Download CWR22: The First Human Prostate Cancer Xenograft with Strongly

[CANCER RESEARCH 56. 3042-3046.
July 1. 1996]
CWR22: The First Human Prostate Cancer Xenograft with Strongly
Androgen-dependent and Relapsed Strains Both in Vivo and in
Soft Agar1
Moolky Nagabhushan, Casey M. Miller, Theresa P. Pretlow, Joseph M. Giaconia, Nancy L. Edgehouse,
Stuart Schwartz, Hsing-Jien Kung, Ralph W. de Vere White, Paul H. Gumerlock, Martin I. Resnick,
Saeid B. Amini, and Thomas G. Pretlow2
Departments of Pathology ¡M.N., C. M. M., T. P. P., J. M. G., N. L E., T. G. P.I Genetics ¡S.S.], Molecular Biology and Microbiology [H-J. K.I, Epidemiology and Biostatistics
¡S.B. A.I. and Urology [M. I. K.. T. G. PJ. Case Western Reserve University Medical Center. Cleveland, Ohio 44106, and Departments of Urology [R. W. d. V. W.¡ and
Hemati/hgy/Oncology
¡P.H. G.]. University of California Davis Medical Center, Sacramento. California 95817
reported prostate cancer xenografts either (a) regress and disappear
never to recur or (b) do not regress significantly after castration. After
castration of animals that bear CWR22, serum PSA has fallen 153080-fold; in all but one animal, >50-fold. Physically measurable
recurrence is preceded by an elevation of serum PSA. The general
availability of this model will offer a unique opportunity to compare
a human prostate cancer that regresses after castration with tumor that
relapses several months to more than 1 year later in the castrated
animals.
ABSTRACT
Most patients' prostate cancers respond to androgen deprivation
but
relapse after periods of several months to years. Only two prostate cancer
xenografts, LNCaP and PC-346, have been reported to be responsive to
androgen deprivation and to relapse subsequently. Both of these tumors
shrink slightly, if at all, and relapse less than 5 weeks after androgen
withdrawal. After androgen withdrawal, the human primary prostate
cancer xenograft CWR22 regresses markedly, and prostate-specific anti
gen (PSA) falls up to 3000-fold in the blood of mice. PSA usually returns
to normal. In some animals, the tumor relapses and is then designated
CWR22R. In these animals, PSA starts to rise approximately 2-7 months,
and tumor begins to grow 3-10 months after castration. Animals with
CWR22 need to be euthanized because of large tumors 6-12 weeks after
the transplantation of CWR22. Androgen
proximately 3-4-fold.
withdrawal
MATERIALS
prolongs life ap
INTRODUCTION
In men, most prostate cancers respond to androgen withdrawal (1)
but relapse after the initial response. Few, if any, prostate cancers in
humans have been cured by hormonal manipulation (2). Models for
the development of experimental therapy are limited. To our knowl
edge, only two xenografts, LNCaP (3, 4) and PC-346 (5), have been
reported previously to show initial responses to androgen withdrawal
followed by resumption of growth. Both of these tumors exhibit
deceleration of their rates of growth after androgen withdrawal by
castration; occasionally, these tumors regress slightly after castration,
usually with less than a 10% reduction in volume. Unlike most
hormonally responsive prostate cancers in men, both of these tumors
resume a more rapid rate of growth less than 5 weeks after castration
(3, 5). PSA3 in the sera of animals bearing LNCaP tumors may
decrease up to 8-fold after castration (3).
In 1994, we (6) described CWR22, an androgen-dependent, serially
transplantable xenograft derived from a primary human prostate can
cer that has been used subsequently in many laboratories. After
castration, CWR22 shows marked regression, not just deceleration or
stabilization of growth, and may regress completely. We now describe
serially transplanted CWR22R tumors, xenografts that have relapsed
several months to more than 1 year after the castration of the hosts. To
our knowledge, this is the first report of a human prostate cancer
xenograft that both regresses markedly for long periods of time after
androgen withdrawal, and, in a proportion of animals, evolves to a
tumor that resumes growth in the castrated animal. All previously
Received 4/1/96: accepted 5/1/96.
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 This work was supported by NIH Grants DK45770. DK51347, CA57179. CA54031,
AND METHODS
The origin (7) and detailed characterization (6) of the serially transplantable
human primary prostate cancer xenograft CWR22 have been described.
CWR22 causes elevations of PSA in mouse peripheral blood that correlate
with growth (size and weight) of the tumors in nude mice. PSA in 50 fil tail
vein blood was assayed at intervals S5 weeks, as shown in the data from
individual experiments, with a slight modification of the method used by us
previously (8). Suspensions of single cells for transplantation and for culture in
soft agar were obtained by digestion of xenografts with 0.1% Pronase (VWR
Scientific. Bridgeport, NJ) as before (6). Nude mice were housed and cared for
as described previously (6, 7). Mice with CWR22 were given 12.5-mg sus
tained release testosterone pellets (Innovative Research of America, Sarasota,
FL) s.c. before receiving tumors and at intervals of 3 months until death.
Cells from freshly dissociated, serially transplanted CWR22 and CWR22R
that were serially transplanted from CWR22 tumors that relapsed after andro
gen deprivation were used in soft agar assays as described by Hamburger and
Salmon (9) with the following modifications: MEM+ (culture medium with
supplements as described in Ref. 10) with 20% normal (Life Technologies,
Inc., Gaithersburg, MD) or charcoal-stripped (Calico Biologicals, Inc., Reamstown, PA) calf serum was used as the solvent for the noble agar layers (Difco,
Detroit, MI). Cell suspensions were filtered through a single layer of Nitex
(Tetko, Inc., Briarcliff Manor, NY) with a48-f¿m porosity immediately before
being used for culture. After preliminary experiments to determine the con
centrations of cells that would allow for the most satisfactory growth for
counting, cells were plated at final concentrations of 0.05 and 0.025 million
cells/ml. Cell suspensions were placed in a volume of 0.4 ml at the tip of a
horizontal 5-ml tube. Dilutions of testosterone in 0.04 ml were placed near the
opening of this horizontal tube, and 3.6 ml 0.33% soft agar at 44°Cwere added
rapidly to the tube. The tube was mixed three times by inversion, and 1 ml soft
agar (final concentration 0.3%) was plated on top of a previously plated 1-ml
layer 0.5% agar. The cells in the several dilutions of testosterone (Sigma
Chemical Co., St. Louis, MO) shown below and the control plates were
cultured and counted in triplicate. The plates were incubated in a humidified
chamber with 5% CO2 in air at 37°C.After 14 days in culture, the number of
clusters per plate was counted and plotted as the mean ±SD. Groups of cells
greater than eight cells per group were scored (11) and termed "clusters."
In preliminary experiments, variables that might affect the soft agar assay
were explored. After filtration through 48-(¿mmesh Nitex as described above,
CA43703, CA57183. and CA55792.
2 To whom requests for reprints should be addressed, at the Institute of Pathology. Case
cells were cultured in soft agar over a wide range of concentrations. Concen
trations of 0.05 and 0.025 million cells/ml gave approximately 50-100 clus
Western Reserve University. 2085 Adelbert Road, Cleveland, OH 44106.
3 The abbreviation used is: PSA, prostate specific antigen.
ters/plate in the absence of testosterone. Cultures observed at intervals of 1
week for 4 weeks gave similar numbers of clusters (more than eight cells per
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ANDROGEN-DEPENDENT
AND RELAPSED
PROSTATE
CANCER
group) of cells at 2 and 3 weeks with decreased numbers of clusters at 4 weeks;
the sizes of the clusters did not increase after 2 weeks. Assays were performed
in each experiment at both concentrations, and the concentration that gave the
number of clusters closest to 100 clusters/plate was selected for evaluation. In
>80% of experiments, the 0.05 million cells/ml satisfied these conditions. At
this concentration, fewer than two clusters per plate were observed in the day
0 control plates in most experiments. In the early phases of this series of
experiments, we occasionally saw more clusters per plate. It became apparent
that experiments with more than two clusters per control plate were experi
ments in which longer intervals of time had elapsed between filtering the cells
through 48-fim Nitex and plating the cells. When more than eight clusters per
plate were observed in any control plate, the entire experiment was discarded.
Viability assays showed that >99% of the clusters were viable at 2 weeks;
viability was assessed with an iodonitrotetrazolium
stain (Sigma Chemical
Co.) as used by Rosenthal et al. (12).
2.5
5
7.5
DAYS POST RESECTION
RESULTS
PSA in the sera of mice declined much more rapidly after the
resection of tumors than after the castration of mice bearing tumors
(Fig. 1). The half-life of PSA after resection (Fig. 1A) appears to be
less than 1 day, a value that is consistent with the values reported for
a study of the LNCaP xenograft (3). Based on only five animals for
which PSA values were obtained at intervals after castration and
removal of sustained release testosterone pellets without resection of
tumors, the half-life of the serum PSA appears to be variable over a
range of approximately 3-8 days. The wide variation observed among
different animals in this experiment may be related in part to the fact
that PSA was elevated 1 day after castration as compared with the
value obtained just before castration in some animals. In the most
extreme example of this, seen in animal 2276 (Fig. Iß),the PSA was
522 ng/ml immediately before castration, 1504 ng/ml at 1 day, 558
ng/ml at 2 days, 417 ng/ml at 5 days, and 289 ng/ml at 7 days after
castration. We speculate that the elevation of PSA in some animals on
the day following androgen deprivation may have been related to
injury of the neoplastic cells. The regression of tumors proceeded
much more slowly than the decrease in PSA, i.e., the PSA usually
declined by 50% in a maximum of 1 week after castration while the
tumor volume usually took more than 1 month to decline by 50%; the
tumor volume often continued to decline for more than 3 months
before becoming stable.
The experiment in which mice have been followed for the longest
period after castration was a part of a separate ongoing study. His
torically, of the 151 animals that have received injections of >1000
CWR22 cells in Matrigel and observed for at least 3 months over the
past 3 years, all but 5 have developed tumors. In our longest (>600
days) experiment, CWR22 cells were transplanted by the injection of
8.3 million cells in 0.05 ml Matrigel into single subscapular sites
bilaterally in nine animals. By design, when the largest tumor reached
7 mm in its smallest dimension, all nine animals were castrated, and
testosterone pellets were removed. The larger of the two tumors was
resected from each of five of the animals 1 day after castration; from
each of the remaining four animals, 4 days after castration. At the time
of castration, PSA values in the blood of these mice ranged between
41 and 282 ng/ml; PSA in the nine animals fell 190-2230-fold after
resection of the larger of their two tumors and castration.
After castration, five of the nine animals failed to develop recurrent
tumors that could be measured with calipers; the remaining four
animals developed relapsed tumors with the first evidence of elevation
of blood PSA seen 92, 151 (two animals), and 221 days after castra
tion (Fig. 2). As detailed below, some of the five mice in which
relapsed tumors were not detected grossly showed evidence of the
persistence of small amounts of residual tumor. To date, these nine
animals have been followed for >600 days after castration or until
g
10
1500-
0
5
10
20
DAYS POST CASTRATION
Fig. 1. A, decline of PSA in serum of tail vein blood after resection of xenografts and
removal of sustained release testosterone pellets. Immediately before resection. PSA
ranged from 398 ng/ml in the mouse with the largest tumor (mouse 2264) to 145 ng/ml
in the mouse with the smallest tumor (mouse 2282). PSA declines with a half-life in the
mouse serum of < 1 day. D, mouse 2262; O. mouse 2264; O. mouse 2266; A, mouse
2280; •¿
mouse 2282. B. decline of PSA after castration of the mice and removal of
sustained release testosterone pellets. In some mice, e.g., mouse 2276. PSA was sharply
elevated 1 day after castration and removal of the exogenous testosterone. One might
speculate that this kind of response to castration may reflect the rapid release of PSA by
injured neoplastic cells. In other mice, e.g., mouse 2272, no such elevation was observed;
however, there may have been release of PSA by the tumor, since PSA consistently fell
more sharply during the second day than during the first day after castration. D, mouse
2270; O, mouse 2272; O, mouse 2274; A, mouse 2276; •¿
mouse 2284.
death. Two of the nine animals, (mice 2144 and 2148) are currently
alive and without recurrence of growing tumors or abnormal serum
PSA. Mice 2146 and 2162 had the lowest PSA values at the time of
resection and had tumor burdens estimated from external measure
ments of 0.95 and 0.72 ml. After resection of the larger of the two
tumors in these two animals, the estimated tumor burdens were 0.29
and 0.27 ml, respectively. Both of these mice died without tumors
detectable by caliper measurements or abnormal circulating PSA. At
autopsy, mouse 2146 showed no tumor at death 362 days after
castration; mouse 2162 died 443 days after castration and was found
to have minute amounts of histologically detectable tumor (estimated
to be <1 mm3) in the organized Matrigel at the site of injection,
despite the normal serum PSA. The fifth animal (mouse 2160) that
died without recurrent growth of tumor that could be detected with
calipers had a slight elevation (0.8 ng/ml) of PSA that developed 359
days after castration. It died 373 days after castration; despite the
abnormal PSA, no tumor could be detected at autopsy including a
thorough histopathological analysis of the site of injection. All other
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ANDROGEN-DEPENDENT
AND RELAPSED
600 -i
0
200
400
PROSTATE
CANCER
measured by calipers for >6 months after castration. Although
CWR22R tumors derived from relapsed tumors (a) cause less eleva
tion of PSA/g tumor in blood and (b) are more heterogeneous than
CWR22 tumors with respect to the amount of PSA/g tumor in blood,
the amount of PSA released into the blood by CWR22R has always
been sufficient to predict the relapse of tumors at least 1 month before
the relapsed growth is detectable with caliper measurements.
In other experiments, small numbers of animals with large tumor
burdens and serum PSA between 369 and 1142 ng/ml have been
castrated, and their sustained release testosterone pellets have been
resected. Animals whose tumors have recurred have never shown
progressive elevations of the PSA in their sera <2 months after
castration. Enlargement of tumors that can be measured with calipers
occurs > 1 month and usually many months after the elevation of PSA
is first detected.
600
DAYS POST CASTRATION
r 1000
Fig. 2. In an experiment designed for a parallel project, animals were injected in each
of two sites, located in the subscapular areas bilaterally, with 8.3 million cells in Matrigel.
i.e.. 8300-fold more cells than are required to produce tumor in >95% of the animals.
When the largest tumor in the 18 injection sites reached 7 mm in its smallest dimension,
animals were castrated, and the sustained release testosterone pellets were resected. The
larger of the two tumors was resected from each mouse 1 or 4 days after castration, jr.
deaths of animals. Four mice developed relapsed tumors after castration and regression of
tumors with the elevation of PSA in the tail vein blood being observed first in mouse 2152
(PSA. 4.7 ng/ml at 92 daysl. next in mice 2142 and 2150 (PSA. 2.0 and 0.6 ng/ml at 151
days), and last in mouse 2154 (PSA. 1.2 ng/ml at 221 days). All four of these mice
developed progressive elevation of their PSAs and growth of their tumors that were
documented histopathologically at the time when they were euthanized 144. 193, 227, and
344 days after castration. D. mouse 2142; < . mouse 2144; O. mouse 2146; A, mouse
2148; •¿
mouse 2150; ».mouse 2152; ».mouse 2154; A. mouse 2160; T. mouse 2162.
animals that have developed blood PSA levels of 0.8 ng/ml after
months of normal blood PSA levels have developed recurrent tumors.
In seven of the nine animals. PSA values dropped to 0.1 or 0.2
ng/ml. The remaining two animals (mice 2152 and 2154) never
developed normal PSA values after castration. In mouse 2152, PSA
dropped from 114 ng/ml just prior to castration to 0.6 ng/ml at 64
days, 4.7 ng/ml at 92 days, 8.8 ng/ml at 123 days, and 73.1 ng/ml at
144 days after castration; weekly measurements with calipers first
suggested relapse 119 days after castration. In mouse 2154. PSA
dropped from 201 ng/ml just prior to castration to 0.4 ng/ml at 92 days
and again at 123 days after castration. In the blood of this mouse, PSA
values were still 0.5 ng/ml at 186 days, 1.2 ng/ml at 221 days, and
13.7 at 254 days before rising progressively to 515.3 ng/ml on the day
when the mouse was euthanized, 344 days after castration; measure
ment with calipers showed relapse 279 days after castration.
Four of the nine animals developed recurrent tumors that could be
measured with calipers and resulted in their being euthanized 144,
193. 227, and 344 days after castration. These results are particularly
interesting in light of the fact that all injection sites received 8.3
million cells, i.e., 8300 times as many cells as are tumorigenic in
>95% of the animals maintained with s.c. sustained release testos
terone. Most animals given 1000 cells and sustained release testos
terone s.c. are euthanized with large tumors <3 months after the 1000
cells are injected.
Fig. 3 shows representative examples of the relationships between
PSA in the sera of mice and the growth of tumors as measured with
calipers. Mouse 2150 (Fig. 3/4) was castrated on day 0; one of its two
tumors was resected on day 4. The tumor that was not resected began
growth as a relapsed tumor, as measured by calipers, approximately
180 days after castration; PSA indicated recurrent tumor 151 days
after castration. Mouse 2274 (Fig. 3ß)was allowed to develop large
tumors bilaterally before castration in an attempt to develop relapsed
tumor after castration. The change in PSA as a function of time after
castration reversed its course 79 days after castration, indicating
relapsed tumor; however, the two tumors did not appear to relapse as
L 100
0)
10£
SS
o.
0.1
O
100
200
DAYS POST CASTRATION
-1000
- 100
1
-100.
0
100
200
DAYS POST CASTRATION
Fig. 3. Mouse 2150 and mouse 2274 were in experiments with different protocols that
involved castration and resection of their sustained release testosterone pellets followed by
the development of relapsed tumors. Although both mice showed elevation of PSA in their
blood sera followed by growth of relapsed tumors as measured with calipers, the temporal
relationships between these two phenomena were quite different in the two mice. A,
animal 2150 had one tumor resected 4 days after castration and resection of the sustained
release testosterone pellet, and the remaining tumor was never large. PSA remained
normal (<0.25 ng/ml) at 64. 92. and 123 days after androgen withdrawal and became
elevated only 1 month prior to the time when the growth of relapsed tumor could be
detected with calipers. D, volume (cm1) of tumor in mouse 2150; •¿.
level of PSA {ng/ml)
in mouse 2150. B, in contrast, mouse 2274, an animal with bilateral large tumors at the
lime of castration, developed its lowest PSA level {6.6 ng/ml) in tail vein blood 51 days
after androgen withdrawal. It showed the first elevation of PSA with the development of
a PSA of 10.4 ng/ml at 79 days after castration, 11.5 ng/ml at 114 days, and progressively
higher levels until death at 225 days after castration. As we have observed in other animals
with large tumors, the tumors continued to shrink long after PSA had begun to become
progressively elevated; and the growth of tumors was not evident from measurement
calipers until approximately 6 months after castration. D. volume (cm3) of tumors in
mouse 2274; •¿
level of PSA {ng/ml) in mouse 2274.
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ANDROGEN-DEPENDENT
AND RELAPSED PROSTATE CANCER
In experiments designed to test the hormonal dependence of
CWR22, three female mice and three male mice were given injections
of the same suspension of minced tumor in 0.5 ml Matrigel. All three
male mice were euthanized 6 weeks later because of large tumors. All
three female mice were euthanized 4 months after injection without
any evidence of tumor. In another experiment, two female mice and
two male mice were given injections of 1.1 million CWR22 cells in
Matrigel. The male mice were euthanized 6 and 9 weeks after trans
plantation with large tumors. Both female mice were euthanized 6
months after they received these cells but failed to develop tumors. In
contrast to CWR22, CWR22R grows in female mice.
We have passaged relapsed tumors serially from five different
mice. The relapsed tumors, CWR22R, have varied widely in their
rates of growth; however, in general, CWR22R tumors have grown
more slowly than CWR22. CWR22 tumors generally reach sizes that
require that the animal be euthanized 6-12 weeks after transplanta
tion. Some of the five strains of relapsed tumors reach similar sizes as
early as 9 weeks; most require 3-7 months after transplantation. The
basis for their slower growth and the marked differences among
different relapsed tumors and their progeny is not yet known.
CWR22 and CWR22R differed in their responses to stimulation
with testosterone in soft agar (Fig. 4). Cluster formation by
CWR22 was increased in the presence of testosterone in a doserelated fashion up to an optimal concentration in the range of
25-35 nvi testosterone. The dose-response curves were parallel in
normal calf serum and in charcoal-stripped
calf serum with the
formation of more clusters in the normal serum than in charcoalstripped serum. In both kinds of sera, cluster formation was in
creased 2-3-fold in the presence of testosterone. The response of
CWR22R to testosterone was less consistent than that of CWR22.
The experiments with CWR22R (Fig. 4) were carried out with
different CWR22R tumors that arose as separate events in different
animals over different intervals of time. In some experiments,
small increases in cluster formation were observed in the presence
of doses of testosterone that were optimal for CWR22. In other
instances, cluster formation of CWR22R appeared to decrease
slightly in the presence of testosterone. The only generalization
about cells from all CWR22R tumors that seems very important in
the light of the available data is that CWR22R cells responded
much less vigorously than did CWR22 cells to testosterone in soft
agar.
A400i
LU
j
E
oc
ni
300H
O. 2000)
CE
LU
100O
i
60
i
20
40
TESTOSTERONE
(nM)
B 250i
LU
^ 200-1
OC
LU
o. 150W
oc
LU
100U
—¿r-» 1
50
0
25
125
TESTOSTERONE
K
r625
(nM)
Fig. 4. A, response of CWR22 lo testosterone in soft agar. G. O, O, and A, CWR22
growth with charcoal-stripped serum in four different trials. •¿
*. •¿,
and A, CWR22
growth with normal serum in four different trials done in the same experiments as the
above trials. B. response of CWR22R to teslosterone in soft agar. O, O, and A, CWR22R
growth with charcoal-stripped serum in three different trials. *, •¿.
and A. CWR22R
growth with normal serum in three different trials done in parallel with the above trials.
CWR22 and the tumors that have relapsed after the withdrawal of androgen. CWR22R.
respond differently to testosterone when cultured in soft agar. The experiments shown
with CWR22R were carried out with tumors that relapsed in different animals at different
times. Although the responses of CWR22R were slightly more heterogeneous than those
of CWR22. the magnitudes of the responses of CWR22R to testosterone were much
smaller than those observed for CWR22 with normal serum and with charcoal-stripped
serum.
DISCUSSION
CWR22R enhances the value of CWR22 as a relatively unique
model of human prostate cancer. The availability of CWR22 and
CWR22R as serially transplanted xenografts provides prostate cancer
researchers, for the first time, a xenograft derived from a primary
prostate cancer that regresses markedly after androgen deprivation
and relapses as new tumor growth in approximately one quarter to one
half of the mice usually 3-10 months after castration, and 1-5 months
after serial measurements of PSA in the tail vein blood has heralded
the impending relapse. Even very large CWR22 tumors shrink by
>50% after tumor-bearing mice are castrated, and tumors of < l g at
the time of castration usually regress to <3 mm in diameter even
when they are fated to recur. Both LNCaP (3, 4) and PC-346 (5) are
reported to be androgen responsive; however, they regress only
slightly, if at all, and only for 3 to 5 weeks after tumor-bearing mice
are castrated (3, 5). The decrease in PSA in the blood of castrated
mice bearing CWR22 is much greater than has been reported for other
prostate cancer xenografts that relapse following regression. PSA
provides a very sensitive indication of the growth of CWR22 (6) that
is independent of other indicators of tumor growth such as size as
measured with calipers.
CWR22 and the relapsed CWR22R provide investigators with an
approach to the investigation of prostate cancer that is relatively
unique. Differences between CWR22 and the two other prostate
cancer xenografts for which relapsed and hormonally responsive
variants are available include the marked physical regression of
CWR22 that is measurable with calipers for months after castration
and the marked drop in PSA in the peripheral blood of animals
bearing CWR22 after castration.
Note Added in Proof
The two animals still alive and without evidence of tumor >600 days after
castration (Fig. 2) were given sustained release testosterone s.c. 603 and 609
days after castration. One died without tumor 3 weeks later; the other was
euthanized with tumor 7 weeks later.
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ANDROGEN-DEPENDENT
AND RELAPSED
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CWR22: The First Human Prostate Cancer Xenograft with
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Vivo and in Soft Agar
Moolky Nagabhushan, Casey M. Miller, Theresa P. Pretlow, et al.
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