Download Model for Orthotopic Propagation of Human

[CANCER RESEARCH 47, 5132-5140, October I, 1987)
Novel Intrapulmonar}' Model for Orthotopic Propagation of Human Lung Cancers
in Athymic Nude Mice
Theodore L. McLemore,1 Mark C. Liu, Penny C. Blacker, Marybelle Gregg, Michael C. Alley, Betty J. Abbott,
Robert H. Shoemaker, Mark E. Bohlman, Charles C. Litterst, Walter C. Hubbard, Robert H. Brennan,
James B. McMahon, Donald L. Fine, Joseph C. Eggleston, Joseph G. Mayo, and Michael R. Boyd
Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892 [T. L. M., M. G., B. J. A., R. H. S., C. C. L.,
W. C. H., R. H. B., J. B. M., J. G. M., M. R. B.]; Departments of Medicine, Radiology, and Pathology, Johns Hopkins University School of Medicine, Baltimore,
Mailand 21205 [M. C. L., M. E. B., J. C. E.]; and Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701
IP. C. B., M. C. A., D. L. FJ
impeded by the lack of optimal animal models for use in these
investigations (4). Animal models which use carcinogens to
A major impediment to the study of human lung cancer pathophysiolinduce primary pulmonary tumors have been developed, but
ogy, as well as to the discovery and development of new specific antitumor
they have not been satisfactory for routine lung cancer studies
agents for the treatment of lung cancer, has been the lack of appropriate
(5-11). Major disadvantages of the carcinogen-induced animal
experimental animal models. This paper describes a new model for the
models for study of primary pulmonary tumors include: they
propagation of human lung tumor cells in the bronchioloalveolar regions
are time consuming (requiring a minimum of 6 mo for comple
of the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.)
tion); and most importantly, they yield a variety of different
implantation procedure. Over 1000 i.b. implantations have been per
histological tumor cell types which might not be directly rele
formed to date, each requiring 3 to 5 min for completion and having a
surgery-related mortality of approximately 5%. The model was used vant to human lung cancer, and thereby limit their usefulness
for meaningful biochemical, cell biology, and drug-testing stud
successfully for the orthotopic propagation of four established human
ies (5-10).
lung cancer cell lines including: an adenosquamous cell carcinoma (NCIHI 25); an adenocarcinoma (A549); a large cell undifferentiated carci
Athymic nude mouse models have recently been used for the
noma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358).
in vivo propagation and study of human pulmonary cancers.
When each of the four cell lines was implanted i.b. using a 1.0 x 10*' The predominant prototypes which have been used for these
tumor cell inoculum, 100 ±0% (SD) tumor-related mortality was ob
studies are the s.c. xenograft (12-16) and the subrenal capsule
served within 9 to 61 days. In contrast, when the conventional s.c. method
(17-20) models. While these offer rapid and relatively simple
for implantation was used at the same tumor cell inoculum, only minimal
methods for in vivo propagation of human lung tumors, they
(2.5 ±5%) tumor-related mortality was observed within 140 days (P <
have limitations, particularly in their use for the study of lung
0.001). Similarly, when a 1.0 x IO5or 1.0 x Ml4cell inoculum was used,
cancer biology as well as for the discovery and development of
a dose-dependent, tumor-related mortality was observed when cells were
effective treatment modalities for human pulmonary malignan
implanted i.b. (56 ±24% or 25 ±17%) as compared with the s.c. method
cies (12, 15, 16, 20).
(5 ±5.7% or 0.0 ±0%) (P < 0.02 and P < 0.05, respectively). Most
A potential disadvantage of both the s.c. and subrenal capsule
(>90%) of the lung tumors propagated by i.b. implantation were localized
models for the propagation and study of human primary pul
to the right lung fields as documented by necropsy and/or high-resolution
monary malignancies is that tumor cells are not orthotopically
chest roentgenography techniques which were developed for these studies.
The intrapulmonar}- model was also used for establishment and propa
implanted (i.e., tumor cells are not implanted in the lung).
Paget originally proposed that human tumor cell populations
gation of xenografts derived directly from enzymatically digested, fresh
require organ site-specific interaction for optimal maintenance
human lung tumor specimens obtained at the time of diagnostic thoraand progression (21). This concept has been widely supported
cotomy and representing all four major lung cancer cell types as well as
a bronchioloalveolar cell carcinoma. Approximately 35% (10 of 29) of by numerous studies using metastatic tumor models (for review,
the fresh primary human lung tumor specimens and 66% (2 of 3) of see Ref. 12) and by recent athymic nude mouse models which
tumors metastatic to the lung were successfully propagated i.b. at a 1.0 have been used to study the orthotopic propagation of selected
x lit1'tumor cell inoculum, whereas only 20% (1 of 5) of the specimens
human solid tumors, such as renal cell carcinoma (12, 16, 20,
were successfully grown in vivo via the s.c. route from a 1.0 x 10" tumor
22), pancreatic carcinoma (23), and certain brain tumors (24).
cell inoculum. Our experience with this in vivo intrapulmonar} model for A similar approach for the propagation of human lung neo
the orthotopic propagation of human lung tumor cells is consistent with plasms in the lungs of athymic nude mice would appear to offer
the view that organ-specific in vivo implantation of human tumors facil
a potentially very practical and relevant model for the study of
itates optimal tumor growth. This new in vivo lung cancer model may
this disease. This paper describes the successful development
substantially facilitate future studies of the biology and therapeutics of
of a novel intrapulmonary model for the orthotopic propagation
this catastrophic disease.
of human lung cancer cells in athymic nude mice using an i.b.2
implantation technique.
INTRODUCTION
ABSTRACT
Currently, lung cancer is the leading cause of cancer-related
adult deaths in the United States, and its incidence continues
to rise, especially in the adult female population (1-3). Ad
vances in the study of the pathophysiology of human lung
cancer as well as the discovery and development of new nonsurgical treatment modalities for this disease may have been
Received 1/5/87; revised 5/21/87; accepted 7/7/87.
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.
' To whom requests for reprints should be addressed, at NCI-Frederick Cancer
Research Facility, Bldg. 428, Rm. 63, Frederick, MD 21701.
MATERIALS AND METHODS
Continuous Human Lung Tumor Cell Lines. Human tumor cell lines
NCI-HI25, NCI-H358, and NCI-H460 were isolated from fresh solid
2 The abbreviations used are: i.b., intrabronchial; HBSS, Hanks' balanced salt
solution; NCI-FCRF, National Cancer Institute-Frederick Cancer Research Fa
cility, Frederick, MD; NCI-H460, NCI-H460 large cell undifferentiated human
lung carcinoma cell line; NCI-H358, NCI-H358 human lung bronchioloalveolar
human lung carcinoma cell line; NCI-HI25, NCI-HI25 adenosquamous cell
human lung carcinoma cell line; A549, A549 human lung adenocarcinoma cell
line; NCr-na/nw mice, NIH subline cancer research athymic nude mice homozygotic for the nude trait (this nomenclature registered with the Institute of Labo
ratory Animal Resources, National Research Council, National Academy of
Sciences, Washington, DC).
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INTRAPULMONARY
MODEL FOR HUMAN LUNG CANCER
Table 1 Histopathological characterization of surgically resected human lung
tumor specimens propagated by i.b. or s.c. implantation
typePrimary*Squamous
Cell
MD), 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT),
and 2 mM L-glutamine at a density of 0.5 x IO6cells/10-ml/flask. Cells
were maintained at 37°Cin a humidified cabinet in an atmosphere of
im
im
propa
propa
plantedS.C."21101050005No.
planted
5% CÜ2in air. Cell monolayers approaching
gated
S.C.*0000101(20)0001(20)
i.b."12355132921332No.
gated
i.b.*33111110
75% confluency were
harvested using the DeLarco formulation of trypsin-EDTA and were
subcultured for a maximum of 8 to 12 serial passages. For i.b. or s.c.
implantation experiments, cells were isolated by centrifugation at 500
x g for 15 min, washed 3 times in 0.9% saline, suspended in a final
volume of HBSS, and adjusted to appropriate inoculation densities. All
cell lines were documented to be of human origin by isoenzyme and
karyotype analyses and were verified to be free of Mycoplasma as well
as pathogenic mouse viruses at the time of study.
Preparation of Fresh Human Lung Tumors. Fresh human lung tumor
specimens obtained at the time of diagnostic thoracotomy were minced
into 0.5- to 1.0-mm3 pieces and enzymatically digested as previously
cell''AdenocarcinomaSmall
cellLarge
cellLarge
cellBronchioloalveolarSubtotalMetastaticiAdenocarcinoma(prostate)Transitional
cell-small
(35)'112(66)12
cell (uri
bladder)SubtotalTotalNo.
nary
(38)No.
" Fresh human tumor specimens were enzymatically digested and then im
planted into athymic nude mice i.b. at a 1.0 x 10' tumor cell inoculum or s.c. at
a 1.0 x 10' tumor cell inoculum as described in "Materials and Methods."
* Tumors were classified as being successfully propagated only if they were
sustained for at least 2 passages where passage 1 represents initial tumor implan
tation into nude mice.
c Primary lung tumors.
'' All diagnoses were histologically confirmed.
' Numbers in parentheses, percentage of propagation efficiencies.
' Tumors metastatic to the lung.
Fig. 1. Diagram of the i.b. implantation technique. The shaded area on the
diagram represents the caudal lobe of the right lung, the area where the majority
of tumor cells are localized following i.b. implantation.
tumor specimens by the NCI-Navy Medical Oncology Branch using
standard procedures (25), cultivated, characterized under a variety of
growth conditions (26), and kindly provided to the NCI-Developmental
Therapeutics Program by Dr. J. Minna and Dr. A. Gazdar. The A549
cell line is a nude mouse ascites-passaged cell line derived at NCIFCRF from the A549 cell line initially described by Giard et al. (27).
The cell lines used for this study were propagated in vitro utilizing
conventional sterile culture techniques after recovery from cryopreserved seed stock prepared and maintained at the NCI-FCRF tumor
repository. Tumor cells were thawed under sterile conditions, washed
3 times with 0.9% saline, and counted with a hemocytometer; cell
counts were corrected for viability by the trypan blue dye exclusion
method. Cells were cultivated in T75 cm2 flasks in conventional culture
medium, consisting of RPMI 1640 (Quality Biologica Is. Gaithersburg,
described (28). Cell number, viability, as well as yield were then deter
mined, and i.b. or s.c. implantations were performed as described below.
All lung tumor diagnoses were histologically confirmed and are sum
marized in Table 1.
Experimental Animals. Pathogen-free, female NCr-nu/nu mice or
BALB/c x DBA/2 F, (hereafter called CD2Fi) immunocompetent
mice, approximately 4 to 6 wk of age, were used. All procedures
involving the animals were performed in a pathogen-free barrier facility
at NCI-FCRF.
Implantation i.b. Animals were anesthetized in a Harvard small
animal anesthesia chamber (Harvard Biosciences, Boston, MA) at
tached to a standard Sonnifer anesthesiology apparatus (Richmond
Veterinarian Supply Co., Richmond, VA) using a 7% flow of nebulized
Metafane (Pitman Moore, Inc., Washington Crossing, NJ)/100% ox
ygen mixture. A 1.0-cm ventral midline incision was made in the neck
over the region of the trachea superior to the supraclavicular notch.
The surgical procedures were facilitated by the use of a binocular x7 to
10 magnification headset. The glandular tissue surrounding the trachea
was separated by blunt dissection, and the trachea was exposed by
dissection of the peritracheal muscle sheath. The tissue between the
trachea! rings was then incised with the bevel of a 25-gauge needle, and
a modified, 27-gauge, blunt-ended 1.2-cm needle was inserted into the
trachea and advanced into the right mainstem bronchus (Fig. 1). An
inoculum of 1.0 x IO6, 1.0 x 10s, or 1.0 x 10" tumor cells, in a 0.1-ml
final volume of HBSS, was then introduced into the right mainstem
bronchus. Animals were maintained in a reverse Trendelenberg maneu
ver during the inoculation to ensure that the fluid containing the cells
remained in the distal airways of the right lung. The skin incision was
then closed with surgical skin clips, and the animals were allowed to
recover under heat lamps for approximately 10 min, were subsequently
returned to their holding cages, and were followed for clinical evidence
of pulmonary tumors.
Implantation s.c. Tumor cells were implanted at a 1.0 x IO7, 1.0 x
IO6, 1.0 x 10s, or 1.0 x 10* inoculum using the previously described
s.c. implantation techniques (12-14) in a 0.1-ml final volume of HBSS.
Chest Roentgenographic Studies. A Senographe 500T mammo
graphie radiological device (Thompson-CGR Medical Corporation,
Columbia, MD) with a 0.1-mm focal spot with compatible screens,
cassettes, and X-ray film (DuPont Lodose system; DuPont, Inc., Tren
ton, NJ) was used for roentgenographic studies. (The 0.1-mm focal
spot is essential for obtaining radiographs with high quality bone and
soft tissue detail). Gastrovist radiopaque contrast material (Berlex
Industries, Wayne, NJ) was used for contrast dye-enhanced radiographic studies to demonstrate localization of i.b.-implanted material
to the various lung fields. The standard i.b. implantation method
(described above) was used for these studies with a 0.1-ml volume of
the contrast material. Animals were then immediately evaluated by
anterioposterior and right lateral chest roentgenography.
Lung Tumor Cell Autoradiography Studies. Monolayer cultures of
NCI-H460 tumor cells were incubated with medium containing 10
mCi/ml [3H]thymidine (specific activity, 18.2 Ci/mmol; New England
Nuclear, Boston, MA) for 48 h prior to implantation. An inoculum of
1.0 x IO6tumor cells was suspended in a 0.1-ml final volume of HBSS
and then administered i.b. to NCr-nu/nu mice as described above.
Control, radiolabeled NCI-H460 cells were incubated on glass cover-
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INTRAPULMONARY
MODEL
FOR HUMAN
LUNG CANCER
Fig. 2. Demonstration of localization of
i.b.-implanted material to the right lung by
contrast dye-enhanced radiography. The chest
radiograph on the left represents an anterioposterior view, and the one on the right, a right
lateral view, of a mouse immediately following
i.b. implantation with radiopaque dye. Note
the localization of the contrast material to the
caudal (lower) lobe of the right lung (arrows).
This corresponds to the highlighted area in
Fig. 1 representing the right lower lobe. Radio
graphs were performed as described in "Ma
terials and Methods."
-*-
l'ß
V
Fig. 3. Localization of tumor cells to the bronchioloalveolar regions of the caudal lobe of the right lung following
i.b. implantation. This light micrograph illustrates a par
affin-embedded, hematoxylin- and eosin-stained histology
section of the caudal lobe of the right lung of an athymic
nude mouse l h post-i.b. implantation with 1.0 x 10" NCIH460 tumor cells. A representative alveolus (a) and airway
(o) are labeled for comparison. Note the clustering of
tumor cells in the bronchioloalveolar regions with filling
of the alveolar spaces by the tumor cells. Representative
tumor cells are demonstrated by arrows. Normal alveoli
are prominent in the lower portion of the micrograph.
X300.
v •
* fi*
Fig. 4. Autoradiographic demonstration of
radiolabeled tumor cell distribution in the cau
dal lobe of the right lung. This light micro
graph illustrates a periodic acid-Schiff-stained,
hematoxylin-counterstained
serial section of
the caudal lobe of the right lung l h following
i.b. implantation with a 1.0 x IO6 NCI-H460
tumor cell inoculum which was labeled for 48
h prior to implantation with [3H]thymidine as
described in "Materials and Methods." Heav
ily radiolabeled tumor cells are easily identified
in the bronchioloalveolar regions (representa
tive cells are demonstrated by arrows). Inset,
control radiolabeled cells, placed on a glass
coverslip for I h. shown for comparison. A
representative alveolus (a) and airway (b) are
also labeled in the figure. Normal alveoli are
present in the upper left and lower right por
tions of the micrograph.
slips at 37°Cin the presence of 5% COj for l h and then processed for
perfusion with 10% low-molecular-weight dextran (Abbott Lab., Chi
autoradiographic evaluation. Mice that received i.b. implants with
cago, IL) (w/v) in 0.9% saline followed by 2% glutaraldehyde in 0.1 M
radiolabeled cells were anesthetized with 0.1 ml of sodium pentobarbital
cacodylate buffer (pH 7.4) as previously described (11). Individual lung
(Richmond Veterinary Supply Company, Richmond, VA) l h after the
lobes were collected and immersed in the above glutaraldehyde solution
i.b. implantation, and lung tissues were then fixed in situ by vascular
for 2 h, followed by further fixation in 10% buffered formalin for 2
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INTRAPULMONARY
MODEL FOR HUMAN LUNG CANCER
"
fl>T>^rV'^V
"X-
Fig. S. Light micrographs of microscopic NCI-H460 tumor nodules in the right lower lobe 7 and 10 days following i.b. implantation. These micrographs
demonstrate NCI-H460 tumor nodules (arrows) at 7 (A) and 10 (lì)
days status posi-i.b. implantation. Note the location of the nodules in the peripheral regions of
the right lower lobe. A, x 500; fi, x 200.
days. Glass slides (Fisherbrand; Fisher Scientific, Fairlawn, NJ) were
precleaned for 5 min with 70% ethyl alcohol containing 1% hydrochlo
ric acid, rinsed well with running distilled water, placed in a 95% ethyl
alcohol rinse for S min, and subsequently air dried. Serial 5 ^ in sections
of each lung lobe were placed onto precleaned glass slides at three levels
approximately 100 urn apart. Duplicate sections were taken at each
level. The fixed lung tissues were subsequently embedded in paraffin,
and autoradiography and staining procedures were performed as pre
viously described (29). Lung tissue sections were then microscopically
evaluated for radiolabeled tumor cell localization.
Lung Tumor Cell Localization Studies. Athymic nude mice were
implanted i.b. with 1.0 x IO6 NCI-H460 cells, and animals were then
Monitoring of Animals for Tumor Development. Animals that received
either i.b. or s.c. implants were carefully followed daily for signs of
tumor development. Throughout the observation period, animals de
veloping respiratory distress were killed by cervical dislocation. All
those animals remaining after 140 days were also killed, and the major
organs (lung, liver, spleen, kidney, brain) were removed, fixed in 10%
buffered formalin, and then processed for histopathological evaluation.
Paraffin sections were stained with hematoxylin and eosin and then
evaluated for the presence of tumor or other histopathology. Mortality
data were based on only those animals that died from direct effects of
tumor growth and/or metastatic disease before 140 days.
fixed in situ by vascular perfusion as described above at 1 h, 7 and 10
days following implantation. Individual lung lobes were removed and
separated, embedded in paraffin, sectioned, stained with hematoxylin
and eosin, and microscopically evaluated for tumor cell localization.
RESULTS
The i.b. implantation method (Fig. 1) provides for the inoc
ulation of human lung tumor cell suspensions into the right
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INTRAPULMONARY
MODEL
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LUNG CANCER
sections of individual mouse lung lobes following i.b. implan
tation of 1.0 x IO6 NCI-H460 cells demonstrated localization
100
20
FOR HUMAN
60
Days Post Implant
Fig. 6. Comparison of tumor-related mortality from i.b. versus s.c. implanta
tion of 4 human lung tumor cell lines in NCr-nu/nu mice. The results of mortality
for i.b. and s.c. implantations are summarized in Fig. 6, where A to D represent
mortality from NCI-HI25, A549, NCI-H460, and NC1-H358 cell lines, respec
tively. »,1.0 X IO6;A, 1.0 x 10s; andO, 1.0 x IO4tumor cell inocula. Each point
represents animal mortality/unit time. Statistical comparisons of the i.b. (n =
251) and s.c. (n - 120) implantation routes for the 4 human lung tumor cell lines
at the different tumor cell inocula are as follows: 1.0 x 10»,
P< 0.001 (nonpaired,
one-tailed Student t test); 1.0 x 10', P < 0.02; and 1.0 x IO4, /»< 0.05. A, n =
20, «= 21, and«= 30 for 1.0 x IO4, 1.0 x 10', and 1.0 x 10*tumor cell inocula,
respectively; B, n = 10, n = 30, and n = 20 for 1.0 x 10", 1.0 x 10*, and 1.0 x
IO4 tumor cell i.b. inocula, respectively. C and D, n = 20 for all three i.b. tumor
cell inocula; n = 10 for the 3 different s.c. tumor cell inocula for the 4 different
cell lines tested. The low mortality for s.c. implantation was related to the low
tumor propagation efficiencies.
of the majority of the tumor cells to the bronchioloalveolar
regions of the caudal lobe of the right lung (Fig. 3). Localization
of tumor cells to this region of the right lung l h after i.b.
implantation was more clearly demonstrated when 1.0 x IO6
radiolabeled NCI-H460 cells were used (Fig. 4). Again, only
the caudal lobe of the right lung consistently contained radiolabeled tumor cells when individual lung lobes were separately
evaluated. In addition, microscopic tumor nodules were ob
served in predominantly the right lower lobe 7 and 10 days
following i.b. implantation when individual lung lobes were
again evaluated at these longer time intervals (Fig. 5). These
different studies all clearly demonstrated that the majority of
tumor cells initially resided in the bronchioloalveolar regions
of the caudal lobe of the right lung following i.b. implantation.
Athymic nude mice implanted i.b. with human lung tumor
cells demonstrated clinical symptoms of cachexia and dyspnea
as the pulmonary xenografts became progressively larger. These
were similar to the symptoms frequently observed in human
lung cancer patients. The tumor cells grew most frequently in
the peripheral regions of the right lung following i.b. implan
tation (Fig. 5); however, certain tumors did grow predominately
in the airways (Fig. 9). Implantation i.b. produced tumors that
ultimately led to the death of the animals (Fig. 6). Lung tumor
progression could also be readily followed roentgenographically
(Fig. 7), thus allowing tumor-bearing animals to be monitored
noninvasively. When the 251 tumor-bearing animals repre
sented in Fig. 6 were studied at necropsy following i.b. implan
tation, 91% of the tumors implanted by this method were
localized to the right lung. Other tumor locations included the
left lung (1%), trachea (2%), or peritracheal (6%) area. Local
mediastinal invasion was observed at necropsy in approximately
90% of the tumor-bearing animals following i.b. inoculation.
In 3% of the i.b.-implanted, tumor-bearing animals, distant
métastaseswere also observed at necropsy in the left lung,
lymph nodes, liver, or spleen.
The histologies of tumors obtained from the i.b. implantation
model were consistently similar to the parent human lung tumor
cell lines from the which they were derived (n = 251). As an
example, a tumor obtained from the right lung of a nude mouse
19 days following i.b. implantation with the NCI-H460 cell line
demonstrated characteristic histopathological features of a hu
man large cell undifferentiated carcinoma of the lung (Fig. 8).
The reproducibility of this model among different cell lines
was demonstrated in that it produced similar pulmonary tumors
and death patterns for the 4 lung tumor cell lines tested (Fig.
6). Reproducibility within cell lines was also demonstrated for
the NCI-H460 cell line when it was implanted i.b. multiple
times over a 6-mo interval (coefficient of variation for mean
survival was 24% for this cell line implanted on 4 separate
occasions).
The intrapulmonar) model was also compared to the conven
tional s.c. model for effectiveness in propagation of human lung
tumor cells. When the 4 different human lung tumor cell lines
were implanted i.b. at 3 different tumor cell inocula, 100%
tumor-related mortality was observed at the 1.0 x IO6 tumor
cell inoculum, and a dose-dependent response was noted with
lower (1.0 x 10s or 1.0 x 10") tumor cell suspensions (Fig. 6).
In contrast, minimal (5%) tumor-related mortality was observed
within 140 days for the s.c. model for the 4 cell lines at all
tumor cell inocula tested (Fig. 6). When tumor cells from the 4
cell lines were implanted i.b. or s.c. in CD2F, immunocompetent mice at a 1.0 x IO6 tumor cell inoculum, no tumors were
mainstem bronchus of athymic nude mice. Over 1000 i.b.
implants have been performed to date, each requiring an average
of 3 to 5 min for completion and having a surgery-related
mortality of approximately 5%. This model provides an orthotopic site for tumor implantation in that the majority of tumor
cells are deposited in the bronchioloalveolar regions of the right
lung, as verified by radiopaque dye-enhanced radiographie stud
ies, routine histology, and autoradiographic evaluation (Figs. 2
to 4).
Radiopaque dye-enhanced radiographie studies were initially
performed, whereby radiopaque contrast material was im
planted i.b., and low-dose, high-resolution chest radiographs
were then immediately performed. These studies clearly dem
onstrated radiographie localization of the radiopaque contrast
material predominantly to the caudal (lower) lobe of the right
lung (Fig. 2). Similarly, microscopic evaluation of histological
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INTRAPULMONARY
MODEL FOR HUMAN LUNG CANCER
Fig. 7. Anterioposterior chest radiographie
comparison of a normal and a lung tumorbearing athymic nude mouse. A normal mouse
radiograph is depicted on the left, and a mouse
19 days status post-i.b. implantation with 1.0
X IO6 NCI-H358 tumor cells is shown on the
right. A large tumor involving the right lung
and mediastinum is easily identified (arrows)
and accounts for the volume loss in the left
lung field. Radiographs were obtained using
the techniques described in "Materials and
Methods."
remarkably similar in histológica! appearance to the original
human lung tumor specimens from which they were derived.
This is exemplified by comparison of the histológica! appear
ance of a superficially invasive squamous cell carcinoma with a
prominent endobronchial exophytic component after its re
moval from the patient's lung and its appearance after being
propagated i.b. in the lung of an athymic nude mouse (Fig. 9).
Note the squamous cell characteristics in both tumor specimens
as well as the striking endobronchial polypoid growth of the
tumor propagated i.b. in the right lung of the nude mouse.
DISCUSSION
Fig. 8. Light micrograph of a large NCI-H460 tumor following i.b. implan
tation. This represents a paraffin-embedded histology section of a large tumor
located in the right lung of a nude mouse 19 days following i.b. implantation with
1.0 X IO6 NCI-H460 tumor cells. The tumor histology is characteristic of the
large cell undifferentiated lung carcinoma cell line from which it was derived. H
& E, X 500.
observed at necropsy after 160 days in either the lungs or the
skin (n = 10 for the number of animals implanted i.b. or s.c.
for each cell line).
Enzymatically dissociated, fresh human lung cancer speci
mens, obtained at the time of diagnostic thoracotomy, were
also successfully propagated using the i.b. technique at a 1.0 x
10* tumor cell inoculum. Approximately 35% (10 of 29) of the
fresh primary lung tumors and 66% (2 of 3) of the tumors
metastatic to the lung were successfully propagated in vivo by
this technique, whereas only 20% (1 of 5) of these same tumors
were successfully established and propagated using the s.c.
method at a 1.0 x IO7 tumor cell inoculum (Table 1). Primary
lung tumors which were successfully propagated i.b. were rep
resentative of all 4 major lung tumor histológica! cell types as
well as one relatively rare human pulmonary tumor, a bronchioloalveolar cell carcinoma. Metastatic lung tumors which
were successfully propagated i.b. included a transitional cell
carcinoma of the urinary bladder as well as an adenocarcinoma
of the prostate (Table 1). Tumors were easily passaged i.b. using
the enzymatic dissociation technique described in "Materials
and Methods."
The tumors which were propagated in the mouse i.b. were
This paper describes a novel intrapulmonary model for the
orthotopic implantation and propagation of human lung can
cers in athymic nude mice. The procedure is relatively easy to
perform, is reproducible, and requires a small number of ani
mals to obtain adequate tumor tissue for experimentation. The
model uses human lung tumor material rather than less ideal
carcinogen-induced animal tumors (5-11) or other non human
lung tumors (30) and potentially affords investigators with an
improved opportunity for the study of human lung cancer.
The present i.b. studies support the concept of the importance
of organ site-specific tumor implantation for optimal tumor
growth which was originally proposed by Paget (21) and sub
sequently supported by numerous other studies using metastatic
tumor models (for review, see Ref. 12) as well as orthotopic
models for certain human cancers (12, 16, 20, 22-24). Positive
and/or negative selection factors which are present in various
mouse tissues may be major determinants of the success or
failure of human tumor cells to propagate in vivo (for discussion,
see Ref. 12). These factors are probably relevant for both
established human lung tumor cell lines as well as for fresh
human lung tumor suspensions implanted orthotopically in
athymic nude mice. Many tissue characteristics, including the
abundant blood supply in the lung, the relatively high compli
ance of lung tissue, and the availability of numerous endogenous
factors in the pulmonary microenvironment (31-37), conceiv
ably might be responsible for the enhanced survival and growth
of tumor cells in the immunodeficient mouse lung. This is
further supported by the observation that lung tumor cells
retained their original histological characteristics when they
were grown i.b. It is also supported by the reduced lung tumor
cell inoculum requirement which was observed for the i.b. model
as compared to the previously described s.c. model which re
quires a tumor cell inoculum of 1.0 x IO7 cells for optimal
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INTRAPULMONARY
MODEL FOR HUMAN LUNG CANCER
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INTRAPULMONARY
MODEL FOR HUMAN LUNG CANCER
tumor development (12-15), and many of these previous s.c.
studies have used human tumor fragments which usually con
tain >1.0 x IO7cells/inoculum. The lower tumor cell inoculum
requirements for the i.b. model appear consistent with an organ
site-specific implantation theory for optimal tumor growth;
however, it is important to note that comparably low lung
tumor cell inocula have been shown to be effective for propa
gation of certain small cell lung carcinoma cell lines implanted
intracranially in immunodeficient rodents (38, 39). Additional
i.b. studies using nonpulmonary human tumors will be neces
sary to further evaluate this organ-site specificity concept.
In addition to its utility for propagating continuous longterm human lung tumor cell lines, the intrapulmonary model
also provides the basis for orthotopic propagation of enzymatically dissociated fresh human lung tumor tissue in athymic
nude mice. This might offer a useful avenue for clinical inves
tigators to expand relatively small surgically excised biopsy
specimens into a tumor volume of sufficient size to provide
fresh tumor tissue for biochemical, histopathological, and cell
biology studies. This procedure might also allow for propaga
tion and expansion of lung tumor tissue from those patients
who are not candidates for surgical resection, but who undergo
diagnostic procedures such as bronchoscopy or needle biopsy
which yield small tumor samples. Since 70 to 80% of all lung
cancer patients are inoperable at the time of diagnosis (40,41),
this would allow potential access to a much larger lung cancer
patient population for a variety of clinical research and/or
diagnostic studies.
The intrapulmonary model also offers a potentially attractive
system for in vivo testing and experimental therapeutics of new
potential antitumor agents. This model might provide pharmacokinetic and pharmacodynamic features more characteris
tic of human lung cancer than s.c. or intrarenal implanted
tumors. In addition, it frequently demonstrates local mediastinal invasion which is characteristic of certain human lung
cancers (41). It also allows for the experimental use of a lethality
end point which might be particularly convenient for large scale
drug-testing studies.
In addition, noninvasive X-ray procedures can be used to
periodically monitor lung tumor size, and this is especially
attractive for experimental therapeutics studies. Implantation
of tumors in the right lung of nude mice is particularly advan
tageous, since no other major anatomical structures (e.g., the
heart) are located in this area which might interfere with the
radiographie evaluation of small human tumor xenografts.
Thus, by using both anterioposterior (or posterioanterior) and
right lateral radiographie views, a semiquantitative, three-di
mensional, radiographie approximation of tumor size can be
obtained (Figs. 2 and 7). Such technology could ultimately
provide an orthotopic in vivo drug-testing model which closely
resembles the clinical setting, in that it makes possible accurate,
periodic, noninvasive monitoring of human lung tumors in an
animal host following treatment with different experimental
therapeutic modalities.
In summary, this intrapulmonary model has several advan
tages over other currently available models and should be of
particular value for future studies of lung cancer biology and
treatment. Advantages include the following, (a) The procedure
provides the first short-term, reproducible, orthotopic in vivo
animal model for the study of human lung cancer, (b) Lung
tumor cells are implanted at the organ site of tumor origin, (c)
The i.b. procedure is relatively easy to perform in a modified
conventional laboratory setting and uses a smaller tumor cell
inoculum than that required by other models, (d) It allows for
propagation of fresh human lung tumor specimens, (e) It poten
tially provides a suitable in vivo model for the study of human
lung tumor biology, and (/) it offers a potentially attractive
and relevant model for in vivo testing of the efficacy of potential
therapeutic agents for the treatment of lung cancer.
ACKNOWLEDGMENTS
The authors express their appreciation to Dr. J. Minna and Dr. A.
Gazdar, NCI Navy Oncology Branch, Bethesda, MD, for providing
tumor lines; to Dr. I. J. Fidler, M.D. Anderson Hospital and Tumor
Institute, Houston, TX, for his helpful advice regarding this manuscript;
and to Kathy Gill for the typing and organization of this text for
publication.
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Novel Intrapulmonary Model for Orthotopic Propagation of
Human Lung Cancers in Athymic Nude Mice
Theodore L. McLemore, Mark C. Liu, Penny C. Blacker, et al.
Cancer Res 1987;47:5132-5140.
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