Preclinical analyses of intravesical chemotherapy for prevention of bladder cancer progression

Abstract

There is a critical need to identify treatment options for patients at high risk for developing muscle invasive bladder cancer that avoid surgical removal of the bladder (cystectomy). In the current study, we have performed preclinical studies to investigate the efficacy of intravesical delivery of chemotherapy for preventing progression of bladder cancer. We evaluated three chemotherapy agents, namely cisplatin, gemcitabine, and docetaxel, which are currently in use clinically for systemic treatment of muscle invasive bladder cancer and/or have been evaluated for intravesical therapy. These preclinical studies were done using a geneticallyengineered mouse (GEM) model that progresses from carcinoma in situ (CIS) to invasive, metastatic bladder cancer. We performed intravesical treatment in this GEM model using cisplatin, gemcitabine, and/or docetaxel, alone or by combining two agents, and evaluated whether such treatments inhibited progression to invasive, metastatic bladder cancer. Of the three single agents tested, gemcitabine was most effective for preventing progression to invasive disease, as assessed by several relevant endpoints. However, the combinations of two agents, and particularly those including gemcitabine, were more effective for reducing both tumor and metastatic burden. Our findings suggest combination intravesical chemotherapy may provide a viable bladder-sparing treatment alternative for patients at high risk for developing invasive bladder cancer, which can be evaluated in appropriate clinical trials.

Full text

Oncotarget 2013; 4: 269-276269
www.impactjournals.com/oncotarget
www.impactjournals.com/oncotarget/
Oncotarget, February, Vol.4, No 2
Preclinical analyses of intravesical chemotherapy for prevention
of bladder cancer progression
Joan C. Delto
1
, Takashi Kobayashi
1
, Mitchell Benson
1,3
, James McKiernan
1,3
, and
Cory Abate-Shen
1,2,3
1
Department of Urology, Columbia University Medical Center, New York, NY, USA
2
Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA
3
Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA
Correspondence to: Cory Abate-Shen, email: cabateshen@columbia.edu
Keywords: Non-muscle invasive bladder cancer, intravesical therapy, genetically engineered mouse models, preclinical studies
Received: February 1, 2013 Accepted: February 24, 2013 Published: February 25, 2013
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT:
There is a critical need to identify treatment options for patients at high risk
for developing muscle invasive bladder cancer that avoid surgical removal of the
bladder (cystectomy). In the current study, we have performed preclinical studies
to investigate the ecacy of intravesical delivery of chemotherapy for preventing
progression of bladder cancer. We evaluated three chemotherapy agents, namely
cisplatin, gemcitabine, and docetaxel, which are currently in use clinically for
systemic treatment of muscle invasive bladder cancer and/or have been evaluated
for intravesical therapy. These preclinical studies were done using a genetically-
engineered mouse (GEM) model that progresses from carcinoma in situ (CIS) to
invasive, metastatic bladder cancer. We performed intravesical treatment in this GEM
model using cisplatin, gemcitabine, and/or docetaxel, alone or by combining two
agents, and evaluated whether such treatments inhibited progression to invasive,
metastatic bladder cancer. Of the three single agents tested, gemcitabine was most
eective for preventing progression to invasive disease, as assessed by several
relevant endpoints. However, the combinations of two agents, and particularly those
including gemcitabine, were more eective for reducing both tumor and metastatic
burden. Our ndings suggest combination intravesical chemotherapy may provide a
viable bladder-sparing treatment alternative for patients at high risk for developing
invasive bladder cancer, which can be evaluated in appropriate clinical trials.
INTRODUCTION
Bladder cancer is the fourth most common cancer
in men and the eighth most common overall, with 74,000
new cases diagnosed and an estimated 15,000 deaths in
2012 (1). Notably, distinct subtypes of bladder cancer
have very different patient outcomes (2-5). In particular,
the lethal form is muscle-invasive disease, for which the
precursor is carcinoma in situ (CIS) (6). The primary
treatment for muscle invasive bladder cancer is cystectomy
(surgical removal of the bladder), which is associated with
signicant morbidity; moreover, progression to metastatic
disease has a particularly low 5-year survival (6-9). At the
other end of the spectrum is non-invasive bladder cancer,
which presents as papillary lesions and generally has good
patient prognosis (6, 9). However, non-muscle invasive
bladder cancer can progress to a high-risk disease that
ultimately gives rise to muscle invasive bladder cancer.
Many patients with recurrent non-muscle invasive
bladder cancer are treated with intravesical delivery of
Bacillus Calmette Guerin (BCG), an immunotherapy
regime (10). Although widely used, there can be signicant
adverse reactions to BCG; moreover, 30% of patients
do not respond and even those that respond have a 20%
chance of progression (10, 11). For patients with high-risk
recurrent non-muscle invasive bladder cancer, including
those who have failed BCG therapy, early cystectomy
with urinary diversion is currently the preferred treatment
option (10). However, cystectomy is associated with
signicant morbidity, which severely impacts quality of

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life, and it may not be a viable option for patients who
are medically unt for surgery. Importantly, patients who
undergo early cystectomy before they progress to invasive
bladder cancer may result in overtreatment.
Thus, there is an urgent need to identify alternative,
bladder-sparing therapies for patients with high-risk
non-muscle invasive bladder cancer. One such option
is intravesical delivery of chemotherapy to prevent
progression to invasive bladder cancer. For example, our
phase I clinical trial using intravesical docetaxel yielded
a 56% complete response rate with 22% durability of
response in three years (12, 13). Other clinical trials
have shown that gemcitabine has promising results for
patients with recurrent non-muscle invasive bladder
cancer in Phase I and Phase II clinical trials (14-16).
Intravesical delivery of selected agents has also been
investigated preclinically in Xenograft models based on
orthotopic implantation of human bladder cancer cells
into immunodecient mouse hosts (17, 18). Although
these clinical and preclinical studies using intravesical
chemotherapy are promising, systemic chemotherapy,
administration of single agents has rarely resulted in
durable long-term remissions. In fact, the most successful
systemic chemotherapy for advanced metastatic bladder
cancer is either a two-drug regimen of gemcitabine
and cisplatin or a four-drug combination regimen of
methotrexate, vinblastine, adriamycin and cisplatin
(MVAC) (8, 9, 19, 20).
We, therefore, reasoned that the design of optimal
intravesical chemotherapy regime(s) for patients with
recurrent non-muscle invasive bladder cancer would
benet from preclinical studies aimed at a direct, side-
by-side comparison of drug regimens involving single
versus double combinations. Toward this end, we have
now performed preclinical studies using a genetically
engineered mouse (GEM) model of progressive
bladder cancer to systematically analyze the efcacy of
chemotherapeutic agents when delivered individually
versus in combination. Our preclinical studies utilize
a GEM model of progressive bladder cancer based on
bladder-specic deletion of two tumor suppressor genes,
p53 and Pten, which are frequently de-regulated in
invasive bladder cancer (21). Following tumor induction
by delivery of Adeno-Cre into the bladder lumen, these
p53
f/f/
; Pten
f/f
mice develop carcinoma in situ (CIS) by 8
weeks, which progresses to invasive bladder cancer with
prevalent metastases by 5 months of age (21, 22). The
bladder tumors from these Adeno-Cre infected p53
f/f/
;
Pten
f/f
mice display similar histologic features as human
muscle invasive bladder cancer, and their metastases arise
in similar tissues, as occurs in humans (21). Our previous
analyses of this GEM model have provided molecular
insights regarding bladder cancer progression and this
model has also provided an effective resource for in vivo
preclinical studies (21, 22). Indeed, we have demonstrated
that bladder tumors arising in these Adeno-Cre infected
p53
f/f/
; Pten
f/f
mice respond to systemic treatment with
rapamycin, while intravesical delivery of rapamycin
prevents progression of CIS to invasive bladder cancer
(21, 22).
In the current study, we have performed preclinical
studies using this GEM model to evaluate the efcacy of
intravesical delivery of chemotherapy for prevention of
progression to invasive bladder cancer. We evaluated three
chemotherapy agents, namely cisplatin, gemcitabine, and
docetaxel, which are standardly administered systemically
for treatment of invasive, metastatic bladder cancer (8,
9, 19, 20). We directly compared these agents to assess
their relative efcacy when delivered individually or in
combination for prevention of progression to invasive
bladder cancer. When tested individually, intravesical
delivery of gemcitabine is most effective among the
three agents for delaying progression to muscle-invasive
bladder cancer; however, each combination of two agents
was more effective for reducing both tumor and metastatic
burden. These ndings suggest that patients at high-risk
for developing invasive bladder cancer may be candidates
for combination intravesical chemotherapy.
Figure 1: Study design for preclinical analyses of intravesical chemotherapy.
Tumor induction was initiated by delivery of
Adeno-Cre directly into the bladder lumen of p53
f/f/
; Pten
f/f
female mice at 8 weeks. Six weeks later (at 14 weeks), mice were imaged
using ultrasound (baseline) and then enrolled into one of 8 treatment arms for the preclinical intravesical treatment: vehicle; gemcitabine,
docetaxel or cisplatin as single agents, or cisplatin + docetaxel, gemcitabine + docetaxel, or gemcitabine + cisplatin in combination. The
treatments were continued for 8 weeks during which time the bladders were imaged every two weeks using ultrasound. At the conclusion
of the treatment period (22 weeks) mice were sacriced for analyses including the endpoints indicated.

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RESULTS
We performed preclinical studies to evaluate the
consequences of intravesical chemotherapy for delaying
progression from CIS to invasive bladder cancer using
a genetically-engineered mouse model that recapitulates
these progression stages. In particular, we initiated
preclinical treatment in p53
f/f/
; Pten
f/f
mice six weeks after
tumor induction with Adeno-Cre (i.e., at 14 weeks of
age; Fig. 1), since, as we have shown previously, by this
time-point these GEM
mice have developed CIS, but have
not progressed to invasive bladder cancer (22). Prior to
initiation of treatment, we conrmed the absence of overt
tumors using ultrasound imaging. Cohorts of mice were
then randomly assigned to the various treatment arms,
which were the Vehicle group, the single agent group
(cisplatin, gemcitabine, or docetaxel), and the combination
group (cisplatin + gemcitabine; gemcitabine + docetaxel;
and cisplatin + docetaxel) (Fig. 1). In initial pilot studies,
we found that the mice were able to tolerate each of these
agents via intravesical delivery, and were able to tolerate
up to two doses of intravesical treatment weekly (data
not shown). Therefore, we performed these preclinical
studies by combining a maximum of two agents for a
biweekly instillation. Each of the single agent and double
combination treatment groups displayed no signicant
weight loss, based on bi-weekly measurements, nor did
they display other overt signs of distress.
Cohorts of mice were treated one time weekly
(for the signal agents) or bi-weekly (for the double
combinations) for a period of 8 weeks (Fig. 1). By this
point following tumor induction (i.e., at 22 weeks), the
vehicle-treated Adeno-Cre-infected p53
f/f/
; Pten
f/f
mice
characteristically develop invasive bladder tumors that
are large in size, have histological features of invasive
bladder cancer, including a high rate of proliferation, and
are highly metastatic (21). Therefore, our analyses focused
on whether intravesical treatment with the individual or
combination chemotherapy regimes prevented progression
to invasive bladder.
Following treatment initiation, the experimental
mice were monitored with ultrasound every two weeks
for detection of tumors (Fig. 1, Fig. 2). During the
8-week treatment period, the vehicle-treated mice
rapidly developed tumors as detected by ultrasound
imaging, as expected (Fig. 2). In contrast, mice treated
with gemcitabine or docetaxel, but not cisplatin, as
single agents developed tumors at a slower rate (Fig.
2). Moreover, mice that received each of the combined
treatments (cisplatin + docetaxel; gemcitabine + docetaxel;
and cisplatin + docetaxel) also displayed a signicant
delay in the formation of overt tumors (p < 0.05; Fig. 2).
These ndings indicate that intravesical chemotherapy
Figure 2: Onset of tumor formation detected by ultrasound imaging. Graphic representation of the percentage of mice tumor-
free over the course of treatment as detected by ultrasound imaging showing the single (top) and combination (bottom) images. The log rank
p-values are indicated for each treatment (compared to Vehicle). Pictures of the ultrasound images are shown in Figure 4.

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with gemcitabine or docetaxel alone or with the three
combinations of agents delays overt tumor formation in
tumor-prone mice.
At the end of the 8-week treatment period, the
cohorts of experimental mice were sacriced and their
bladders and other tissues were evaluated. As expected,
all of the vehicle-treated mice displayed large bladder
tumors (~2 grams) that were readily evident upon gross
inspection (Table 1, Fig. 3A, 4; Supplementary Fig. 4).
In contrast, mice in each of the treatment groups treated
with intravesical chemotherapy displayed a reduction
in the size of the bladder, although the extent differed,
particularly for mice treated with the single agents (Table
1, Fig. 3A). In particular, the bladder weights of mice
treated with gemcitabine alone were signicantly reduced
(5.4 fold reduced, p = 0.0271), whereas those treated
with docetaxel or cisplatin were reduced in weight (1.8
and 3.73 fold reduced, respectively) the mean values
were not signicantly different compared to the Vehicle-
treated mice (Table 1, Fig. 3A, 4, Supplementary Fig. 1).
In combination, the reduction in bladder size was further
augmented for gemcitabine in combination with either
docetaxel (7.6 fold reduced, p = 0.0194) or cisplatin (6.0
fold reduced, p = 0.0250), and the combination of cisplatin
+ docetaxel also resulted in a signicant reduction
in bladder weight albeit to a lesser extent than the
combinations with gemcitabine (4.53 fold; p = 0.0.0311;
Table 1, Fig. 3A, 4, Supplementary Fig. 1). Notably, by
gross inspection, bladders treated with the combination
agents, and to a lesser extent gemcitabine alone, were
similar in size and appearance to normal bladder (~0.20-
0.30 grams), consistent with the interpretation that there
was minimal tumor volume (Fig. 4, Supplementary Fig. 1).
Therefore, intravesical treatment with either gemcitabine
alone or with the various combinations of agents resulted
in a signicant reduction of tumor burden at the end of the
treatment period.
We evaluated the histopathology of the bladders
following intravesical chemotherapy by examining
multiple H&E sections throughout the bladder using
whole slide imaging (Table 1, Fig. 4). As evident in
representative high power images (Fig. 4), the vehicle
treated mice display invasive bladder cancer as has
been described previously (21). The experimental mice
treated with the single chemotherapy agents displayed
varying degrees of histopathology that were consistent
with their gross bladder phenotypes. In particular, the
bladder epithelium of most of the mice treated with either
gemcitabine or docetaxel was multilayered with areas
of hyperproliferation, reminiscent of to CIS, but did not
display evidence of invasion, whereas the epithelium of
most of the mice treated with cisplatin alone, which had
evident bladder tumors, displayed histological evidence of
invasion (Table 1, Fig. 4). Moreover, the histology of the
bladders of most of the experimental mice treated with
each of the double combinations had some evidence of
CIS, but were otherwise relatively normal in appearance
with little or no evidence of invasion (Table 1, Fig. 4).
These ndings further support the efcacy of combination
chemotherapy for prevention of bladder cancer
progression.
As an additional parameter of disease progression,
we evaluated the proliferation of the bladder epithelial
and/or tumor cells of the experimental mice following
treatment with intravesical chemotherapy. As we have
shown previously (21), the vehicle treated mice display
a high rate of proliferation (~40%) (Fig. 3B, Table 1).
Consistent with the tumor size and histological phenotype,
Figure 3: Endpoint analyses for preclinical studies. Summary of data from experimental mice following treatment with Vehicle or
with the single or combination agents as indicated. A) Summary of Bladder weights. B) Summary of cellular proliferation as evaluated by
Ki67 immunostaining of the treated bladders.

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which was signicantly abrogated by the intravesical
chemotherapy, the single agents, and particularly
gemcitabine and docetaxel, displayed a signicant
reduction in proliferation (17-20%; p = 0.012 to 0.04).
Moreover, mice treated with the combination agents
displayed a more profound reduction in proliferation
(10.9% to 13.7%; p = 0.01 to 0.03). These ndings further
underscore the efcacy of combination intravesical
chemotherapy for prevention of bladder cancer
progression.
Finally, we evaluated whether treatment with
intravesical chemotherapy affected the development of
metastases. As we expected based on previous analyses
(21), at the conclusion of the treatment period the majority
of the vehicle-treated mice (15/18) had prominent overt
metastases to several tissues including lymph nodes, liver,
and pancreas (Table 1). The incidence of metastases was
consistently reduced for the experimental mice treated
with the single agents, although not signicantly. However,
mice treated with each of the combination chemotherapy
regimes displayed a statistically signicant reduction in
metastases (2/12 to 2/15, p = 0.003 to 0.004; Table 1).
Taken together, these ndings demonstrate the efcacy
of intravesical delivery of combination chemotherapy for
prevention of progression to invasive, metastatic bladder
cancer.
DISCUSSION
Our study addresses the need to identify alternative,
bladder-sparing treatment options for patients at risk for
developing invasive bladder cancer. Toward this end, we
investigated the efcacy of intravesical chemotherapy in
a preclinical model of the disease. Several key aspects of
our study advance the ndings of previous clinical studies
(12, 14), as well as preclinical analyses using an orthotopic
xenograft model of invasive bladder cancer (18). First, we
have used a GEM model that reliably exhibits progression
from pre-invasive lesions to overt invasive disease, and
therefore our study has enabled us to specically address
the efcacy of these treatments in the context of disease
progression. Additionally, we have performed a direct,
side-by-side comparison of intravesical delivery of three
chemotherapy agents, namely cisplatin, gemcitabine, and
docetaxel. This has enabled us to compare their efcacy
when delivered alone or in combination. Lastly, we
have evaluated multiple endpoints, which proved to be
important when considering potentially clinically-relevant
endpoints, such as the development of the metastases, in
which the combination treatments outperformed any of the
single agents.
The major conclusion of the current study is that
while the single agents, and particularly gemcitabine,
may be somewhat effective for reducing tumor burden,
Table 1: Summary of treatment endpoints
Bladder Weight Phenotype Proliferation Metastases
Treatment N
Mean
weight
(gr)
SEM
Fold
Change
P value Description
% Ki67
P value
Cases
w/ Mets
P value
Vehicle
PBS
(DMSO in PBS)
22
2.13
(N = 10)
0.64 - -
Large tumors
(18/22); Small
tumors (4/22)
39.1
(N = 4)
-
15/18
(85%)
-
Single Agents
Cisplatin 15
1.15
(N = 7)
0.30 1.85 NS
Small tumors
(12/15); CIS
(3/15)
20.4
(N = 4)
0.038
6/14
(42%)
NS
Gemcitabine 14
0.39
(N = 5)
0.20 5.46 0.0271
Small tumors
(3/14); CIS
(11/14)
17.5
(N = 4)
0.019
4/10
(40%)
NS
Docetaxel 14
0.57
(N = 5)
0.48 3.73 NS
Small tumors
(5/14); CIS
(11/14)
19.2
(N = 4)
0.013
4/10
(40%)
NS
Combinations
Cis + Doce 22
0.47
(N = 13)
0.16 4.53 0.0311
Small tumors
(3/22);
Normal/CIS
(19/22)
10.9
(N = 4)
0.032
2/12
(16%)
0.004
Gem + Doce 23
0.28
(N = 13)
0.18 7.60 0.0194
Small tumors
(2/23);
Normal/CIS
(21/23)
13.7
(N = 4)
0.017
1/13
(8%)
0.001
Cis + Gem 26
0.36
(N = 14)
0.14 6.00 0.0250
Small tumors
(4/26);
Normal/CIS
(22/26)
12.6
(N = 4)
0.011
2/15
(13%)
0.003

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when considering all of the endpoints examined, the most
effective regime for preventing progression to invasive
bladder cancer is a combination agents, particularly
those including gemcitabine. The further implication
of our ndings is that while intravesical treatment
with gemcitabine, or with the other single agents, may
have an immediate benet for delaying progression,
sustained effects that may impact overall survival, such
as metastases, may require combination treatments. The
additional implication of our study is that more than
one combination is likely to be effective for preventing
progression, which may leave open several options for
patients who do not respond or tolerate one treatment
regime but may benet from an alternative.
We propose that our ndings provide the rationale
for evaluation of combination intravesical chemotherapy
for patients with non-muscle invasive bladder cancer who
are at high risk for progressing to invasive disease. Such
treatment would be particularly effective for management
of patients who are unable to undergo cystectomy, but
may be extended to others who are also at high risk of
progressing to muscle invasion and wish to avoid early
removal of the bladder. Thus, combination intravesical
chemotherapy may provide an alternative to cystectomy
that improves the quality of life.
MATERIALS AND METHODS
Mouse model of progressive bladder cancer
All animal experiments were performed according
to protocols approved by the Institutional Animal Care
and Use Committee at Columbia University Medical
Center. The genetically engineered mouse (GEM)
model of progressive bladder cancer used in this study
was developed and characterized in our laboratory and
has been described previously (21). Briey, these mice
are based on bladder-specic deletion of oxed alleles
of p53 and Pten (i.e., p53
ox/ox
; Pten
ox/ox
) in a C57/Bl6
strain background. Tumors are induced by delivery of
an Adeno-virus expressing Cre recombinase (hereafter
referred to as Adeno-Cre) directly into the bladder lumen
at 8 weeks of age (21). Ultrasound imaging using a
Vevo 2100® Imaging System (Visual Sonics, Toronto,
Ontario, Canada) was performed to detect bladder tumors,
following the instructions of the manufacturer. Following
tumor induction, mice were monitored on a daily basis
for body condition (i.e., muscle tone and weight) and
sacriced when their body condition score was <1.5, as
per guidelines of the Institutional Animal Care and Use
Committee.
Intravesical drug treatment
For these studies we used female Adeno-Cre-
injected p53
ox/ox
; Pten
ox/ox
mice because they are
amenable to intravesical treatment (22). Under anesthesia,
mice were placed in supine position and the external
urinary orice cleansed with betadine. A 24G Jelco
angiocatheter (~10-15 mm in a typical 20 gram female
mouse) was inserted through the urethra to the bladder
lumen. Irrigation with sterile PBS was performed to ensure
proper placement of the catheter tip, and the remaining
urine was aspirated with a 1 cc syringe.
Chemotherapy agents (50 µl) were delivered into
Figure 4: Phenotype of mice treated following intravesical treatment. Shown are representative images of bladders from Adeno-
Cre infected p53
f/f/
; Pten
f/f
mice following treatment with Vehicle or with the single or combination agents as indicated. The top panels show
whole mount images of the dissected bladder at the time of dissection. The upper middle panels show representative ultrasound images and
representative H&E images of bladder histology at the conclusion of the treatment. The bottom panels show images of representative of
Ki67-immunostaining for quantication of proliferation.

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the bladder lumen and a 5-0 silk suture was tied around
the urethral meatus to prevent expulsion; the installation
time was 2 hours. Agents used were as follows: Cis-
Diamineplatinum (II) Dichloride (Cisplatin; #479306)
was purchased from Sigma; Gemcitabine (#NC0063515)
and Docetaxel (#D-1000) were purchased from LC Labs.
Optimal dosages for each agent were estimated based
on prior literature (18, 23-25) and then conrmed in
pilot studies. Cisplatin and Gemcitabine were dissolved
in sterile PBS and diluted to working concentrations of
0.5 mg/ml and 25 mg/ml, respectively. Docetaxel was
reconstituted to a concentration of 12.5 mg/ml in DMSO
and diluted in sterile PBS to 0.5mg/ml. Each drug was
delivered 1 time per week using the following dosage
schedule: Vehicle (PBS) was delivered on Monday and/
or Wednesdays. (Note that the Vehicle group included
mice in which DMSO was diluted into PBS; these were
not appreciably different than the PBS group). Cisplatin
was delivered on Monday; Docetaxel was delivered on
Wednesdays; Gemcitabine was delivered on Friday. When
two agents were delivered, these were given on two days,
rather the same day. To control for the enhanced efcacy
of the combination versus the second treatment, a cohort
of the single agent mice were delivered drug at twice the
dose, which was not appreciably different than the single
dose.
Cohorts of mice were enrolled randomly into
the various treatment arms. The size of the cohorts
was determined using standard power analyses, with
bootstrapping from pilot studies. In particular, based on
the phenotype and response to drug treatment from pilot
studies, we estimated that a minimum of 6 mice would
provide statistical power for analyses; however, each of
experimental groups had a minimum of 10 mice in each
condition. Attrition, due either to death from tumor size or
infection from the catheterization, or other was less than
10% overall; the mice reported on only included those that
survived to the end of the study.
Analyses of mouse phenotypes
Following eight cycles (8 weeks) of drug treatment,
mice were sacriced and autopsied to evaluate the overall
bladder phenotype and to quantify metastatic lesions.
Bladders were harvested and processed for histological
analysis as described (21). Metastases to distant organs
were scored by visual inspection upon sacrice, and
conrmed by histological analyses; a mouse was indicated
to be positive for metastases if we observed a minimum
of two overt lesions. Metastases were observed mainly in
the lymph nodes, pancreas, liver, and GI tract, as we have
reported previously (21). Indications for sacrice prior to
eight cycles of treatment included tumor size of 1.5 cm or
greater, hematuria, or weight loss of greater than 15% of
initial body weight.
Immunohistochemical staining was performed
on parafn-embedded tissues as described (26).
Quantication of cellular proliferation was performed by
immunostaining with Ki67 (Lieca # NCL-Ki67p) using at
least three independent sections on 4 independent mice/
group (26). As we have reported previously, the percentage
of Ki67-positively stained cells in the bladder epithelium
or tumors were in comparison to the unstained cells
(26). Images were captured using a whole slide scanner
(Olympus VS120-S5).
Statistical Analysis
Statistical analysis were performed using Welch
t-test and Fisher’s Exact test as appropriate. GraphPad
Prism software (Version 4.0) was used for statistical
analysis and to generate data plots.
ACKNOWLEDGEMENTS
Ultrasound imaging was performed in the Herbert
Irving Comprehensive Cancer Center Small Animal
Imaging Shared Resource. This work was supported by
grants CA084294 (to CAS), funding from the Alexander
and Margaret Stewart Trust provided to the Institute for
Cancer Genetics, and from the T.J. Martell Foundation
for Leukemia, Cancer and AIDS Research (to MB). TK
was supported by post-doctoral training grants from the
American Urological Association Foundation and the
American Association for Cancer Research. CAS is an
American Cancer Society Research Professor supported
in part by a generous gift from the F.M. Kirby Foundation.
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