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An orthotopic metastatic prostate cancer
model in SCID mice via grafting of a
transplantable human prostate tumor line
Yuwei Wang
1
, Hui Xue
1
, Jean-Claude Cutz
1,2
, Jane Bayani
3
, Nasrin R Mawji
4
,
Wilfred G Chen
5
, Lester J Goetz
6
, Simon W Hayward
7
, Marianne D Sadar
4
,
C Blake Gilks
8
, Peter W Gout
1
, Jeremy A Squire
3
, Gerald R Cunha
9
and Yu-Zhuo Wang
1
1
Department of Cancer Endocrinology, BC Cancer Agency, Vancouver, British Columbia, Canada;
2
Department of Anatomical Pathology, St Joseph’s Hospital—Hamilton Health Sciences, Hamilton, Ontario,
Canada;
3
Department of Laboratory Medicine and Pathobiology, Ontario Cancer Institute, Toronto, Ontario,
Canada;
4
Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada;
5
Urology Research Unit, Carlton Centre, San Fernando, Trinidad;
6
Department of Urology, Gulf View Medical,
La Romaine, Trinidad;
7
Departments of Urologic Surgery and Cancer Biology, Vanderbilt University Medical
Center, Nashville, TN, USA;
8
Department of Pathology, Vancouver General Hospital, University of British
Columbia, Vancouver, British Columbia, Canada and
9
Departments of Anatomy and Urology, University
of California San Francisco, San Francisco, CA, USA
Metastasis is the major cause of prostate cancer deaths and there is a need for clinically relevant in vivo models
allowing elucidation of molecular and cellular mechanisms underlying metastatic behavior. Here we describe
the development of a new in vivo model system for metastatic prostate cancer. Pieces of prostate cancer tissue
from a patient were grafted in testosterone-supplemented male NOD-SCID mice at the subrenal capsule graft
site permitting high tumor take rates. After five serial transplantations, the tumor tissues were grafted into
mouse prostates. Resulting tumors and suspected metastatic lesions were subjected to histopathological and
immunohistochemical analysis. Samples of metastatic tissue were regrafted in mouse anterior prostates and
their growth and spread examined, leading to isolation from lymph nodes of a metastatic subline, PCa1-met.
Orthotopic grafting of PCa1-met tissue in 47 hosts led in all cases to metastases to multiple organs (lymph
nodes, lung, liver, kidney, spleen and, notably, bone). Histopathological analysis showed strong similarity
between orthotopic grafts and their metastases. The latter were of human origin as indicated by
immunostaining using antibodies against human mitochondria, androgen receptor, prostate-specific antigen
and Ki-67. Spectral karyotyping showed few chromosomal alterations in the PCa1-met subline. This study
indicates that transplantable subrenal capsule xenografts of human prostate cancer tissue in NOD-SCID mice
can, as distinct from primary cancer tissue, be successfully grown in the orthotopic site. Orthotopic xenografts
of the transplantable tumor lines and metastatic sublines can be used for studying various aspects of
metastatic prostate cancer, including metastasis to bone.
Laboratory Investigation (2005) 85, 1392–1404. doi:10.1038/labinvest.3700335; published online 12 September 2005
Keywords:
human prostate cancer; metastatic model; orthotopic; xenograft; subrenal capsule
Metastatic prostate cancers have a poor prognosis
with limited responses to palliative therapies such
as androgen ablation and chemotherapy.
1,2
The
development of new, effective therapies for meta-
static prostate cancer is considered to be critically
dependent on the availability of clinically relevant
in vivo models.
3
Current models used for studying
prostate cancer metastasis and drug evaluation
generally consist of xenografts in immuno-deficient
mice of well-established human prostate cancer cell
lines that have adapted to in vitro growth, for
example, LNCaP and PC-3.
4–6
Such models have
been useful for identifying cellular and molecular
mechanisms underlying metastasis and develop-
ment of new therapeutics. However, they have
profound shortcomings: the highly anaplastic
Received 3 March 2005; revised 8 July 2005; accepted 11 July
2005; published online 12 September 2005
Correspondence: Dr Y-Z Wang, PhD, Department of Cancer
Endocrinology, BC Cancer Agency—Research Centre, 675 West
10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3.
E-mail: ywang@bccrc.ca
Laboratory Investigation (2005) 85, 1392 –1404
&
2005 USCAP, Inc All rights reserved 0023-6837/05
$30.00
www.laboratoryinvestigation.org
prostate cancer cells used represent the extreme end
of highly advanced cancers and are not associated
with original tumor stroma, now recognized as a
crucial factor in the pathogenesis of cancer meta-
stasis.
4,5
These limitations severely restrict the
predictive power of such models with regard to
responses of patients’ tumors to anticancer drugs in
the clinic.
6
In view of this, various groups have
embarked on developing more relevant models
based on xenografting of primary prostate cancer
tissue in immuno-deficient mice, mainly using
advanced tumors and the subcutaneous graft
site.
3,7–11
These xenograft models retain growth and
histopathological features characteristic of the ori-
ginal cancers and have been used for rapid screening
of potential therapeutics. Unfortunately, xenograft-
ing to the subcutaneous site was associated with
exceedingly low tumor take rates and only success-
ful in case of highly advanced malignancies.
Furthermore, the subcutaneous compartment is a
nonorthotopic graft site. It does not mimic the
original tumor microenvironment, nor does it allow
full expression of metastatic potential. These defi-
ciencies reduce the usefulness of such models for
studies of prostate cancer metastasis.
12
Recently, we have developed a procedure for
successfully grafting and serially transplanting
primary human prostate cancer tissues in SCID
mice.
13
Using the subrenal capsule graft site, we
have consistently achieved high tumor take rates
(495%). This was a consequence of utilizing: (i)
SCID mouse hosts, (ii) adjustment of the hormonal
status of the host, if required and (iii) meticulous
surgical technique. In the present study, we found
that transplants of subrenal capsule xenografts of
human prostate cancer tissue, as distinct from
primary tumor tissue, were able to grow in the
orthotopic site (prostate) of SCID mice. The meta-
static behavior of the grafts at this site led to
isolation of a transplantable, metastatic tumor sub-
line, designated PCa1-met. Orthotopic xenografts in
SCID mice of the transplantable tumor and its
metastatic subline led to tumors showing histo-
pathological, tissue invasive and metastatic features,
which are similar to those normally encountered in
the clinic. Such xenografts could therefore be useful
as orthotopic models for human metastatic prostate
cancer.
Materials and methods
Materials and Animals
Chemicals, stains, solvents and solutions were
obtained from Sigma-Aldrich Canada Ltd, Oakville,
ON, Canada, unless otherwise indicated. Male 6- to
8-week old nonobese diabetic, severe combined
immuno-deficient (NOD-SCID) mice were bred by
the BC Cancer Research Centre Animal Resource
Centre, BC Cancer Agency, Vancouver, Canada.
Cell Cultures
The human prostate cancer cell line, LNCaP, was
obtained from the American Type Culture Collection
(Manassas, VA, USA). Cells were maintained in
RPMI-1640 medium (Stem Cell Technologies, Van-
couver, BC, Canada), supplemented with 10% fetal
bovine serum (Gibco-BRL, Burlington, ON, Canada),
penicillin (50 units/ml) and streptomycin (50 mg/ml)
(Stem Cell Technologies) in a humidified atmo-
sphere of 95% air and 5% CO
2
at 371C. Subculturing
was carried out using standard techniques, includ-
ing trypsinization using 0.25% trypsin/1.0 mM
EDTA.
Prostate Cancer Tissue Acquisition
Prostate cancer tissue was obtained via prostatect-
omy from a 75-year-old male, with informed
consent, at the Urology Research Unit, Carlton
Centre, San Fernando, Trinidad. The patient, diag-
nosed with advanced prostate cancer, had not
received neoadjuvant therapy prior to prostatect-
omy. The specimens were examined, sectioned and
selected by pathologists for histological analysis and
xenografting. The tumor sections selected for graft-
ing were shipped overnight, immersed in cold
Hanks’ balanced salt solution supplemented with
antibiotics to Vancouver, Canada.
Subrenal Capsule Grafting and Development of
Transplantable Tumor Lines
Within 24 h of its arrival, a minor portion of the
tumor was fixed for histological analysis. The
remainder of the tumor was cut into approximately
300 pieces with each piece about 1 3 3mm
3
in
size. A total of 70 NOD-SCID mice were supple-
mented with testosterone (25 mg) via subcuta-
neously implanted testosterone pellets.
14,15
The
tumor pieces were grafted to the subrenal capsule
sites of the mice. Under sterile conditions, a skin
incision of approximately 2 cm was made along the
dorsal midline of an anesthetized mouse. With the
animal lying on its side, an incision was then made
in the body wall slightly shorter than the long axis of
the kidney. The kidney was slipped out of the body
cavity by applying pressure on the other side of the
organ using forefinger and thumb. After exterioriza-
tion of the kidney, #5 fine forceps were used to
gently pinch and lift the capsule from the renal
parenchyma to allow a 2–4 mm incision in the
capsule using fine spring-loaded scissors. A pocket
between the kidney capsule and the parenchyma
was then created by blunt dissection. Care was taken
not to damage the parenchyma and thus prevent
bleeding. The graft was transferred to the surface of
the kidney using blunt-ended forceps. The cut edge
of the renal capsule was lifted with fine forceps, and
the graft was inserted into the pocket under the
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capsule using a fire-polished glass pipette. Two or
three grafts per kidney could be placed under the
renal capsule. The kidney was then gently eased
back into the body cavity and the body wall and skin
incisions sutured. Mice were housed in groups of
three in microisolators with free access to food and
water and their health was monitored daily. Animal
care and experiments were carried out in accor-
dance with the guidelines of the Canadian Council
on Animal Care.
After 60 or 90 days of growth (or earlier if required
by the health status of the hosts), the animals were
killed in a CO
2
chamber for necropsy. Tumors were
harvested, measured, photographed and fixed for
histopathological analysis. Some of the rapidly
growing tumors were maintained for up to eight
transplant generations by serial subrenal capsule
transplantation into testosterone-supplemented
male NOD-SCID mice. One of these transplantable
lines, PCa1, was used for the study presented here.
Orthotopic Grafting and Isolation of a Metastatic
Tumor Subline, PCa1-met
Tumor tissue of the transplantable PCa1 line was
harvested from a kidney and cut into pieces of about
2mm
3
. These were grafted into the anterior prostates
of 12 male, testosterone-supplemented NOD-SCID
mice (two per mouse). Briefly, a transverse incision
was made in the lower abdomen and the bladder,
seminal vesicles and prostate were partially pulled
out of the abdominal cavity to expose the anterior
prostate. A 2–3 mm incision was made through the
capsule of the anterior prostate between the two
main ducts. A 2 mm
3
PCa1 tumor graft was inserted
into the pocket created under the prostatic capsule.
The exteriorized organs were then returned into the
body cavity, and the incisions in the body wall and
skin closed using a running suture of 4-0 silk. After
6 weeks of growth, the host mice were killed for
gross examination of lymph nodes. Swollen lymph
nodes were bisected, and one-half subjected to
histological examination as frozen sections. Con-
firmed lymph node metastases were designated
‘PCa1-met’. Pieces of fresh lymph node containing
metastatic deposits were transplanted into prostates
of new hosts and the emerging PCa1-met line was
subsequently maintained by serial transplantation
both in prostates and under renal capsules of NOD-
SCID mice. The procedure for selecting PCa1-met is
illustrated in Figure 1.
Development of Multiorgan Metastases
PCa1-met tissue, freshly isolated from subrenal
capsule sites of NOD-SCID mice, was regrafted into
anterior prostates of 47 male, testosterone-supple-
mented NOD-SCID mice (divided into five groups).
At intervals of 2, 3, 4, 6 and 8 weeks after grafting,
mice were killed and lymph nodes, lungs, livers,
kidneys, spleens and bone (femur) were fixed for
histological and immunohistochemical (IHC) exam-
ination or frozen in liquid nitrogen for RT-PCR
analysis.
Histological and IHC Staining
The original tumor specimen, its transplants and
metastases were fixed in 10% neutral-buffered
formalin and embedded in paraffin; for bone
metastases, specimens were treated, after fixing,
with a sterile decalcification solution (15% EDTA/
0.5% paraformaldehyde in PBS, pH 8.0) for 3 weeks
prior to paraffin embedding. Serial sections (5 mm
thick) were cut on a microtome and mounted on
glass slides. Approximately 80 sections were cut
from each paraffin block. For histopathological
examination, every fourth section was de-waxed in
Histoclear (National Diagnostic, Atlanta, GA, USA)
and hydrated in graded alcohol solutions and
distilled water for H&E staining and examined
under a light microscope. For IHC staining, endo-
genous peroxidase activity was blocked with 0.5%
hydrogen peroxide in methanol for 30 min followed
by washing in PBS pH 7.4. In all, 5% normal goat or
donkey serum in PBS was applied to the sections for
30 min to block nonspecific sites. The sections were
then incubated with primary antibodies overnight at
41C or with control IgG from nonimmunized mice.
Rabbit polyclonal anti-androgen receptor (AR) anti-
bodies (PA1-111A) were purchased from Affinity
BioReagents (Golden, CO, USA) and mouse anti-
human mitochondria monoclonal antibodies (MAB-
1273) from Chemicon International (Temecula, CA,
USA). An anti-CK8 mouse monoclonal antibody
preparation (LE41) was generously provided by
Dr EB Lane, University of Dundee, UK. Polyclonal
rabbit antibodies against human prostate specific
antigen (PSA) and mouse anti-human Ki-67 anti-
bodies (m7240) were purchased from Dako (Carpin-
teria, CA, USA); these two antibodies reacted
with human, but not with mouse tissues. Purified
rabbit and mouse IgGs were obtained from Zymed
Corp. (So. San Francisco, CA, USA). Biotinylated
anti-rabbit and anti-mouse IgGs were obtained
from Amersham International (Arlington Heights,
IL, USA). Biotinylated anti-goat antibodies were
purchased from Sigma. Peroxidase-linked avidin/
biotin complex reagents were obtained from Vector
Laboratories (Burlingame, CA, USA). Following
incubation with the primary antibodies, sections
were washed with PBS and incubated for 30 min at
room temperature with the appropriate biotinylated
secondary anti-mouse immunoglobulins diluted
with PBS 1:200. After incubation with the secondary
antibodies, sections were washed in PBS (three
10-min washes), and then incubated with avidin–
biotin complex (Vector Laboratories, Foster City, CA,
USA) for 30 min at room temperature. Following a
further 30 min of washing in PBS, immunoreactivity
was visualized using 3,3
0
-diaminobenzidine tetra-
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hydrochloride (DAB) in PBS and 0.03% hydrogen
peroxide. Sections were counterstained with hema-
toxylin and dehydrated in graded alcohols. Control
sections were processed in parallel with mouse or
rabbit nonimmune IgG (Dako) used at the same
concentrations as the primary antibodies.
RT-PCR
Total RNA was isolated from tumor tissue using
TRIZOL reagent (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer’s instructions. A 1 mg
quantity was used for one-step RT-PCR (Invitrogen)
with the following thermal cycling conditions: one
cycle at 501C for 30 min, 941C for 2 min with an
additional 29 cycles for both GAPDH and mouse b2-
microglobulin at 941C for 15 s, 621C for 30 s and 721C
for 1 min and a final extension at 721C for 5 min. For
the nested PSA RT-PCR, 1 mg of total RNA was
subjected to one-step RT-PCR (Invitrogen) using the
external primers under the following conditions:
one cycle at 501C for 30 min, at 941C for 2 min and 25
cycles at 941C for 15 s, at 621C for 30 s and 721C for
1 min and a final extension at 721C for 5 min. In all,
1 ml from this PCR product was subjected to a second
PCR run using the internal primers with the
following conditions: 941C for 2 min and 25 cycles
at 941C for 15 s, 621C for 30 s and 721C for 1 min and
a final extension at 721C for 5 min. Sequences of the
primers used are as follows. External primers for
PSA: sense-GAT GAC TCC AGC CAC GAC CT,
antisense-CAC AGA CAC CCC ATC CTA TC; inner
primers for PSA: sense-GCA AGT TCA CCC TCA
GAA GG, antisense-GAT ATG TCT CCA GGC ATG
GC; primers for human GAPDH: sense-CCG AGC
CAC ATC GCT CAG A, antisense-CCC AGC CTT
CTC CAT GGT G; primers for mouse b2-microglo-
bulin: sense-CAC GCC ACC CAC CGC AGA ATG
GGA AGC C, antisense-CTG CGT GCA TAA ATT
GTA TAG CAT ATT AG.
Western Blot Analysis
The mouse and xenograft tissues were ground up
with the aid of mortar and pestle using liquid
nitrogen and then homogenized in ice-cold RIPA
Figure 1 Establishing an orthotopic metastatic prostate cancer model. Primary human prostate cancer tissue (a) was grafted under renal
capsules of testosterone-supplemented male NOD-SCID mice (b). After successful serial subrenal capsule transplantations of tumor grafts
(up to 8 times), tumor tissue was regrafted into anterior prostates of NOD-SCID mice (c, arrows). These orthotopic grafts gave rise to
lymph node metastases (d, arrow), which were successfully regrafted into mouse prostates, giving rise to metastases (e) in the lymph
node (H&E), lung (Ki-67 staining), liver (H&E) and bone (Ki-67 staining).
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buffer (40 mM Tris-HCl, pH 7.0/1 mM EDTA/4%
glycerol/10 mM DTT/0.2% SDS/20 mM Na molyb-
date/50 mM NaF/complete protease inhibitors
(Roche)) using a polytron homogenizer. Protein
concentrations were determined by RC DC assay
(BioRad). In all, 50 mg of protein was resolved on
an 8.5% SDS-polyacrylamide gel and transferred
to nitrocellulose membrane. The membrane was
blocked for 1 h in 5% (w/v) nonfat dry milk in
20 mM Tris (7.6), 150 mM NaCl and 0.1% Tween-20
and then probed with anti-AR441 at 1:500 (Santa
Cruz) overnight and subsequently with goat anti-
mouse HRP-conjugated secondary antibody. The
protein bands were detected using the enhanced
chemiluminescence kit (Amersham).
Spectral Karyotyping Analysis
PCa1-met tumor tissue was harvested to produce
metaphase spreads via primary cell cultures. To this
end, tumor tissue fragments (about 3–5 mm in
greatest dimension) were finely minced into a paste
and incubated in tissue culture medium at 371C, as
described above. When cultures grew to about 40%
confluency, cells were incubated with 0.1 mg/ml
colcemid (Gibco/BRL) for 30 min followed by 10%
trypsin (Invitrogen) for 5–10 min. Cell swelling, cell
fixation, metaphase slide preparation and pretreat-
ment were performed as previously described.
16,17
Spectral karyotyping (SKY) painting and detection
were performed using the SKYt kit probe cocktail
according to the manufacturer’s recommendations
(Applied Spectral Imaging, Carlsbad, CA, USA).
Pretreated slides were denatured in 70% forma-
mide/2 SSC at 751C for 1–2 min. The SKY probe
was denatured for 7 min at 751C and reannealed at
371C for 1 h. After hybridization for 16–40 h at 371C
in a humid dark chamber, posthybridization washes
and detection were performed as per the manufac-
turer’s directions. Acquisition of hybridization sig-
nals and corresponding DAPI-stained chromosome
spreads was carried out using the SD 200 bio-
imaging system and software (ASI Ltd, MigdalHae-
mek, Israel) attached to a Zeiss Axioplan-2 Micro-
scope (Carl Zeiss, Canada). SKY was performed
using SKYview software version 1.6.2 (ASI, Ltd) as
previously described and in keeping with ISCN
conventions.
16,17
Results
Characterization of a Patient-Derived Prostate Cancer
Line, PCa1
The transplantable PCa1 tumor line was derived
from a subrenal capsule xenograft of primary
prostate cancer tissue in a NOD-SCID mouse (see
Figure 1), supplemented with testosterone to over-
come xenograft atrophy due to intrinsically low
levels of the hormone in SCID mice.
13
It was
propagated by serial subrenal capsule xenografting
in testosterone-supplemented male NOD-SCID mice
for up to eight transplant generations. The tumor
volume doubling time in such mice was approxi-
mately 7 days. Similar to the primary tumor, the
PCa1 xenografts were poorly differentiated, gener-
ally lacking glandular differentiation (Figures 2a
and d). The primary tumor cells expressed human
AR and PSA as indicated by IHC staining using anti-
human AR and PSA antibodies (Figures 2b and c)
with high specificity as indicated by positive and
negative controls (Figures 2h and j); likewise, the
PCa1 xenografts expressed human AR and PSA,
although weakly but still detectable by IHC (Figures
2e and f), thus confirming their human prostatic
origin. Taken together, the data indicate that the
PCa1 tumor line represents an actively growing
human prostatic adenocarcinoma.
Development of a Metastatic PCa1 Subline, PCa1-met
PCa1 tissue could be grown successfully in anterior
prostates of testosterone-supplemented NOD-SCID
mice, in contrast to primary prostatic cancer tissue
whose grafting at this site was less effective.
13
At 6
weeks after orthotopic grafting of PCa1 tissue,
lymph nodes of two out of 12 mice were found to
contain metastatic foci. Metastatic cancer in one of
the hosts, designated PCa1-met, was further deve-
loped by grafting metastases-containing lymph node
tissue in anterior prostates and subsequent trans-
plantation of PCa1-met tissue in prostates or under
the renal capsule (see Figure 1). Figure 3 shows
lymph node metastases of PCa1-met at gross (Figure
3a) and microscopic (Figure 3b) levels. The meta-
static cells stained positively for human mitochon-
dria (Figure 3c) and human Ki-67 (Figure 3d). The
metastatic deposits also weakly expressed AR
(Figure 3e) and PSA (Figure 3f). Detection of
expression of human Ki-67, PSA and mitochondria
provide evidence that the PCa1-met cells are of
human prostatic origin.
Development of Multiorgan Metastases from
Orthotopic PCa1-met Tissue Xenografts
Grafting of PCa1-met tissue into anterior prostates of
NOD-SCID mice produced tumor take rates 495%
(260 out of 270 grafts). The grafts showed poorly
differentiated features and local invasion of host’s
prostate tissue (see Figure 4a). An adjacent section
(Figure 4b) stained positively with anti-human
mitochondria antibody. Furthermore, the orthotopic
grafts weakly expressed AR as observed with PCa1
cells (data not shown). After approximately 2 weeks
of grafting the mice developed multiorgan meta-
stases. As shown in Table 1, the animals developed
metastases in lymph nodes, lung, liver, kidney,
spleen and bone. The histology (H&E staining) of
the metastases in the lung (Figure 4c), liver (Figure
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5a), kidney (data not shown) and spleen (Figure 6a)
was similar to that of the original PCa1-met (Figure
3). Metastases in the lung (Figure 4d), liver (Figure
5b), kidney (data not shown) and spleen (Figure 6b)
were positively stained with anti-human mitochon-
dria antibodies. Similarly, human-specific Ki-67
staining was detected in metastases to lung (Figure
4e), liver (Figure 5c), kidney (data not shown) and
spleen (Figure 6c). This demonstrates that the
metastases were highly proliferative. IHC analyses
confirmed weak expression of AR in metastases in
the lung (Figure 4f), liver (Figure 5d), kidney (data
not shown) and spleen (Figure 6d); in contrast, AR
expression was negative in host tissues.
Using light microscopy of sections of host’s femur,
micrometastases of human origin in the marrow
were detected via staining with specific anti-human
mitochondria antibody (Figure 7a, arrows). Gross
bone metastases, however, were not observed in any
of the engrafted animals. The human origin of the
micro-metastases was also confirmed by RT-PCR for
human GAPDH (data not shown). Supporting
evidence for their human prostatic origin was
obtained via nested RT-PCR showing that human
PSA mRNA was associated with PCa1-met tissue as
well as with bone marrow (Figure 7d). Similarly,
human AR protein was found to be associated with
PCa1-met tissue (Figure 7e). At high magnification,
H&E-staining (Figure 7b) shows that the metastatic
human prostate cancer cells in a bone lesion had
histologic and cytologic features of the original
tumor cells, including a solid growth pattern,
moderately pale to clear cytoplasm, oval nuclei
and nucleoli and, importantly, produced osteolytic
lesions as demonstrated by replacement of marrow
and resorption of boney trabeculae (Figure 7b). An
adjacent section, stained with anti-human mito-
chondria antibody (Figure 7c), shows cytoplasmic
immunoreactivity within metastatic tumor cells
whereas the remaining bone tissue is negative.
Figure 2 Sections of human primary prostate cancer before grafting (a–c) and of the transplantable PCa1 tumor line developed by serial
subrenal capsule transplantation (d–f). The primary cancer is poorly differentiated and lacks glandular differentiation (a, H&E staining),
but is positive for AR (b) and PSA (c). The transplantable PCa1 tumor line retains poor differentiation and lack of glandular structure (d),
and is weakly positive for ARs (e, arrows) and PSA (f, arrowheads), indicating that it was derived from human prostate cancer. All IHC
staining experiments were carried out using both positive (g, AR; i, PSA) and negative (h, AR; j, PSA) controls using benign human
prostate tissue.
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SKY Analysis of PCa1-met
Primary PCa1-met tissue cultures were harvested after
5 days of incubation and subsequent 30-min treat-
ment with colcemid. The number of metaphases
obtained was very low (o1% metaphase count), but
sufficient to obtain a consensus SKY karyotype. PCa1-
met cells were diploid with a modal chromosome
Figure 3 Lymph node metastases from orthotopic xenografts of the PCa1-met prostate cancer tissue subline. Gross metastasis (a, arrow)
and microscopic sections of PCa1-met lymph node metastases (b, H&E), showing histological similarity with the parental subrenal
capsule xenografts (see Figure 2). Immunostaining is positive for human mitochondria (c), Ki-67 (d), AR (e) and PSA (f), indicating that
the lymph node metastases are derived from human prostate cancer.
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number of 45 and loss of the intact Y chromosome.
Sporadic (ie nonclonal, nonrecurring) chromosomal
aberrations were infrequent and the number of
clonal abnormalities was small. Figure 8 shows the
PCa1-met karyotype which is identical to that of PCa1
(not shown). The most frequent clonal changes
observed consisted of three unbalanced translocations
involving six chromosomes (#4, 6, 12, 13, 22 and Y).
Figure 4 Sections of an orthotopic graft of PCa1-met human prostate cancer tissue and its lung metastases. Tumor cells have grown
around a mouse host prostatic duct (a, H&E, arrow), staining positively with anti-human mitochondria antibody, while the host’s
prostatic ductal epithelial cells are negative (b). Two metastatic foci are present in the lung parenchyma (c, H&E, arrows). The tumor cells
stain positively with anti-human mitochondria (d), anti-Ki-67 (e) and anti-AR (f) antibodies.
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Discussion
Therapy-resistant metastasis is the leading cause of
human prostate cancer death.
1
Currently, there are
no effective therapies for metastatic prostate cancer,
and this deficiency is in part a reflection of a lack of
understanding of the mechanisms involved in the
progression of the disease.
2
Research in this area has
been hampered by a lack of clinically relevant,
experimental models.
3
For example, certain models
are based on injection of highly anaplastic human
prostate cancer cells into the bloodstream or into
target organs (eg, tibia) of immuno-deficient mice and
produce ‘experimental metastatic foci’.
18
Such foci are
not a consequence of the normal metastatic behavior
of cancer cells (intravasation, migration, extravasa-
tion). Instead, they rather reflect the ability of the
injected cells to proliferate in certain distant locations.
In contrast, models based on implantation of prostate
cancer tissue, as distinct from cells, into prostates of
experimental animals appear clinically more appro-
priate, since an implantation site is used that is more
representative of the original tumor environment.
19
There is accumulating evidence that metastatic
behavior of a neoplasm is governed not only by
intrinsic tumor factors, but also to a large extent by
multiple interactions between cancer cells and their
microenvironment promoting metastasis of discrete
tumor subpopulations.
4
Tumor-associated macro-
phages, for example, can induce matrix breakdown,
tumor cell motility and angiogenesis.
20,21
In esta-
blishing a model that represents a patient’s tumor, it
Table 1 Incidence of prostate cancer metastases in host
tissues (%)
Weeks after
grafting
(# of mice)
Lymph
nodes
Lung Liver Spleen Kidney Bone
2 (10) 80 30 0 0 0 0
3 (10) 100 100 30 30 10 0
4 (10) 100 100 80 90 40 0
6 (10) 100 100 90 90 100 0
8 (7) 100 100 100 100 100 43
Figure 5 Sections of liver metastases from orthotopically engrafted PCa1-met human prostate cancer tissue. Tumor cells are present in
the liver parenchyma (a, H&E, arrow), staining positively with anti-human mitochondria (b), anti-Ki-67 (c) and anti-AR (d) antibodies.
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is therefore essential that the grafted specimen
contains all of the crucial components of the original
tumor and that these are retained in the grafts. In
this regard, it is notable that the initial subrenal
capsule xenografts of prostate cancer tissue indeed
contained human stroma which, however, was
gradually replaced by mouse stroma.
22
Primary prostatic neoplasms are thought to con-
sist of a variety of carcinoma subpopulations
showing differences in invasive and metastatic
properties.
7
The present study shows that prostate
cancer tissue from a patient can be readily grown in
prostates of NOD-SCID mice following its establish-
ment as subrenal capsule xenografts. It appears that
the high tumor take rate achievable with the
subrenal capsule grafting technique, for both low
and higher grade cancers, minimizes the chance of
losing subpopulations of the original prostatectomy
specimen.
13
Potential loss of such subpopulations
appears much greater when other grafting sites
with much lower engraftment rates are used
(eg the subcutaneous compartment). The PCa1 line,
developed in this study, represents an actively
growing human prostatic adenocarcinoma line with
poorlydifferentiated features and general lack of
glandular differentiation (Figure 2). PCa1-met, the
metastatic subline derived from lymph node metas-
tases of the PCa1 line, resembles its parental line
with regard to poorly differentiated features, as well
as its human origin and expression of AR and PSA
(Figures 3 and 4). The metastases in the mice
receiving orthotopic transplantation of PCa1-met
tissue were located in lymph nodes, lung, liver,
kidney, spleen and bone (Figures 3–7), which are
common metastatic sites in prostate cancer patients.
The finding in mouse bone of orthotopically grafted
cancer cells of prostatic origin and exhibiting
osteolytic action is particularly interesting, since
this phenomenon is not observed with other
currently available metastatic prostate cancer
models.
23
It appears therefore that a subline such
as the PCa1-met, produced by subrenal capsule
and subsequent orthotopic xenografting, is highly
suitable for use in NOD-SCID mice as a model for
Figure 6 Sections of spleen metastases from orthotopically engrafted PCa1-met human prostate cancer tissue. Foci of tumor cells are
present in splenic parenchyma (a, H&E, arrows), staining positively with anti-human mitochondria (b), anti-Ki-67 (c) and anti-AR (d)
antibodies.
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Figure 7 Sections of bone marrow containing metastases from orthotopically engrafted PCa1-met human prostate cancer tissue. Low-
power photomicrograph (produced by IHC staining using anti-human mitochondria) showing replacement of marrow elements by
metastatic carcinoma (a, arrows) with thinning of boney trabeculae resulting in osteolytic appearance. Higher power photomicrographs
showing sheets of tumor cells and a single thinned trabecula (arrows) (b, H&E staining; c, staining with anti-human mitochondria
antibodies). (d) RT-PCR products for PSA (human) and b2-microglobulin (mouse; control) using total RNA harvested from tissues.
(e) Western blot of human AR from various tissues.
Figure 8 Spectral karyotype composite of the PCa1-met human prostate cancer line. DAPI-banded preparation of metaphase
chromosomes from a cell following short-term culture (a), hybridized to SKY paints (b) and after pseudocolor classification (c). Resultant
karyotype table is presented in (d). A SKY profile of PCa1-met: 45, X,Y,der(6)t(6;13)(q24;q22), der(12)t(Y;12)(q11;p11),
der(22)t(4;22)(?;p11).
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metastatic prostate cancer. It may be noted that the
PCa1-met line was found to be androgen-indepen-
dent, growing readily in mice following androgen
ablation (unpublished observation).
SKY analyses of three widely used prostate can-
cer cell lines (LNCaP, DU-145, PC-3) have demon-
strated aneuploid karyotypes with many chromo-
somal alterations including complex chromosomal
rearrangements and a high degree of karyotypic
instability. No common chromosomal rearrangement
or translocation breakpoint was identified.
24,25
In
contrast to these highly anaplastic cell lines, early-
stage prostatic tumors are typically diploid with
some propensity to progress to aneuploidy and
karyotypic complexity in advanced stages.
26
The
karyotype of PCa1-met is comparable to the latter,
with surprisingly few chromosomal alterations
produced by as few as six breakpoints involving
chromosomes 4, 6, 12, 13, 22 and Y. Common
involvement of chromosome 8, typically a loss of 8p
with gain of 8q, reported previously,
27
was not seen
in the present study using SKY. However, smaller
but significant chromosomal alterations, such as
small interstitial deletions, insertions/duplications
and inversions cannot be revealed by whole-chro-
mosome painting and would require higher-resolu-
tion techniques such as locus-specific-FISH, Mband
FISH or array CGH. Loss of the Y chromosome is
frequently associated with aging. However, it has
also been suggested that loss of certain Y chromo-
some specific genes may play a role in the
pathogenesis of prostate cancer.
28
The finding of
simple and identical karyotypes in PCa1 (data not
shown) and PCa1-met, its metastatic subline, sug-
gests that significant chromosome and karyotypic
instability are not characteristics of these tumor
lines, in contrast to commonly used prostate cancer
cell lines such as DU-145.
29
It is also likely that
conservation of such few chromosomal changes is
of biological significance, by offering selective
advantage to the tumor cells and maintenance of
malignant and/or metastatic properties.
In conclusion, the orthotopic prostate cancer
metastasis model developed in this study should
be useful for investigations of mechanisms under-
lying prostate cancer metastasis and therapeutic
applications. The methodology used to develop this
model may also be suitable for developing ortho-
topic metastatic cancer models of other types of
human cancer. Thus, the first step of the methodo-
logy, that is, subrenal capsule grafting, has been
successful for a variety of primary human cancer
tissues, including cancers of the ovary,
30
kidney,
22
lung, pancreas and lymphoid tissue (unpublished
observations).
Acknowledgements
We thank Rebecca Wu and Lily Wei for excellent
technical assistance. Paula Marrano is thanked for
technical expertise and help in SKY analyses. This
study was supported in part by grants awarded
to YZW by NCI Canada (#014053), the US Army
Department of Defense, USAMRMC W81XWH-04-1-
0290, the Prostate Cancer Foundation of Canada
and to GRC by the National Cancer Institute
(# CA89520). J-C Cutz was a CIHR fellow in
Molecular Oncologic Pathology.
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