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The Prostate 39:182–186 (1999)
High-Malignancy Orthotopic Nude Mouse Model
of Human Prostate Cancer LNCaP
Xiaoen Wang, Zili An, Jack Geller, and Robert M. Hoffman*
AntiCancer, Inc., San Diego, California
BACKGROUND. An animal model of human prostate cancer LNCaP demonstrating high
rates of spontaneous metastasis from the orthotopic site after tumor implantation would be
very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice
in order to develop high metastatic mouse models of human cancer.
METHODS. Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP,
was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological
examination of primary tumors and relevant organs was done to identify and quantitate
micrometastasis.
RESULTS. Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean
primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the
transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis.
Mean survival time was 72 days, indicating a high degree of malignancy of the tumor.
CONCLUSIONS. The extensive and widespread lung metastasis as well as lymph node
metastasis following orthotopic implantation of LNCaP in nude mice and the short survival
time provide a high-malignancy nude model of the LNCaP human prostate tumor. Prostate
39:182–186, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
LNCaP; orthotopic implantation; lung metastasis; prostate cancer;
lymph node metastasis; nude mouse model
INTRODUCTION
LNCaP is a cell line derived from a supraclavicular
lymph node metastasis of human prostate carcinoma.
LNCaP exhibits increased proliferation in response to
androgens. When LNCaP cells were mixed with the
reconstituted basement membrane Matrigel and injected subcutaneously in nude mice, tumors grew in
the mice by 12 weeks without observed metastases [1].
The tumorigenicity and incidence of metastasis of
LNCaP after orthotopic (intraprostate) or subcutaneous injection in male nude mice were compared by
Stephenson et al. [2]. LNCaP cells produced tumors
only when orthotopically implanted in the prostate.
In a subsequent study by Pettaway et al. [3], 56% of
mice injected orthotopically with a suspension of
LNCaP cells formed prostate tumors. Twelve of 43
mice had microscopic metastases in the regional
lymph nodes.
In another study, orthotopic injection of LNCaP in
© 1999 Wiley-Liss, Inc.
nude mice resulted in local growth in 7 of 10, and
lymph node metastasis in 4 of 10 nude mice [4].
Nude and SCID mice were injected either subcutaneously or orthotopically with LNCaP cell suspensions in a comparative study by Sato et al. [5]. LNCaP
tumor incidence after subcutaneous injection was
100% in SCID mice and 80% in nude mice. No lymph
node or distant metastases were observed with subcutaneous tumors. After orthotopic injection, 89% of
SCID mice and 60% of athymic mice had prostate tumors. Retroperitoneal or mediastinal lymph node metastases were found in 100% of SCID mice, and microscopic pulmonary metastases were identified in 40%.
Grant sponsor: U.S. National Cancer Institute; Grant number: R44
CA53963.
*Correspondence to: Robert M. Hoffman, AntiCancer, Inc., 7917
Ostrow St., San Diego, CA 92111. E-mail: all@anticancer.com
Received 28 August 1998; Accepted 24 December 1998
Orthotopic Nude Mouse Model of LNCaP Tumors
The nude mice had retroperitoneal lymph node metastases in 25% of the animals, but no other metastasis
was found.
Injection of tumor cells into the prostate gland of
nude mice, nevertheless, requires delicate, hard-tocontrol technical conditions, and spillage outside the
prostate may generate rather variable results. Furthermore, some studies showed that artificial dissemination rather than spontaneous metastasis might occur
following tumor cell injection, even when cells were
not directly injected into the vasculature [6–8]. It has
been shown that tumor cells can enter the draining
lymph or blood circulation within 10 min–3 hr of injection, which will eventually form distant artificial
metastases [9,10]. Most importantly, a cell suspension
lacks the tissue architecture which seems critical for
the full expression of the spontaneous metastatic potential of transplanted human tumors.
Recently, we introduced a new transplantation
technique of surgical orthotopic implantation (SOI) of
histologically intact tumor tissue for developing metastatic models of human cancer in immunodeficient rodents [11–16]. We previously demonstrated orthotopic
growth of prostate cancer line PC-3 with subsequent
lymph node metastases by using the SOI technique
[16]. In a subsequent study, a large series of animals
was implanted with PC-3 with improved SOI techniques; a high frequency of lymph nodes and lung
metastases was found [17].
In the present study, we applied SOI to LNCaP to
develop a high-malignancy nude mouse model.
Lymph node and lung metastases occurred frequently, with a mean survival time of 72 days for the
host after transplantation.
MATERIALS AND METHODS
Animals
Athymic male nu/nu CD-1 mice (Charles River
Laboratories, Wilmington, MA), 4–5 weeks old, were
used in the study. They were maintained in a specific
pathogen-free environment, in compliance with
USPHS guidelines governing the care and maintenance of experimental animals (Assurance No. A38731). Mice were fed with an autoclaved laboratory rodent diet (Teklad LM-485, Western Research Products,
Orange, CA).
Surgical Orthotopic Implantation
LNCaP tumor tissue used for surgical orthotopic
implantation was derived from stock tumor growing
subcutaneously in nude mice maintained in the immunodeficient-rodent facility of AntiCancer, Inc. Tissue from the periphery of the tumor was harvested in
183
log phase, and necrotic tissue was carefully removed
under a dissecting microscope to maximize the
amount of viable tissue for implantation. The viable
tissue was then cut into small cubes of 1 mm3 in standard tissue culture medium under sterile conditions.
To minimize variation in subsequent tumor growth
and metastasis, these tumor pieces were randomly
mixed and an equal amount of five pieces was implanted in each mouse, as described below.
Mice were anesthetized by isoflurane (Ohmeda
Caribe, Inc., Guayama, PR) and positioned supinely.
An opening was made right above the pubis symphysis to expose the prostate gland. The fascia surrounding the ventral portion of the prostate was carefully
isolated, and the two ventral lateral lobes of the gland
were separated by a small incision, using a pair of fine
surgical scissors. Five of the above tissue pieces were
sutured into the incision using an 8-0 nylon suture.
The two parts of the separated lobes were then sutured together, with the tumor pieces wrapped within.
The surrounding fascia was then used to wrap this
portion of the gland to consolidate the incision. The
abdomen was closed using a 6-0 suture [7,16].
Evaluation of Tumor Growth and Metastasis
All dead mice were immersed in 10% formalin for
subsequent autopsy and microscopic examination. Regional and distant lymph nodes, lungs, livers, and
other organs where metastasis could occur were routinely embedded, sectioned, and stained with hematoxylin and eosin, using standard techniques for microscopic examination.
RESULTS AND DISCUSSION
Orthotopic Growth of LNCaP in the Nude
Mouse Prostate
LNCaP grew extensively after surgical orthotopic
implantation (SOI) in the ventral portion of the prostate. Eighteen of 20 implanted mice had orthotopic
growth. Tumors in the prostate had a mean weight of
9.2 g (Table I), which disfigured the shape of the lower
abdomen (Fig. 1A). Autopsy demonstrated that the
orthotopically-growing tumors usually protruded into
the abdominal cavity and very frequently invaded the
lower abdominal wall. Distended urinary bladders
and hydronephrosis due to blocked urethras were also
frequently seen.
Invasion and Metastasis
The seminal vesicles, the bladder, and the lower
abdominal wall were often invaded by orthotopic pri-
184
Wang et al.
TABLE I. Tumor Growth, Metastases, and Survival of SOI Model
of LNCaP
Mouse
number
Survival
time
(days)
Tumor
size
(mm)
Tumor
weight
(g)
Lymph
node
metastasis
Lung
metastasis
67
56
44
68
63
66
71
78
120
88
71
137
119
46
48
46
50
54
28.6 × 31.4
24.1 × 32
14.1 × 22.4
25 × 29.9
23.8 × 32.3
29.8 × 31.3
28.7 × 31.3
25 × 29.4
26.3 × 32.3
24.6 × 29.8
21.1 × 31.8
31 × 38.4
27.4 × 31.1
23.9 × 35.7
25.1 × 36.9
25.3 × 31.4
25.6 × 27.1
20 × 23.2
11.72
9.08
2.02
9.68
10.48
9.04
9.57
11.85
7.74
9.35
6.3
14.29
8.89
10.7
11.05
9.47
9.4
5.65
+
−
−
−
+
−
+
+
+
−
+
−
−
+
+
+
+
+
+
−
−
−
+
−
−
−
−
−
+
−
+
−
+
+
+
+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
TABLE II. Comparison of Orthotopic Injection of Cell Suspensions and Surgical Orthotopic Implantation of Tumor
Fragments of LNCaP in Nude Mice
Reference
Stephenson et al. [2]a
Pettaway et al. [3]b
Sato et al. [5]
Rembrink et al. [4]
Present study
Orthotopic
implantation
method
Mean size of
primary tumor
Lymph node
metastasis (number of
positive animals/total)
Lung metastasis
(percent of
positive animals)
Average
survival
time
Cell suspension
Cell suspension
Cell suspension
Cell suspension
Fragment
0.23 g
1.2 g
ND
0.71 gc
9.24 g
0
12/43
3/12
5/10
11/18
0
0
0
0
44
ND
100 days
ND
ND
72 days
a
Study with orchiectomized animals not included.
Study with selected metastatic variants not included.
c
Includes prostate and seminal vesicles + bladder. Normal average weight was 0.35–0.45 g.
b
mary tumors. Microscopic examination of the tissue
sections demonstrated that 8 of 18 animals had lung
metastases, and 11 of 18 had periaortic lymph node
metastases (Fig. 1, Table I).
Microscopic examination of lung specimens
showed that the metastases were disseminated in
small nests of tumor cells (Fig. 1D).
Survival
Histopathology
Histopathology of the primary tumor showed
densely packed, poorly differentiated cells. The prostate gland was almost replaced by tumor cells, and
very few glandular structures could be seen (Fig. 1B).
Microscopically, lymph node metastases involved
almost the whole node (Fig. 1C).
The mean survival time of the animals was 72 days
(Fig. 2).
Our studies with human bladder and stomach cancer demonstrated that orthotopic implantation of histologically intact tumor tissue by the techniques of
SOI, when directly compared to cell suspensions, resulted in much greater metastatic expression of the
implanted tumor after SOI [18–20].
Orthotopic Nude Mouse Model of LNCaP Tumors
185
Fig. 1. Gross and histologic appearance of LNCaP after orthotopic implantation in the ventral prostate lobes of nude mice as intact tissue
fragments. A: Gross photograph of primary tumor of LNCaP 60 days after implantation (arrow). The tumor has protruded into the
abdominal cavity. B: Histologic appearance of primary tumor showed a group of anaplastic epithelial cells with pleomorphic nuclei and
prominent nucleoli. C: Microscopic appearance of regional lymph node metastases. The metastatic tumor cells (indicated by solid arrow)
replaced most of the normal lymph node structure (indicated by open arrow). D: Microscopic appearance of lung metastases. Arrow
indicates the metastatic tumor, which is near a vascular structure.
The histologically intact tumor tissue used in SOI
possesses a large amount of supportive stromal cells,
which are essential for maintaining the threedimensional tumor architecture. The tissue architecture is believed to contribute to the difference in metastatic expression resulting from SOI compared to orthotopic injection of tumor cell suspensions, which
have no three-dimensional architecture.
In this study, we report on the high rate of spontaneous lung and lymph node metastasis and short survival time of nude mice after SOI of LNCaP in nude
mice. The high incidence of spontaneous metastasis
and malignancy of LNCaP after SOI observed in this
study demonstrates a high-malignancy model of androgen-dependent human prostate cancer. The model
is thus a unique and valuable tool for the study of and
for development of new therapeutics for metastatic,
hormone-dependent prostate cancer.
Pettaway et al. [3] demonstrated that cells derived
from the metastatic lesions of the orthotopically injected parental line of LNCaP became increasingly
metastatic after multiple selection cycles of orthotopic
injection, isolation of metastatic cells, and orthotopic
reinjection in nude mice. Even so, after a number of
cycles of selection, LNCaP still did not metastasize to
the lung from the orthotopic site. Lung metastasis was
186
Wang et al.
4.
5.
6.
7.
Fig. 2. Survival curve of LNCaP model after orthotopic implantation in the ventral prostate lobes of nude mice as intact tissue
fragments. The average survival time for this group was 72 days.
only achieved by iv injection of the selected metastatic
cells.
Our techniques of SOI eliminate the need to select
highly metastatic cells through complex procedures
from a parental tumor for the purpose of establishing
metastatic animal models of human cancer. Moreover,
the SOI metastatic model more closely mimics the
clinical pattern, thus providing a better tool to study
the biology of cancer metastasis and for evaluating
novel cancer therapeutics.
8.
9.
10.
11.
12.
13.
CONCLUSIONS
This report demonstrates a high malignancy and
short survival model of LNCaP after orthotopic implantation in nude mice. The technique of SOI of histologically intact tumor tissue preserves the stromal
tissue and overall tumor architecture during the implantation. We believe that the stromal cells in the
implanted tissue fragments play a very important role
in the subsequent metastasis and high malignancy.
The metastatic model of LNCaP reported here
should enable us to study the whole clinical course of
metastatic prostate cancer, from orthotopic growth, to
local invasion, to distant metastasis of an androgendependent prostate cancer.
14.
15.
16.
17.
18.
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