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The Prostate 34:169–174 (1998)
Surgical Orthotopic Implantation Allows High
Lung and Lymph Node Metastatic Expression of
Human Prostate Carcinoma Cell Line PC-3 in
Nude Mice
Zili An,1 Xiaoen Wang,1 Jack Geller,1* A.R. Moossa,2 and
Robert M. Hoffman1,2
1
AntiCancer Inc., San Diego, California
Department of Surgery, University of California San Diego School of Medicine,
San Diego, California
2
BACKGROUND. Prostate cancer is the second leading cause of male death in the United
States. When diagnosed, nearly half the cases have metastatic lesions. An animal model of
human prostate cancer demonstrating spontaneous metastasis from the orthotopic site after
tumor implantation should be of great help for us to understand the disease and to formulate
treatment strategy. We report here a high metastatic model of human prostate cancer PC-3.
METHODS. We developed microsurgical techniques, termed surgical orthotopic implantation (SOI), to implant histologically intact tumor tissues orthotopically in immunodeficient
mice. In this study intact tissue of the human prostate cancer cell line PC-3, harvested from a
subcutaneous tumor in a nude mouse, was implanted to the ventral lateral lobes of the
prostate gland in a series of nude mice. Mice were sacrificed when found moribund, and
autopsy and histology were performed subsequently.
RESULTS. A high frequency of lymph node and lung metastasis was noted upon histological examination. The extensive and widespread lung metastasis following orthotopic implantation of PC-3 is, to the best of our knowledge, the first report in the literature.
CONCLUSIONS. In contrast to orthotopic injection of cell suspensions, no multiple metastatic
cell selection was necessary after SOI for significant expression of the metastatic potential of
PC-3. We conclude that the stromal tissue architecture maintained in the implanted tumor
played a critical role in tumor growth and progression. Prostate 34:169–174, 1998.
© 1998 Wiley-Liss, Inc.
KEY WORDS:
surgical orthotopic implantation; histologically intact tumor tissue;
prostate cancer; PC-3; metastasis; nude mice
INTRODUCTION
Prostate cancer is the second leading cause of male
death in the United States, accounting for about 40,000
deaths per year [1]. A clinical report of over 600 patients suffering from D2 prostate cancer indicated an
overall mean survival of 36 months in patients treated
with combined androgen blockade [2]. At the time of
initial presentation approximately 30–50% of prostate
cancer patients have evidence of metastatic disease [3].
The mechanism controlling prostate cancer progression and effective therapeutic strategies for meta© 1998 Wiley-Liss, Inc.
static prostate cancer are poorly understood. This is in
part due to the lack of a suitable animal model that can
mimic the clinical patterns of human prostate cancer
growth and metastasis.
Nonhuman mammals have very rare incidence of
prostate cancer [4], which limit the possibilities of us-
This work was performed at AntiCancer Inc.
*Correspondence to: Dr. Jack Geller, AntiCancer Inc., 7917 Ostrow
Street, San Diego, CA 92111. E-mail: antica@ix.netcom.com
Received 18 October 1996; Accepted 10 February 1997
170
An et al.
ing nonhuman mammal model to study human prostate cancer. With the first introduction of immunodeficient rodents in cancer research in the late 1960s,
xenografted human cancer models are now widely
used. The inability to reject many of the xenografts
implanted in these animals due to defect(s) in their
immune system make them the only known tool for
studying in vivo human cancer growth and metastasis
outside the human body [45].
Studies in a number of laboratories have shown
that orthotopic injection of human tumor cells in immunodeficient mice can produce relevant metastatic
patterns in comparison to ectopic transplantation [5–
11]. 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 [12–14]. It has
been shown that tumor cells can enter the draining
lymph or blood circulation within 10 minutes to 3
hours of injection, which will eventually form distant
artificial metastases [15,16]. Most importantly, a cell
suspension lacks the tissue architecture which seems
critical for the full expression of the spontaneous
metastatic potential of the 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 [17–21]. We previously demonstrated orthotopic
growth of prostate cancer line PC-3 with subsequent
lymph node metastases by using SOI technique [22].
In the present study, a large series of animals was
implanted with PC-3 by improved SOI techniques.
The patterns of growth and high frequency of metastasis are reported here. The advantages of this model
for the study of prostate cancer and the possible
mechanisms that underlie them are also discussed.
MATERIALS AND METHODS
Animals
Twenty male athymic 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. Mice were fed with
autoclaved laboratory rodent diet (Teklad LM-485,
Western Research Products, Orange, CA).
Surgical Orthotopic Implantation
PC-3 cells were obtained initially from the American Tissue Type Culture Collection (Rockville, MD).
Tumor tissue used for surgical orthotopic implantation was derived from a tumor growing subcutaneously after injection of PC-3 cells in a nude mouse.
Tissue from the periphery of the tumor was harvested
in log phase and necrotic tissue was carefully removed
under a dissecting microscope to minimize 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 5 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.
Evaluation of Tumor Growth and Metastasis
Mice were euthanized if found moribund during
the observation period. All mice were humanely sacrificed using CO2 inhalation three months after tumor
implantation and then immersed in 10% formalin for
subsequent autopsy and microscopic examination. Regional and distant lymph nodes, the lung, the liver as
well as other organs suspected of metastasis were routinely embedded, sectioned, and stained with hematoxylin and eosin using standard techniques for microscopic examination. The skeletal system was carefully examined grossly under a dissecting microscope
(7×) with the removal of the soft tissue for possible
bone metastasis.
RESULTS
Orthotopic Growth of PC-3 in the Nude
Mouse Prostate
The well established human prostate carcinoma
line PC-3 grew extensively after surgical orthotopic
Metastasis of PC-3 After Surgical Orthotopic Implantation
implantation (SOI) in the ventral portion of the prostate. The take rate was 95% with only 1 of 20 mice
having no orthotopic growth upon autopsy. Within
the 3-month period following implantation, all tumors
in the prostate reached more than 2 cm in diameter
and usually disfigured the shape of the lower abdomen. The survival time of the animals ranged from 9
to 12 weeks. At time of euthanization, severe signs of
cachexia could be observed in all the mice. Autopsy
demonstrated that the orthotopically growing tumor
usually protruded into the abdominal cavity and very
frequently invaded the lower abdominal wall. Distended urinary bladder and hydronephrosis due to
blocked urethra were also frequently seen (Fig. 1A,B).
Invasion and Metastasis
The seminal vesicles, the bladder and the lower abdominal wall were often invaded by the orthotopic
primary tumors (Fig. 1-A). Microscopic examination
of the tissue sections demonstrated that 5 of 19 animals had lung metastases and 13 of 19 had periaortic
lymph node metastases (Table I). Gross examination
of the skeletal system did not demonstrate bone metastasis and subsequent histology study was not performed.
Histopathology
Histopathology of the primary tumor showed
sheets of densely packed, anaplastic epithelial cells
with abundant, foamy, eosinophilic cytoplasm. The
nuclei were pleomorphic and had varying amounts of
unevenly dispersed chromatin. Many abnormal mitoses could be seen. The prostate gland was almost replaced by tumor cells and very few glandular structures could be seen (Fig. 1-C).
Microscopically, lymph node metastases were characterized by widespread infiltration of tumor cells in
the subcapsular, the cortical and medullary area. In
many of the lymph nodes analyzed, tumor cells occupied the whole node and lymphatic cells could barely
be seen (Fig. 1-D).
Microscopic examination of lung specimens
showed that the metastases were disseminated. Small
nests of tumor cells could be spotted in almost every
high power field. When large tumor nests were seen,
they often resided around the airway structures (Fig.
1-E).
DISCUSSION
Surgical orthotopic implantation of histologically
intact tumor tissue into nude mice preserves the supportive stromal tissue and therefore maintains during
171
implantation, the overall tissue architecture, which refers to the three dimensional histological structure of
the tumor blocks.
Previous studies showed that the proliferation of
tumor cells implanted in nude mice was preceded by
the penetration of host stromal cells into the tumor
[23,24]. Based on this important role of stroma in tumor growth, some investigators coinjected cultured
fibroblasts with tumor cells and observed significant,
growth-stimulating effects of the fibroblasts [25–27].
Also, many studies demonstrated enhanced growth of
tumor cells following co-injection of Matrigel [28–31],
an extract of basement membrane components, which
primarily consists of laminin, collagen IV and heparan
sulfate proteoglycan [32]. Current concepts suggest
that Matrigel serves as a supportive matrix for the
tumor cells. It collects tumor cells, allows paracrine
factors to take effect, activates the release of angiogenic factors and stimulates the production of proteases and motility of tumor cells, thus facilitating
growth and progression [33,34].
Our studies with human bladder and stomach cancer, on the other hand, demonstrated that orthotopic
implantation of histologically intact tumor tissue by
the techniques of SOI, rather than cell suspensions,
resulted in much greater metastatic expression of the
implanted tumor in a head-to-head comparison [35–
37].
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 and orthotopic
injection of tumor cell suspensions, which have no
three-dimensional architecture.
Stephenson et al. [38] reported lymph node metastasis but no lung metastasis of PC-3 after orthotopic
injection of cell suspension in nude mice. Since as
proven by other previous studies that the injected tumor cells may enter the lymphatic stream very shortly
after the injection [12–16], there even exists a question
of whether the lymph node metastasis is an artificial
dissemination. Shevrin et al. [39] demonstrated lung
metastasis of PC-3 cells, but this was achieved by injecting tumor cells directly into the tail vein of the
nude mice, in which several steps of the metastatic
process that naturally occur in patients were bypassed
[40,41,42]. More recently, Waters et al. [43] reported
rather high lung metastasis of PC-3 cells after urinary
bladder wall injection of cell suspensions. However,
they concluded, on the other hand, that lung metastasis of PC-3 from orthotopic tumor cell injection was
infrequent (1 of 10 mice).
In this study, we report, for the first time, the high
172
An et al.
Fig. 1. A: Gross picture of primary tumor of PC-3 growing in
the prostate gland of a nude mouse after orthotopic implantation
(SOI) (solid arrow). The hollow arrow indicates the distended
bladder due to urethra obstruction from tumor growth. Note that
the bladder wall was invaded by the primary tumor. B: Solid arrow
shows the hydronephrosis from urinary tract obstruction. Hollow
arrows indicate enlarged periaortic lymph nodes due to metastasis
(The primary tumor was removed at autopsy). C: Histopathology
of primary tumor of PC-3 after SOI. a: Tumor cells; b: the capsule
of the prostate gland. Arrows indicate the compressed glandular
acini of the prostate gland. D: Lymph node metastasis of PC-3
after SOI. a: Residual cluster of lymphocytes; b: tumor cells. Arrow
indicates the capsule of the lymph node. E: Lung metastasis of
PC-3 after SOI. a: Metastatic tumor nests; b: airway structure.
rate of spontaneous lung and lymph node metastasis
of human prostate cancer line PC-3 from the orthotopic site. Although it is possible that genetic drift and
differences in individual laboratory technique may influence the comparative result between cell suspensions and intact-tissue transplantation, such a high incidence of spontaneous metastasis from the orthotopic
site after SOI observed in this study still brings this
model of human prostate cancer closer to the clinical
profile. The model is thus a unique and valuable tool
for the study of and for development of new thera-
peutics for metastatic, hormone-independent prostate
cancer. Future development will include a bonemetastasis model.
In a very recent report, Pettaway et al. [44] demonstrated that cells derived from the metastatic lesions of
the orthotopically injected parental PC-3, as well as
the androgen-dependent cell line LNCaP, became increasingly metastatic after multiple selection cycles of
orthotopic injection, isolation of metastatic cells and
orthotopic reinjection. Even so, after a number of
cycles of selection PC-3 still did not metastasize to the
Metastasis of PC-3 After Surgical Orthotopic Implantation
TABLE I. Metastatic Sites of Human Prostate Cancer
Cell Line PC-3 After Surgical Orthotopic Implantation*
No. of mice
autopsied
19
Incidence and sites
of metastasis
Lymph node†
Lung†
13/19
5/19
*PC-3 tumor tissue fragments derived from a subcutaneous tumor in nude mice, which grew after cell suspension injection
were implanted in the ventral lateral lobes of the prostate of
nude mice by surgical orthotopic implantation (SOI) (see text for
details). Lymph nodes and the lungs, collected during autopsy,
were routinely fixed, embedded, sectioned, and stained by using standard procedures of H&E staining for microscopic examination.
†
Number of positive mice/number of mice explored.
lung from the orthotopic site. Lung metastasis was
eventually achieved by iv injection of the selected
metastatic cells.
Our techniques of SOI should eliminate such a 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.
CONCLUSIONS
To our knowledge, this is the first report demonstrating high lung metastasis of PC-3 after orthotopic
implantation. 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 tumor growth and progression. Our large series of studies with nude mice models of various human cancers constructed by SOI suggest that
preservation of tumor architecture allows the greater
expression of the metastatic potential of the implanted
tumors than other techniques.
The metastatic model of PC-3 reported here should
enable us to study, simultaneously, the whole clinical
course of metastatic prostate cancer, from orthotopic
growth, local invasion, to distant metastasis. Subsequent studies will focus on the surgical orthotopic implantation of patient prostate cancer specimens to further understand the disease process for developing
new treatment strategies.
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