E., Conning, D. M., and Corrin, B. Autoradiographical and
morphological evidence for apocrine secretion of dipalmitoyl lecithin in the
terminal bronchiole of mouse lung. Am. J. Anal., 138: 11-36, 1979.
71. Pack, R. J., Al-Ugaily, L. H., and Morris, G. The cells of the tracheobronchial epithelium of the mouse: a quantitative light and electron micro
scope study. J. Anat., 132: 71-84, 1981.
72. Lauweryns, J.-M., Cokelaere, M., and Boussauw, L. L'ultrastructure de
('epithelium bronchique et bronchiolaire de la souris. C. R. Assoc. Anat.,
146: 548-560, 1971.
73. Smith, M. N., Greenberg, S. D., and Spjut, H. J. The Clara cell: a comparative
ultrastructural study in mammals. Am. J. Anat., 755: 15-30, 1979.
74. Flaks, B., and Flaks, A. Electron-microscope observations on the formation
of the cytoplasmic lamellar inclusion bodies in murine pulmonary tumors
induced in vitro. J. Pathol., 70«:211-217, 1972.
75. Brooks, R. E., and Adkinson, B. Intracellular components of neoplastic and
normal alveolar cells from mouse lungs: quantitative ultrastructural compar
ison. J. Nail. Cancer Inst., 47: 639-644, 1971.
76. Smith, G. J., Le Mesurier, S. M., De Montfort, M. L., and Lykke, A. W. J.
Development and characterization of type II pneumocyte-related cell lines
from normal adult mouse lung. Pathology, 16:401-405, 1984.
77. Stoner, G. D., Kikkawa, Y., Kniazeff, A. J., Miyai, K., and Wagner, R. M.
Clonal isolation of epithelial cells from mouse lung adenoma. Cancer Res.,
55:2177-2185, 1975.
78. Adamson, I. Y. R., and Bowden, D. H. The type II cell as progenitor of
alveolar epithelial regeneration. Lab. Invest., 30: 35-42, 1974.
79. Williams, M. C., and Mason, R. J. Development of the type II cell in the
fetal rat lung. Am. Rev. Respir. Dis., 7/5: 37-47, 1977.
80. Le Beux, Y., Hetenyi, G., and Phillips, M. J. Mitochondria! myelin-like
figures: a non-specific reactive process of mitochondria! phospholipid mem
branes to several stimuli. Z. Zellforsch., 99:491-506, 1969.
81. Reasor, M. J. Phospholipidosis in the alveolar macrophage induced by
cationic amphiphilic drugs. Fed. Proc., 43: 2478-2581, 1984.
82. Beckmann, H.-J., and Dierichs, R. Extramembraneous particles and struc
tural variations of tubular myelin figures in rat lung surfactant. J. Ultrastruct.
Res., 86: 57-66, 1984.
83. Nakamura, H., Tonosaki, A., Washioka, H., Takahashi, K., and Yasui, S.
Monomolecular surface film and tubular myelin figures of the pulmonary
surfactant in hamster lung. Cell Tissue Res., 241: 523-528, 1985.
84. Nichols, B. A. The vacuolar apparatus of alveolar macrophages and the
turnover of surfactant. In: R. van Furth (ed.), Mononuclear Phagocytes.
Functional Aspects, Part I, pp. 5-152. The Hague: Martinus Nijhoff Pub
lishers, 1980.
85. Fasske, E., and Morgenroth, K. Functional morphology of phagocytosing
alveolar macrophages. Virchows Arch., 49:195-208, 1985.
86. Heppleston, A. G., and Young, A.E. Population and ultrastructural changes
in murine alveolar cells following 23'PuOz inhalation. J. Pathol., 146: 155166, 1985.
87. Fasske, E., and Morgenroth, K. Experimental bleomycin lung in mice. A
contribution to the pathogenesis of pulmonary fibrosis. Lung, 767:133-146,
1983.
88. Klarner, P., and Gieseking, R. Zur Ultrastruktur des Lungentumors der
Maus. Z. Krebsforsch., 64:7-21, 1960.
89. Brooks, R. E. Ultrastructure of mouse pulmonary adenomas. In: P. Nettesheim, M. G. Hanna, and J. W. Deatherage (eds.), Morphology of Experimental
Respiratory Carcinogenesis, No. 21 AEC Symp. Ser., pp. 185-202. Oak
Ridge: USAEC Technical Information Center, 1970.
90. Shimura, S., Aoki, T., Tomioka, M., Shindoh, Y., and Takishima, T. Con
centrically arranged endoplasmic reticulum containing some lamellae (barlike structure) in alveolar type II cells of rat lung. J. Ultrastruct. Res., 93:
116-128, 1985.
91. Singh, G., Katyal, S. L., and Torikata, C. Carcinoma of type II pneumocytes.
Immunodiagnosis of a subtype of "bronchioloalveolar carcinomas." Am. J.
Pathol., 702: 195-208, 1981.
92. Singh, G., Scheithauer, B. W., and Katyal, S. L. The pathobiologic features
of carcinomas of type II pneumocytes. An immunocytologic study. Cancer
(Phila.), 57: 994-999, 1986.
93. Dairaku, M., Sueishi, K., Tanaka, K., and Morie, A. Immunohistological
analysis of surfactant-apoprotein in the bronchiole-alveolar carcinoma. Vir
chows Arch., 400: 223-234, 1983.
94. Fasske, E. Das bronchopulmonale Adenocarzinom: Gewebliche Differenzi
erung und zellulare Herkunft. Pathologe, 5: 3-12, 1984.
95. Ghadially, F. N., Harawi, S., and Khan, W. Diagnostic ultrastructural mark
ers in alveolar cell carcinoma. J. Submicrosc. Cytol., 17: 269-278,1985.
96. Hattori, S., Matsuda, M., and Wada, A. An electron microscopic study of
pulmonary adenomas in mice. Gann, 56: 275-280, 1965.
160
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
Mouse Papillary Lung Tumors Transplacentally Induced by N
-Nitrosoethylurea: Evidence for Alveolar Type II Cell Origin by
Comparative Light Microscopic, Ultrastructural, and
Immunohistochemical Studies
Sabine Rehm, Jerrold M. Ward, Ank A. W. Ten Have-Opbroek, et al.
Cancer Res 1988;48:148-160.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/48/1/148
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.