[CANCER RESEARCH 59, 1987–1993, April 15, 1999]
Severe Combined Immunodeficient-hu Model of Human Prostate Cancer Metastasis
to Human Bone1
Jeffrey A. Nemeth, John F. Harb, Ubirajara Barroso, Jr., Zhanquan He, David J. Grignon, and Michael L. Cher2
Program in Urologic Oncology (J. A. N., D. J. G., M. L. C.) and Cancer Biology (X. H.), Barbara Ann Karmanos Cancer Institute, and Departments of Urology (J. A. N., J. F. H.,
U. B., M. L. C.) and Pathology (D. J. G, M. L. C.), Wayne State University School of Medicine, Detroit, Michigan 48201
ABSTRACT
INTRODUCTION
Commonly used in vivo models of prostate cancer metastasis include
syngeneic rodent cancers and xenografts of human cancer in immunodeficient mice. However, the occurrence of osseous metastases in these
models is rare, and in xenograft models, species-specific factors may limit
the ability of human cells to metastasize to rodent bones. We have modified the severe combined immunodeficient (SCID)-human model to test
the ability of circulating human prostate cancer cells to home to macroscopic fragments of human bone and other organs previously implanted
into SCID mice. We have also compared the growth of human prostate
cancer cells in various human and mouse tissue microenvironments in
vivo. Macroscopic fragments of human fetal bone, lung, or intestine
(16 –22 weeks gestation) or mouse bone were implanted s.c. into male
CB.17 SCID mice. Four weeks later, human prostate cancer cells were
injected either i.v. via the tail vein (circulating cell colonization assay) or
directly into the implanted tissue fragments transdermally (end organ
growth assay). Tumor growth was followed for 6 weeks by palpation and
magnetic resonance imaging. After 6 weeks, tumors were enumerated in
implanted human and mouse organ fragments and native mouse tissue.
Tumors were characterized by histology, immunohistochemistry, and
chromosomal analysis. After i.v. injection, circulating PC3 cells successfully colonized implanted human bone fragments in 5 of 19 mice. Tumors
were easily followed by palpation and imaging and had an average volume
of 258 mm3 at autopsy. Histological examination revealed osteolysis and a
strong desmoplastic stromal response, which indicated intense stromalepithelial interaction. Bone tumors were subcultured, and chromosomal
analysis demonstrated that the tumors were derived from the parental
prostate cancer cell line. Microscopic tumor colonies were also found in a
few mouse lungs after i.v. injection of PC3, DU145, and LNCaP cells,
however the volume of the lung nodules was less than 1 mm3 in all of the
cases. No colonization of human lung or intestine implants, the mouse
skeleton, or other mouse organs was detected, demonstrating a speciesand tissue-specific colonization of human bone by PC3 cells. Direct injection of 104 prostate cancer cells into human bone implants resulted in
large tumors in 75–100% of mice. PC3 and DU145 bone tumors were
primarily osteolytic, whereas LNCaP bone tumors were both osteoblastic
and osteolytic. PC3 and LNCaP bone tumors showed a desmoplastic
stromal response, which indicated intense stromal-epithelial interaction.
All three of the cell lines formed tumors in implanted human lung tissue;
however, the tumors were all <10 mm3 in volume and showed minimal
stromal involvement. No tumors formed after either s.c. injection or
injection of cells into implanted mouse bone demonstrating both speciesand tissue-specific enhancement of growth of human prostate cancer cells
by human bone. The severe combined immunodeficient-human model
provides a useful system to study species-specific mechanisms involved in
the homing of human prostate cancer cells to human bone and the growth
of human prostate cancer cells in human bone.
Bone has long been recognized to be the most common target organ
of prostate cancer metastasis (1), and the phenomenon of osseous
metastasis signals the final, incurable stage of disease. The reasons
underlying the proclivity of prostate cancer to metastasize to bone
remain unclear; however, the competing hypotheses can be divided
into two categories: anatomical/hemodynamic factors (Batson’s
plexus) and “seed and soil.” In 1940, Batson (2) described a venous
portal system between the plexus of veins draining the prostate and
the plexus of veins surrounding the lumbar spine. In animal experiments and in human cadavers, Batson demonstrated that dye injected
into the deep dorsal vein of the penis passed through the prostatic
plexus and continued through longitudinal, valveless veins to the
sinusoidal venous structures of the lumbar spine (2, 3). These experiments suggested that passive hemodynamic patterns may contribute
to the tendency of prostate cancer to metastasize to the lumbar spine.
The alternative hypothesis has been attributed to Paget, who surmised
that the metastatic pattern of a given type of carcinoma is dependent
on the interaction of individual tumor cells with the microenvironment
of the target tissue (4). Thus the high frequency of osseous metastases
in prostate cancer is not due to passive hemodynamic patterns but to
a molecular affinity between prostate cancer cells and the bone
marrow microenvironment.
The phenomenon of prostate cancer metastasis to bone has been
difficult to mimic in animal models. In syngeneic animal prostate
cancer models such as the Dunning rat model (5) and in the transgenic
mouse model TRAMP (6), lymph node and pulmonary metastases are
common, but the occurrence of bone metastasis from a primary tumor
site is rare. It has also been difficult to mimic clinical patterns of
osseous metastasis in immunodeficient murine xenograft models of
human prostate cancer. For example, i.v. injection of up to 1 3 106
PC3 or LNCaP human prostate cancer cells does not typically result
in the development of bone metastases (7, 8). Increased osseous
metastasis of prostate cancer cell lines has been achieved by various
experimental manipulations, including coinjection of tumor cells with
bone stromal cells (9), orthotopic injection of prostate tumor cells
(10), or brief occlusion of the vena cava during i.v. injection of cells
(11). Nonetheless, these xenograft models all share the fact that
human cells must metastasize to and grow in mouse organs. It is
possible that many of the molecules involved in the metastatic process
such as proteinases, adhesion molecules, chemotactic factors, and
growth factors and their receptors, are species-specific, which may
contribute to the difficulty in achieving bone metastases in xenograft
models of prostate cancer.
Recent reports have described the use of the SCID-hu3 system to
study the behavior of metastatic human tumor cells. It was found that
small cell lung carcinoma cells that did not grow in murine organs
after i.v. injection specifically colonized human fetal lung and bone
marrow tissues implanted in SCID mice (12). Application of the
SCID-hu3 system to the study of human colon carcinoma has also led
Received 10/9/98; accepted 2/16/99.
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.
1
Supported by the American Cancer Society CRTG-97-044-01-EDT (to M. L. C.).
2
To whom requests for reprints should be addressed, at Departments of Urology and
Pathology, Wayne State University School of Medicine, 540 East Canfield Road, Scott
Hall #9112, Detroit, MI 48201. Phone: (313) 577-2879; Fax: (313) 577-0057; E-mail:
[email protected].
3
The abbreviations used are: SCID-hu, severe combined immunodeficient-human;
MR, magnetic resonance; MRI, magnetic resonance imaging; FOV, field of view; CGH,
comparative genomic hybridization; PSA, prostate-specific antigen.
1987
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
to the identification of adhesion molecules that may be important in
colon cancer metastasis (13). These studies clearly illustrate the importance of species- and tissue-specific interactions in the design of in
vivo metastasis models. Using the SCID-hu system, we have found
that circulating human prostate cancer cell lines preferentially colonized implanted human bone tissue. In addition, growth of human
prostate cancer cells was specifically enhanced by direct interaction
with the human bone marrow microenvironment.
MATERIALS AND METHODS
receive MR signals because it produced images with increased signal:noise
ratios compared with the whole-body coil. The surface coil and whole-body
coil were actively decoupled to avoid signal interference. High-resolution
T1-weighted spin echo images (TE 5 15 ms, TR 5 545 ms, flip angle 5 90`,
8.0 cm FOV, and 256 3 256 matrix size) were acquired in coronal sections to
serve as the detail anatomical reference for tumor position. However, tumor
cannot be easily distinguished from surrounding normal tissue in these T1weighted images. To enhance contrast between tumor and normal tissue, a
multislice T2-weighted spin echo sequence (TE 5 60 ms, TR 5 1225 ms, flip
angle 5 90`, 2 averages) with fat saturation was used. T2-weighted images of
10 contiguous slices covering the whole tumor were obtained in two sections
(axial and coronal), yielding in-plane resolution ,0.1 mm2 with a slice
thickness of 1.0 mm (6.4-cm or 8.0-cm FOV, 256 3 256 matrix size).
Data Analysis and Histology. Mice were killed 6 weeks after injection of
tumor cells by cervical dislocation while under methoxyflurane anesthesia.
Human tissues and certain mouse tissues (lumbar vertebrae, ribs, visible lymph
nodes, and lungs) were dissected, photographed, and measured with calipers.
Tumor volume was estimated by the formula a 3 b2/2, where a is the longest
dimension and b is the width. All of the other organs and the mouse skeleton
were visually inspected. Mouse lungs were stained in Bouin’s fluid to reveal
tumor nodules. Portions of each tissue were fixed in 10% buffered formalin,
decalcified (bone specimens), embedded in paraffin, sectioned, and stained
with H&E for routine histological examination. Immunohistochemical staining
was also performed to confirm the presence of tumor cells in bone tissues.
Briefly, sections were deparaffinized and rehydrated through graded alcohols.
For antigen retrieval (15), slides were placed in 10 mM citrate buffer and boiled
by microwave heating for 5 min. Nonspecific sites were blocked by incubation
with Superbloc (ScyTek, Logan, UT). Sections were incubated with either
monoclonal antipan cytokeratin antibody (C-2562, Sigma, St. Louis, MO) or
nonspecific mouse immunoglobulins at 1:200 dilution. Positive immunoreactive sites were detected using a Vectastain alkaline phosphatase kit (Vector,
Burlingame, Ca) and Sigma Fast Red substrate, and nuclei were briefly
counterstained with hematoxylin.
Mean tumor volumes were calculated for each cell line test group and tumor
site, and statistical comparisons of mean tumor volumes between groups were
performed using Student’s t test (one-tailed analysis).
Subculture of Tumors and Genetic Analysis. Tumors arising in implanted human bone tissues after i.v. injection of tumor cells were analyzed to
confirm the cell of origin. Small portions were minced under sterile conditions
and cultured in Keratinocyte-SFM (Life Technologies, Inc.) containing EGF,
bovine pituitary extract, and 10% fetal bovine serum until a confluent monolayer of cells was established. This medium was chosen to retard the growth of
fibroblastic cells. After the first and second passages, cultures were stained for
cytokeratin as described above (“Histology”) to assess the purity of the tumor
cell populations. By the second passage, the cultured tumor cells were nearly
100% cytokeratin-positive, suggesting minimal stromal cell survival in culture.
At this point, cells were analyzed by CGH.
CGH analysis was performed essentially as described previously (16, 17).
DNA was prepared separately from bone tumor subcultures and parental cell
lines by proteinase K digestion and phenol/chloroform extraction. One mg of
tumor DNA or reference normal DNA was nick-translated in the presence of
FITC-12-dUTP (green fluorescence) or Texas Red-5-dUTP (red fluorescence,
DuPont NEN, Boston, MA), respectively. Labeled tumor and normal DNA
were hybridized together with 30 mg of Cot-1 DNA (Life Technologies, Inc.)
onto normal male metaphase spreads (Vysis, Downers Grove, IL) for 2–3 days.
After washing, slides were counterstained with 0.1 mM DAPI. Ten to 15
metaphase images were acquired for each tumor-normal hybridization, of
which the best images were chosen for quantitative analysis. Quantitative
image analysis was performed as described previously using the QUIPS CGH
system (Vysis Inc., Downers Grove, IL; Ref. 18).
Serum PSA Measurements. Serum samples were taken from mice bearing
LNCaP tumors in implanted human bone fragments at the time the mice were
killed. Total PSA levels were measured by routine methods at the Clinical
Chemistry Laboratory of the Detroit Medical Center.
Cell Lines and Cell Culture. DU145, LNCaP, and PC3 human prostate
cancer cell lines were purchased from the American Type Culture Collection.
DU145 cells were maintained in culture in DMEM-10% fetal bovine serum,
and LNCaP and PC3 cells were maintained in RPMI 1640 –10% fetal bovine
serum. All of the culture reagents were purchased from Life Technologies, Inc.
(Gaithersburg, MD). Cell lines were used at low passage number in all of the
experiments.
Animal Care and Human Tissue Implantation. Male homozygous
C.B-17 scid/scid mice, aged 5 weeks, were purchased from Taconic Farms
(Germantown, NY). Mice were maintained according to the NIH standards
established in the “Guidelines for the Care and Use of Experimental Animals,”
and all of the experimental protocols were approved by the Animal Investigation Committee of Wayne State University. Human male fetal tissues were
obtained with informed consent according to regulations issued by each state
involved and the federal government. Methoxyflurane anesthesia was used
during all of the surgical procedures. Implantation of 6-week-old mice with
human fetal lung fragments (SCID-hu-L) or human fetal intestine fragments
(SCID-hu-I) was performed as described by Shtivelman (12). Briefly, human
fetal lungs or intestines of 16 –22 weeks of gestation were cut into 2 3 2 3 2
mm fragments and implanted s.c. in the ventral surface of the mouse via a
small skin incision. Implantation of mice with human fetal human bone
fragments (SCID-hu-B) was performed as described previously(14). Briefly,
human fetal femurs and humeri of 16 –22 weeks of gestation were divided in
half longitudinally and then again in half transversely into four fragments
approximately 1 cm long and 3 or 4 mm in diameter. These bone fragments
were implanted s.c. in the flank through a small skin incision with the opened
marrow cavity against the mouse muscle. As a control for species-specific
growth of prostate cells in human bone, femurs were harvested from normal
SCID mice, and bone fragments were implanted in the flank of recipient mice
in an identical fashion (SCID-m-B). All of the mice received at least two
different tissue implants; human bone in the flank and human lung or intestine
in the abdomen or human bone and mouse bone in opposite flanks.
Circulating Cell Colonization Assay. Cultured prostate cancer cell lines
were harvested by incubation with trypsin/EDTA and washed twice in Hank’s
balanced salt solution. Cell number and viability were measured by trypan blue
exclusion. For in vivo circulating cell colonization experiments, 1 3 106 cells
in a volume of 200 ml were injected into SCID-hu mice via lateral tail vein
using a 27-g needle. In all of the experiments, cancer cells were injected 4
weeks after tissue implantation because this time point was found to be optimal
for colonization of human tissues by carcinoma cells in previous reports (12).
End Organ Tumor Growth Assay. For experiments measuring growth of
human prostate cancer cells in human metastatic organ microenvironments in
vivo, 1 3 104 cells in a volume of 20 ml were injected directly into implanted
human or mouse tissue fragments through the skin using a 27-g needle. Again,
injections were performed 4 weeks after tissue implantation. For the bone
fragments, the opened marrow surface was targeted with the needle. Some
control mice also received the same number of cells injected s.c. in the
shoulder region as a further control for tumor growth. For the LNCaP cell line,
a second group of mice received a larger inoculum of 5 3 104 cells (see
“Results”).
MRI. MR images were acquired on a Bruker AVANCE spectrometer
equipped with 4.7-T horizontal-bore magnet and actively shielded gradients.
During MR observation, the mouse was anesthetized (1.5% v/v halothane) and
immobilized on a bed that was heated by circulating temperature-controlled RESULTS
water. A whole-body RF transceiver coil (70-mm diameter) was used to
Implantation of Human Fetal Tissues. SCID-hu mice were used
transmit homogeneous RF excitation and hold the animal bed. A RF surface
for circulating cell colonization and end organ growth experiments
transceiver coil (30-mm diameter) was placed on the back of the mouse to
1988
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
3– 4 weeks after the implantation of human tissues. Previous studies
demonstrated higher rates of colonization of implanted human tissues
when cells were injected at early time points, before the recovery of
hematopoiesis in the bone marrow compartment (12). To assure
viability of implanted human fetal tissues at the time of injection, four
SCID-hu mice bearing different human tissue implants were killed 4
weeks after implantation, and the tissues were examined histologically. The marrow compartment of human bone implants contained
mainly stromal elements and some residual hematopoietic cells, and
evidence of new bone formation could be seen (Fig. 1A). Small blood
vessels containing RBCs were clearly visible in the tissue sections.
Human lung implants grew in size, and the tissue appeared to become
more differentiated with evidence of alveolar structures, columnar
epithelium lining bronchioles, and cartilage (Fig. 1B). Likewise, implanted human fetal intestine appeared healthy, with a well-differentiated epithelial lining (Fig. 1C). These results are consistent with
previous reports describing the biology of implanted human fetal
tissue fragments (12, 14) and suggest that the implanted tissues were
healthy and adequately vascularized.
Colonization of Human Tissue Implants by Circulating Tumor
Cells. The ability of circulating prostate cancer cells to colonize
human tissues in vivo was tested by the injection of DU145, LNCaP,
or PC3 cells into SCID-hu mice via the lateral tail vein. Three weeks
after the injection of cancer cells, 5 (26%) of 19 mice receiving PC3
cells developed grossly palpable and measurable tumors in implanted
human bone fragments (Table 1). Six weeks after injection, tumors
were confirmed and measured at autopsy. Histological analysis revealed large solid tumors composed of cohesive nests of tumor cells
intermingled with a cellular desmoplastic stromal response (Fig. 2B).
Close examination showed areas of bone destruction and resorption,
which indicated an osteolytic tumor phenotype (Fig. 2C).
Immunohistochemical staining for cytokeratin confirmed the presence of epithelial tumor cells (not shown). DU145 and LNCaP cells
did not form macroscopic palpable tumors in human bone tissues after
tail vein injection (Table 1), and this observation was confirmed
histologically and by immunohistochemical staining for cytokeratin
(not shown). Histological evaluation of implanted human lung and
intestine tissues did not reveal any evidence of colonization by any of
the cell lines tested (Table 1), which suggested that circulating PC3
cells prefer to colonize human bone as opposed to other human
tissues, and the colonization of human bone by PC3 cells was not
merely an effect of the implantation procedure.
Because cells injected i.v. via the tail vein must pass through
the mouse lung before entering the general circulation, we examined
the mouse lungs for signs of colonization. In a small number of mice,
the three cell lines formed between 5 and 15 colonies visible on the
lung surface (Table 1), however these colonies were always less than
1 mm3 in volume and did not cause any adverse respiratory effects. Of
Table 1 Circulating cell colonization assay
Tumor incidence was measured in implanted human tissues and host mouse lungs 6
weeks after i.v. injection of 1 3 106 prostate cancer cells.
Target organ
Cell line
Human
bone
Human
lung
Human
intestine
Mouse lunga
(average no. of colonies/
mouse)
DU145
LNCaP
PC3
PC3-HB1
0/12
0/12
5/19b
0/12
0/12
0/6
0/16
0/12
0/7
0/3
0/5
NDc
5/12 (12.5)
1/12 (6)
2/19 (4.5)
0/12
a
Volume of individual prostate cancer cell lung nodules was less than 1 mm3 in all of
the cases.
b
Average PC3 bone tumor volume 5 258.0 mm3.
c
ND, not done.
the two mice with PC3 tumors in the mouse lungs, one occurred in a
mouse that also had a human bone tumor. A representative lung
colony formed by circulating PC3 tumor cells is shown in Fig. 2D.
None of the mice experienced limb paralysis, which was consistent
with a lack of spinal metastases, and all of the mouse tissues examined
were clear of tumor deposits by gross and histological inspection. The
absence of metastasis to mouse bone suggested that species-specific
factors favored targeting to the human bone environment.
To confirm the origin of tumors arising in implanted human bone
tissues after i.v. injection of PC3 cells, explant cultures of these
tumors were established. After the second passage, the cultures were
greater than 98% epithelial cells, as judged by cellular morphology
and positive staining for cytokeratin (not shown), confirming that the
tumors were of epithelial origin. One of the resulting cell populations,
termed PC3-HB1, was characterized by CGH analysis. We found
gains in the regions of chromosome 7p, 8q, and 11q, and losses in the
regions of chromosome 4q, 9p, and 10p (not shown). Analysis of the
parental PC3 cells revealed the same pattern of chromosomal alterations as PC3-HB1. This pattern of gains and losses was consistent
with the recent findings of Nupponen et al. (19). Genetic analysis thus
confirmed that the bone tumors that were formed in the circulating
cell colonization assay originated from circulating PC3 cells.
The ability of circulating PC3-HB1 cells to form tumors in
SCID-hu mice was also tested. Injection of 1 3 106 cells into
SCID-hu mice via the lateral tail vein did not result in any detectable
tumors in human bone or lung tissues nor in the mouse lungs (Table
1). The reason for this result was unclear.
End Organ Tumor Growth Assay. To measure the effects of
various human tissue microenvironments on the in vivo growth of
human prostate cancer cells, we used a direct injection approach to
ensure uniform seeding of the tissues with the tumor cells. The
injection of 1 3 104 DU145, PC3, or PC3-HB1 cancer cells directly
into implanted human bone fragments resulted in grossly evident
Fig. 1. SCID-hu tissue implants. Histological appearance of human fetal tissues 4 weeks after implantation into SCID mice. A, the marrow compartment of human bone implants
contained mainly stromal elements and some residual hematopoietic cells, and evidence of new bone formation could be seen (arrow). 340. B, implanted human lung with alveolar
structures (arrow) and columnar epithelium lining bronchioles (arrowhead). 340. C, implanted human fetal intestine appeared healthy, with a well-differentiated epithelial lining
(arrow). 340. No evidence of necrosis was seen in any tissue examined.
1989
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
Fig. 2. Circulating cell colonization assay. Representative tumor formed in SCID-hu mouse after i.v.
injection of PC3 cells. A, gross appearance of tumor in
human bone fragment. Tumor appeared as a smooth
mass (bracket) extending from residual normal bone
tissue. Bar, 1 cm. B, low power view of tumor mass,
showing residual bone tissue at the periphery (arrow).
320. C, close examination revealed cohesive nests of
tumor cells in a desmoplastic stromal response. Evidence of osteolytic activity could also be seen (arrowhead). 3100. D, representative colony of PC3 tumor
cells in mouse lung, consisting of a small number of
cells (arrow). 3100.
tumors in a majority of mice (Table 2). The bone tumors were easily
palpable through the skin and could be measured with calipers.
To test the accuracy of tumor measurement before sacrifice, MRI
was performed on two mice bearing PC3 tumors in human bone
tissue. The tumors appeared as regions of high signal intensity on T2
weighted images (Fig. 3). Residual bony tissue could be discerned as
dark punctate areas within the tumor mass. Measurements taken from
these images agreed with direct measurement of the same bone tumors
harvested the following day.
The average size of bone tumors formed by PC3 cells was larger than
tumors formed by DU145 (P , 0.05), which indicated more rapid growth
of PC3 cells (Table 2). Injection of 1 3 104 LNCaP cells did not result
in detectable bone tumors (Table 2), however increasing the inoculum
size to 5 3 104 cells resulted in tumors in 6 (75%) of 8 mice. The bone
tumors formed by LNCaP cells were also large and similar in size to the
PC3 and PC3-HB1 bone tumors (P . 0.5 for both comparisons). In
contrast, no tumors were formed in any group after s.c. injection of the
same low number of cancer cells, which indicated that the human bone
environment enhanced prostate tumor growth in vivo.
Histological examination of the bone tumors formed by each of the
cell lines showed large, poorly differentiated tumors with a variable
degree of stromal-epithelial interaction (Fig. 4, A, B, and C). DU145
cells formed solid, cellular tumor masses without central necrosis
(Fig. 4A). A limited desmoplastic stromal response was present at the
tumor-normal tissue interface. In contrast, the tumors formed by PC3
cells showed a striking desmoplastic stromal response with large areas
of central necrosis (Fig. 4C). Fragments of necrotic bone could be
seen within the tumor masses. The differential distribution of tumor
and stromal cells was more clearly demonstrated by immunostaining
for cytokeratin (Fig. 4, D, E, and F). Tumors formed by DU145 and
PC3 also appeared to be osteolytic in nature. In DU145 tumors, areas
of bone resorption could clearly be seen adjacent to tumor cell nests
(Fig. 4A, arrow); and in many of the PC3 tumors, the calcified bone
tissue was almost totally replaced by tumor cells and fibrous stroma
(Fig. 4C). LNCaP cells formed solid tumors in bone with a moderate
desmoplastic stromal response (Fig. 4B). The tumors formed by
LNCaP cells appeared to be of a mixed osteoblastic/osteolytic type
(Fig. 4B), and regions of bone resorption and deposition of new bone
matrix (arrow) could be seen. The level of total PSA was also
measured in serum samples taken from two mice bearing LNCaP
tumors in human bone tissues and was found to average 4.5 ng/ml.
This result further confirmed the presence of LNCaP tumor in these
mice and suggested that LNCaP cells retained the ability to produce
PSA while growing in a human bone microenvironment in vivo.
To control for the possibility that any implanted human tissue could
enhance tumor growth, prostate cancer cell lines were also injected
into implanted human lung tissues. Tumor deposits formed in a
portion of implanted human lung tissues directly injected with prostate tumor cells, and in all of the cases, the tumor nodules were
extremely small in comparison with tumors formed in the bone
implants (Table 2). For all of the three cell lines, the tumors that grew
in implanted lung tissue consisted of solid masses of tumor cells with
intervening vasculature (Fig. 4, G, H, and I). Very little interaction
with stromal cells could be seen, and many of the tumor nodules were
surrounded by a thin capsule.
To control for species specificity, prostate cancer cell lines were
injected into s.c. implanted mouse bone fragments. For each cell line,
four mouse bone implants were injected, and in no case did a bone
tumor result (Table 2). This provided further evidence of the importance of human-human stromal-epithelial interactions with regard to
prostate cancer cells growing within bone.
Table 2 End organ tumor growth assay
Tumor incidence and mean tumor volume were measured 6 weeks after the direct injection of prostate cancer cells into implanted human or mouse tissues.
Injection site
Cell Line (no. of cells injected)
DU145 (1 3 104)
LNCaP (1 3 104)
LNCaP (5 3 104)
PC3 (1 3 104)
PC3-HB1 (1 3 104)
a
b
a
a
Human bone
Human lung
Mouse bone implant
Subcutaneous
7/9 (113.5 6 26.0)
0/7
6/8 (305.5 6 62.5)
16/18 (407.5 6 106.5)
10/10 (235.5 6 92.0)
4/9 (4.0 6 1.0)
5/10 (10.5 6 1.5)
NDb
3/14 (2.5 6 1.0)
3/10 (1.0 6 0.5)
0/4
0/4
ND
0/4
ND
0/7
0/8
0/6
0/8
ND
Tumor volume (in parentheses) expressed in mm3 6 SE.
ND, not done.
1990
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
Fig. 3. MRI. T2-weighted spin echo MR images (TE 5 60
ms, TR 5 1225 ms) of a mouse bearing a PC3 tumor in
implanted human bone tissue. A, coronal section, B, axial
section. Arrows, tumor location.
DISCUSSION
When designing animal models of human prostate cancer metastasis, it is important to take into account the possible contribution of
human-human stromal-epithelial interactions to the establishment and
growth of prostate tumors in the target bone tissue. The results of this
study demonstrate that circulating human prostate cancer cells were
able to specifically and preferentially colonize implanted human bone
tissue in SCID mice, and that human bone provided a more favorable
growth environment for human prostate cancer cells than either human lung or mouse bone tissue.
Circulating PC3 cells formed tumors in 5 (26%) of 19 human bone
implants but in only 2 (11%) of 19 mouse lungs. In addition, the
human bone tumors were very large compared with the microscopic
mouse lung tumors, and we found no other metastatic deposits in the
native mouse organs. This is remarkable for several reasons: (a)
i.v.-injected tumor cells must pass through the mouse lung capillary
beds before entering the general circulation; and (b) the mass of
human tissue available to the circulating cancer cells is quite small in
comparison with the mouse skeleton and other organs. These observations further support the notion that the colonization of human bone
involved species- and tissue-specific mechanisms and was not due to
the passive lodging of tumor cells in the bone. Studies on other tumor
cell lines have clearly demonstrated that selective colonization of
specific target organs is not simply the result of nonspecific trapping
Fig. 4. End organ growth assay. Representative photomicrographs demonstrating histological features of tumors formed in human bone tissue (A-F) and human lung tissue (G-I)
6 weeks after the direct injection of human prostate cancer cell lines. A, DU145 cells formed solid tumors with evidence of osteolytic activity (arrow). 3100. A mild desmoplastic
stromal response was visible in some areas of bone tumors and was revealed by red staining of tumor cells with a pancytokeratin antibody (D). 3200. B, bone tumors formed by LNCaP
also showed a desmoplastic stromal response, and evidence of both osteoblastic (arrow) and osteolytic activity (arrowhead) could be seen by routine staining. 3100. E, the intermingling
of red-stained LNCaP cells and stromal cells could be better seen after cytokeratin immunostaining. 3200. C, PC3 cells formed solid osteolytic tumors, and bone tissue was often
completely destroyed. 3100. A strong desmoplastic stromal response was visible in these tumors, revealed both by routine H&E staining (C) and by red staining for cytokeratin (F).
3200. DU145, LNCaP, and PC3 cell lines all formed small solid tumors in human lung tissue and demonstrated little stromal-epithelial interaction (G, H, and I, respectively). 3100.
1991
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
of tumor cells by the organ vasculature. One of the best studied
examples is the B16 melanoma cell line, in which sublines have been
isolated that preferentially metastasize to the lung and liver (20).
Similarly, in vitro selection combined with repeated in vivo passaging
of the PC3 human prostate cancer cell line has resulted in sublines that
reliably colonize distinct organ sites (7), and sublines of the LNCaP
cell line with the ability to metastasize to bone have been isolated (9).
A natural selection process in the original patients may also account
for the differential ability of the three cell lines used in our study to
colonize human bone implants; PC3 cells were derived from a metastatic bone lesion (21), whereas the LNCaP and DU145 cell lines
were derived from lymph node and brain metastases, respectively
(22, 23).
The ability of circulating PC3 cells to colonize human bones in
25% of our mice is also remarkable considering that we used routinely
cultured PC3 cells at a low passage number after purchase, without
any prior selective pressure. In previous studies, metastasis of PC3
cells to bone has been achieved, but only after repeated rounds of in
vivo selection (7, 24) or through forced seeding of the lumbar vertebrae by simultaneous tail vein injection and caval occlusion (11).
Taken in this light, our observed tumor rate in human bone clearly
exceeds what would be expected for the metastasis of PC3 cells to
mouse bone.
The colonization of bone marrow by prostate cancer cells is likely
to involve adhesion to specific ligands found either on endothelial or
parenchymal cells or within the extracellular matrix of the bone
marrow environment. It has been demonstrated that various bone
components can stimulate tumor cell adhesion and chemotaxis (25),
and recently Kostenuik et al. (26) found that prostate cancer cells
adhere to type I collagen in osteoblast extracellular matrix via the
a2b1 integrin. In addition, Pasqualini and Ruoslahti (27) have suggested the existence of organ-selective vascular address molecules.
Using in vivo screening of random peptides, they identified amino
acid sequences that specifically target the vascular endothelium of
target organs, through binding to either integrins or other undefined
molecules. It is possible that prostate tumor cells use a similar mechanism to adhere specifically to endothelium in human bone, allowing
colonization and eventual tumor formation. Such a mechanism has
been demonstrated for the homing of lymphocytes and granulocytes to
specific tissue sites (28). Conversely, a lack of expression of these
important molecules by DU145 and LNCaP could explain the inability of these circulating cells to colonize the human bone implants. We
are currently investigating specific cell-surface molecules that may
allow PC3 cells to colonize human bone.
The failure of circulating prostate cancer cells to form gross skeletal
tumors in the mouse skeleton could be related to an inability of the
cells to adhere to molecular targets in the mouse marrow compartment
via mechanisms mentioned above. Alternatively, single tumor cells or
small cell aggregates may have been present in the marrow but were
unable to proliferate and form detectable skeletal tumors because of a
lack of favorable environmental factors that are present in human
bone tissue. In fact, results of earlier studies have shown that whether
injected via intraarterial or i.v. routes, tumor cells could be detected in
organ sites throughout the test animals, regardless of the ultimate
formation of metastatic deposits (29). Rapid growth of human prostate
cancer cells in mouse bone has been demonstrated after intrafemoral
injection (30); however, larger numbers of prostate cancer cells were
used in this study, making it difficult to determine whether tumor
growth in that system was actually dependent on the bone marrow
environment. Our observations show that a relatively small inoculum
of 1 3 104 prostate cancer cells failed to grow when seeded directly
into implanted mouse bone but formed tumors reliably in human bone
implants. Therefore, both specific adhesion mechanisms and species-
specific factors may help explain the difficulty in obtaining skeletal
metastasis in other rodent models.
Once metastatic cells arrive in the bone marrow compartment, they
must grow to form a tumor. Results from our end organ growth assay
clearly showed that the three human prostate cancer cell lines preferred to proliferate and form tumors in the human bone environment
in comparison with the human lung, mouse bone, and mouse s.c.
environments. In 1889, Paget (4) noted that breast cancer consistently
metastasized to specific organs. This observation led to the seed-andsoil hypothesis of metastasis, whereby certain organs provide a favorable environment for the growth of certain tumor cells. Our observations are consistent with the hypothesis of Paget and suggest that
human bone provides a supportive environment for both the targeting
and growth of human prostate cancer cells. Results of a number of
studies have demonstrated the inductive capacity of bone and bonederived factors on the growth of prostate cancer cells. Gleave et al.
(31) noted that soluble factors produced by bone fibroblasts could
induce tumor formation by LNCaP in nude mice, suggesting that
paracrine interactions might be involved. Tissue culture studies have
also shown that soluble factors derived from bone marrow (32) or
osteoblast-like cells (33) could stimulate growth of various prostate
cancer cell lines. Using coculture techniques, Lang et al. (34) found
that close contact with bone marrow stromal cells in culture can
stimulate the growth of cells derived from primary prostate tumors.
Close physical interaction with bone cells has also been shown to
enhance tumorigenicity of normally nontumorigenic LNCaP prostate
cancer cells (35). s.c. tumors formed in nude mice after coinjection of
LNCaP cells with either bone or prostate fibroblasts but not with
kidney or lung fibroblasts. Our present observations also suggest that
human bone provides a more supportive environment than human
lung. A combination of the SCID-hu system and in vitro studies is
likely to be useful in defining the factors important to prostate cancer
growth in bone in vivo.
The choice to use fetal bones rather than adult bones in this study
was based on the success of previous studies using this system (12,
13) and the observations of Heike et al. (36), who found that implanted adult human bone fragments did not stimulate an angiogenic
response and required constant cytokine infusion to prevent degeneration and fibrosis of the marrow compartment. It is possible that some
biological factors are different between adult and fetal bones. For
example, hematopoietic cells from implanted human marrow retain
some fetal characteristics (14). Nonetheless, the SCID-human bone
system is still a valuable tool to begin addressing specific humanhuman interactions involved in bone metastasis, including growth
factors, adhesion molecules, extracellular matrix components, and
cell-cell contact. In addition, the surgical procedures are simple,
measurements are easy and accurate, and the resulting tumors cause
minimal discomfort to the animals, allowing longer-term studies. Use
of the SCID-hu model may also allow more accurate prediction of the
response of metastatic cancer cells in the bone to therapeutic agents
(37) and will be helpful in the development of new therapies targeted
at sites of interaction between prostate cancer cells and the human
bone environment.
ACKNOWLEDGMENTS
We thank Dr. Jeffrey L. Evelhoch for valuable advice concerning MRI analysis.
REFERENCES
1. Abrams, H. L., Spiro, R., and Goldstein, N. Metastases in carcinoma: analysis of 1000
autopsied cases. Cancer (Phila.), 3: 74 – 85, 1950.
2. Batson, O. V. Function of vertebral veins and their role in the spread of metastases.
Ann. Surg., 112: 138 –149, 1940.
1992
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
SCID-hu MODEL OF PROSTATE CANCER BONE METASTASIS
3. Batson, O. V. The role of vertebral veins in metastatic processes. Ann. Intern. Med.,
16: 38 – 45, 1942.
4. Paget, S. The distribution of secondary growths in cancer of the breast. Lancet,
1: 571–573, 1889.
5. Isaacs, J. T., Isaacs, W. B., Feitz, W. F. J., and Scheres, J. Establishment and
characterization of seven Dunning rat prostatic cancer cell lines and their use in
developing methods for predicting metastatic abilities of prostatic cancers. Prostate,
9: 261–281, 1986.
6. Gingrich, J. R., Barrios, R. J., Morton, R. A., Boyce, B. F., DeMayo, F. J., Finegold,
M. J., Angelopoulou, R., Rosen, J. M., and Greenberg, N. M. Metastatic prostate
cancer in a transgenic mouse. Cancer Res., 56: 4096 – 4102, 1996.
7. Wang, M., and Stearns, M. E. Isolation and characterization of PC-3 human prostatic
tumor sublines which preferentially metastasize to select organs in S. C. I. D. mice.
Differentiation, 48: 115–125, 1991.
8. Dumont, P., Petein, M., Lespagnard, L., Tueni, E., and Coune, A. Unusual behaviour
of the LNCaP prostate tumour xenografted in nude mice. In Vivo (Athens), 7:167–
180, 1993.
9. Thalmann, H. N., Anezinis, P. E., Chang, S. M., Zhau, H. E., Kim, E. E., Hopwood,
V. L., Pathak, S., von Eschenbach, E. A., and Chung, L. W. K. Androgen-independent
cancer progression and bone metastasis in the LNCaP model of human prostate
cancer. Cancer Res., 54: 2577–2581, 1994.
10. Sato, N., Gleave, M. E., Bruchovsky, N., Rennie, P. S., Beraldi, E., and Sullivan,
L. D. A metastatic and androgen-sensitive human prostate cancer model using
intraprostatic inoculation of LNCaP cells in SCID mice. Cancer Res., 57: 1584 –1589,
1997.
11. Shevrin, D. H., Kukreja, S. C., Ghosh, L., and Lad, T. E. Development of skeletal
metastasis by human prostate cancer in athymic nude mice. Clin. Exp. Metastasis,
6: 401– 409, 1988.
12. Shtivelman, E., and Namikawa, R. Species-specific metastasis of human tumor cells
in the severe combined immunodeficiency mouse engrafted with human tissue. Proc.
Natl. Acad. Sci. USA, 92: 4661– 4665, 1995.
13. Sampson-Johannes, A., Wang, W., and Shtivelman, E. Colonization of human lung
grafts in SCID-hu mice by human colon carcinoma cells. Int. J. Cancer, 65: 864 – 869,
1996.
14. Kyoizumi, S., Baum, C. M., Kaneshima, H., McCune, J. M., Yee, E. J., and
Namikawa, R. Implantation and maintenance of functional human bone marrow in
SCID-hu mice. Blood, 79: 1704 –1711, 1992.
15. Shi, S-R., Key, M. E., and Kalra, K. L. Antigen retrieval in formalin-fixed, paraffin
embedded tissues: an enhancement method for immunohistochemical staining based
on microwave oven heating of tissue sections. J. Histochem. Cytochem., 39: 741–748,
1991.
16. Cher, M. L., Bova, G. S., Moore, D. H., Small, E. J., Carroll, P. R., Pin, S. S., Epstein,
J. I., Isaacs, W. B., and Jensen, R. H. Genetic alterations in untreated metastases and
androgen independent prostate cancer detected by comparative genomic hybridization
and allelotyping. Cancer Res., 56: 3091–3098, 1996.
17. Cher, M. L., Lewis, P. E., Banerjee, M., Sakr, W., Grignon, D. J., and Powell, I. J.
Similar pattern of chromosomal alterations in prostate cancers from African Americans and Caucasian Americans. Clin. Cancer Res., 4: 1273–1278, 1998.
18. Moore, D. H., Pallavicini, M., Cher, M. L., and Gray, J. W. A t-statistic for objective
interpretation of comparative genomic hybridization (CGH) profiles. Cytometry, 28:
183, 1997.
19. Nupponen, N. N., Hyytinen, E. R., Kallioniemi, A. H., and Visakorpi, T. Genetic
alterations in prostate cancer cell lines detected by comparative genomic hybridization. Cancer Genet. Cytogenet., 101: 53–57, 1998.
20. Fidler, I. J., and Hart, I. R. Biological diversity in metastatic neoplasms: origins and
implications. Science (Washington DC), 217: 998 –1003, 1982.
21. Kaighn, M. E., Narayan, K. S., Ohnuki, Y., Lechner, J. F., and Jones, L. W.
Establishment and characterization of a human prostatic carcinoma cell line (PC-3).
Invest. Urol., 17: 16 –23, 1979.
22. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P, Rosenthal, H., Chu, T. M.,
Miranda, E. A., and Murphy, G. P. LNCaP model of human prostatic carcinoma.
Cancer Res., 43: 1809 –1818, 1983.
23. Mickey, D. D., Stone, K. R., Wunderli, H., Mickey, G., and Paulson, D. F. Characterization of a human prostate adenocarcinoma cell line (DU-145) as a monolayer
culture and a solid tumor in athymic mice. Progr. Clin. Biol. Res., 37: 67– 84, 1980.
24. Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R., Killion, J. J., and
Fidler, I. J. Selection of highly metastatic variants of different human prostatic
carcinomas using orthotopic implantation in nude mice. Clin. Cancer Res., 2: 1627–
1636, 1996.
25. Magro, C., Orr, F. W., Manishen, W., Sivananthan, K., and Mokashi, S. Adhesion,
chemotaxis, and aggregation of Walker carcinosarcoma cells in response to products
of resorbing bone. J. Natl. Cancer Inst., 74: 29 –34, 1985.
26. Kostenuik, P. J., Sanchez-Sweatman, O., Orr, F. W., Singh, G. Bone cell matrix
promotes the adhesion of human prostatic carcinoma cells via the a2b1 integrin. Clin.
Exp. Metastasis, 14: 19 –26, 1996.
27. Pasqualini, R., and Ruoslahti, E. Organ targeting in vivo using phage display peptide
libraries. Nature (Lond.), 380: 364 –366, 1996.
28. Springer, T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration:
the multistep paradigm. Cell, 76: 301–314, 1994.
29. Potter, K. M., Juacaba, S. F., Price, J. E., and Tarin, D. Observations on organ
distribution of fluorescein-labelled tumour cells released intravascularly. Invasion
Metastasis, 3: 221–233, 1983.
30. Soos, G., Jones, R. F., Haas, G. P., and Wang, C. Y. Comparative intraosseal growth
of human prostate cancer cell lines LNCaP and PC-3 in the nude mouse. Anticancer
Res., 17: 4253– 4258, 1997.
31. Gleave, M., Hsieh, J-T., Gao, C., von Eschenbach, A. C., and Chung, L. W. K.
Acceleration of human prostate cancer growth in vivo by factors produced by prostate
and bone fibroblasts. Cancer Res., 51: 3753–3761, 1991.
32. Chackal-Roy, M., Niemeyer, C., Moore, M., and Zetter, B. R. Stimulation of human
prostatic carcinoma cell growth by factors present in human bone marrow. J. Clin.
Invest., 84: 43–50, 1989.
33. Lang, S. H., Miller, W. R., and Habib, J. K. Stimulation of human prostate cancer cell
lines by factors present in human osteoblast-like cells but not in bone marrow.
Prostate, 27: 287–293, 1995.
34. Lang, S. H., Clarke, N. W., George, N. J. R., Allen, T. D., and Testa, N. G. Interaction
of prostate epithelial cells from benign and malignant tumor tissue with bone-marrow
stroma. Prostate, 34: 203–213, 1998.
35. Gleave, M., Hsieh, J-T., Gao, C., von Eschenbach, A. C., and Chung, L. W. K.
Acceleration of human prostate cancer growth in vivo by factors produced by prostate
and bone fibroblasts. Cancer Res., 51: 3753–3761, 1991.
36. Heike, Y., Ohira, T., Takahashi, M., and Nagahiro, S. Long-term human hematopoiesis in SCID-hu mice bearing transplanted fragments of adult bone and bone marrow
cells. Blood, 86: 524 –530, 1995.
37. Wilmanns, C., Fan, D., O’Brian, C. A., Bucana, C. D., and Fidler, I. J. Orthotopic
and ectopic organ environments differentially influence the sensitivity of murine
colon carcinoma cells to doxorubicin and 5-fluorouracil. Int. J. Cancer, 52:
98 –104, 1992.
1993
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.
Severe Combined Immunodeficient-hu Model of Human
Prostate Cancer Metastasis to Human Bone
Jeffrey A. Nemeth, John F. Harb, Ubirajara Barroso, Jr., et al.
Cancer Res 1999;59:1987-1993.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/59/8/1987
This article cites 33 articles, 12 of which you can access for free at:
http://cancerres.aacrjournals.org/content/59/8/1987.full.html#ref-list-1
This article has been cited by 29 HighWire-hosted articles. Access the articles at:
/content/59/8/1987.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1999 American Association for Cancer Research.