Prevention of age-related spontaneous mammary tumors in outbred

Cancer Detection and Prevention 32 (2008) 65–71
www.elsevier.com/locate/cdp
Prevention of age-related spontaneous mammary tumors in outbred
rats by late ovariectomy
Maricarmen D. Planas-Silva PhD*, Tina M. Rutherford BSc, Michelle C. Stone BSc
Department of Pharmacology, Penn State College of Medicine, MCH078, 500 University Drive, Hershey, PA 17033, USA
Accepted 25 January 2008
Abstract
Background: Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)positive tumors. In Sprague–Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous agedependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent
mammary tumor development. Methods: In this study we used Hsd:Sprague–Dawley1 SD1 (Hsd) rats to determine the effect of late
ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or
over their life span. In some experiments, palpable tumors were surgically removed upon presentation. Results: Removal of ovaries before
middle age onset (5–7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in
19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8
to 5.3% (relative risk = 0.05, 95% CI = 0.0072–0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was
one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by
surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival
rate and maximum life span of Hsd rats. Conclusion: Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This
animal model may be useful for evaluating novel interventions in breast cancer prevention.
# 2008 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.
Keywords: Cancer prevention; Breast cancer; Aging; Ovarian function; Estrogen receptor; Cyclin D1; Tumor multiplicity; Animal model; Overall survival;
Life span
1. Introduction
One in eight women develop breast cancer over their life
span. A key risk factor in breast cancer is age, although not
all types of breast cancer are affected similarly by aging.
Most breast cancers in older women (70–80%) express
estrogen receptor (ER) alpha, whereas only 50% of breast
tumors in younger women are ER-positive [1]. This agedependent increase in the incidence of ER-positive, but not
ER-negative, breast tumors is well documented [1]. ERpositive tumors are estrogen-dependent and can respond to
hormonal treatments, such as treatment with the selective
estrogen receptor modulators tamoxifen or with aromatase
inhibitors, that block estrogen synthesis. Moreover, breast
* Corresponding author. Tel.: +1 717 533 0860.
E-mail address: [email protected] (M.D. Planas-Silva).
cancer prevention trials have shown that the selective
estrogen receptor modulators, such as tamoxifen and
raloxifene, can efficiently reduce ER-positive breast tumors
in high-risk women [2–7].
To investigate new prevention strategies for age-related
breast cancer, an animal model that develops spontaneous
age-dependent ER-positive breast cancer is desirable. The
rat has been useful in studies of estrogen-responsive breast
tumors due to the presence of ER in most rat mammary
tumors while absent in most mouse mammary tumors [8]. In
particular, Sprague–Dawley rats have been used successfully to study the role of hormones in breast cancer
development and the protective effects of hormonal
therapies in preventing carcinoma formation after carcinogen treatment [8–11]. Like humans, the incidence of
spontaneous mammary tumors in Sprague–Dawley rats is
age-dependent [12–14].
0361-090X/$30.00 # 2008 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cdp.2008.01.004
66
M.D. Planas-Silva et al. / Cancer Detection and Prevention 32 (2008) 65–71
Tumor incidence in outbred Sprague–Dawley rats
depends on several variables including type of diet, amount
of caloric intake, environment and source of the Sprague–
Dawley stock [10,15–17]. To our knowledge, the first study
showing the requirement of intact ovarian function in
spontaneous formation of mammary tumors in Sprague–
Dawley rats was published over 40 years ago [13] using rats
ovariectomized at a young age (3 months). A more recent
study, confirmed the observation that early ovariectomy (3
months) reduces spontaneous mammary tumor formation in
aging Sprague–Dawley rats [18]. However, as intervention
strategies in women are typically started later in life we
sought to determine the effect of late ovariectomy in
spontaneous mammary tumor development using Sprague–
Dawley rats. For this study, we used the Harlan (Hsd:Sprague–Dawley1 SD1) stock (Hsd). This stock was selected
because Hsd rats have lower body weight with aging and a
lower incidence of pituitary tumors than rats from the
Charles River (Crl:CD1 (SD) Br) stock, commonly used for
toxicological studies [19,20]. We observed that late
ovariectomy efficiently reduced the development of spontaneous age-related mammary tumors in Hsd rats, without
decreasing overall survival.
2. Materials and methods
2.1. Animal treatments
Two different experimental designs (protocols) were used
in this study. The goal of the first protocol was to compare
the cumulative incidence of spontaneous mammary tumors
in virgin female Sprague–Dawley rats in the presence of
ovarian function or its absence since middle age onset. Two
independent experiments were conducted following this
protocol. The first experiment had a total of 23 animals (12
intact, 5 sham-ovariectomized and 6 ovariectomized) and
animals were observed until 110 weeks of age. The second
experiment had a total of 14 animals (7 sham-ovariectomized and 7 ovariectomized). In this second experiment
from the first protocol animals were followed through their
life span. The goal of the second protocol was to compare
tumor multiplicity and overall life span in the presence or
absence of ovarian function (earlier or later in life). In this
second protocol, mammary tumors or other subcutaneous
tumors were surgically removed to allow animals to
continue living after mammary tumor presentation [18].
A total of 30 animals were used in this study (6 intact, 12
sham-ovariectomized and 12 ovariectomized).
Intact, sham-ovariectomized and ovariectomized virgin
female Sprague–Dawley rats were obtained from Harlan
Sprague–Dawley Inc. (Indianapolis, IN). Surgeries were
performed by the vendor either earlier in life (1 month of
age) or later in life, before middle age onset (between 4.5
and 7 months of age). The average number of rats per group
was six. For the first experiment, all rats were followed until
110 weeks of age. At that time, all surviving animals were
euthanized. In all other experiments, rats were followed over
their entire life span.
All rats were housed in an animal facility that is
accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Rats were
caged in groups of six and housed in a controlled
environment of temperature (70 2 F), humidity
(50 10%) and lighting (12 h light/dark cycle, 07:00–
19:00 h). Animals were fed Teklad Rodent Diet 8604
(Harlan, Madison, WI) and water ad libitum. All animal
studies were performed following the guidelines outlined in
Guide for the Care and use of Laboratory Animals.
Rats with mammary or other palpable tumors exceeding
2.0 cm in diameter were either euthanized (first protocol,
110-week and life span experiment) or underwent surgery
for tumor removal (second protocol, overall survival
experiment). If surgery was successful, the rat was housed
alone for a period of 2 or 3 weeks for observation and then
placed back in its original cage. In all experiments, animals
were euthanized if they were deemed ill, off-feed or
moribund according to standard guidelines [21]. The
mammary glands of the euthanized animals were analyzed
for the presence of mammary tumors. To assess tumor
multiplicity, mammary tumors were considered distinct only
when observed in different mammary glands. Upon death of
the animal, several tissues such as uterus, ovaries and
pituitary were removed for further analysis when possible
and ovariectomy was confirmed at this stage. Complete
necropsy was done only if the cause of death of the animal
could not be readily determined.
2.2. Histology and immunohistochemical analyses
Mammary tumors were immersion-fixed in 10% neutral
buffered formalin for 24 h and paraffin embedded. Serial
6 mm paraffin sections were stained with hematoxylin and
eosin for histopathological diagnosis. Mammary epithelial
tumors were classified as fibroadenomas, adenomas or
adenocarcinomas as appropriate. For immunohistochemistry, slides were baked for 1 h at 60 8C and deparaffinized.
Sections were then subjected to antigen retrieval by boiling
in 0.01 M sodium citrate buffer (Vector Laboratories,
Burlingame, CA) for 10 min and allowed to cool for
10 min. Endogenous peroxidase was quenched by incubating slides for 10 min in 3% H2O2 in dH2O. Nonspecific
binding was blocked by incubating sections for 30 min in
5% bovine serum albumin/0.5% Tween. In addition, to block
endogenous biotin present in mammary tissue, sections were
then incubated in the Avidin portion of ABC kit (Vector
Laboratories) for 30 min. After rinsing in phosphate buffer
saline, sections were incubated with the specific primary
antibody diluted in 5% bovine serum albumin, overnight at
4 8C. Mammary carcinomas were immunostained for ER
(MC-20, Santa Cruz Biotechnology, Santa Cruz, CA), cyclin
D1 (Ab-3, NeoMarkers, Fremont, CA) and progesterone
M.D. Planas-Silva et al. / Cancer Detection and Prevention 32 (2008) 65–71
receptor (PR) (Santa Cruz). The following day, the slides
were rinsed in phosphate buffer saline and exposed to the
appropriate Vector Elite ABC kit as per manufacturer’s
instructions. Sections were counterstained with hematoxylin
(Sigma–Aldrich, St. Louis, MO).
2.3. Statistical analyses
For analysis of mammary tumor-free survival curves, the
Kaplan–Meier method was used with log-rank tests for
group differences. To compare the numbers of animals with
tumors or 110-week survival, the Fisher’s exact test was
used. For comparison of tumor multiplicity, the unpaired
two-tail t-test was used. For all statistical analyses the
PRISM software was used (GraphPad Inc., San Diego, CA).
3. Results
3.1. Age and hormone-dependent mammary tumor
incidence on Hsd rats
To determine the effect of late ovariectomy on
spontaneous mammary tumor development we first carried
out a 110-week study with female Hsd rats, in the presence
or absence of ovarian function. Removal of ovaries was
performed before rats entered into middle age (i.e., <7
months old). This time was chosen because age-dependent
alterations in estrous cycles are observed in Sprague–
Dawley rats during middle age (7–9 months old) [22]. Fig. 1
shows the incidence of mammary tumors as percent of
mammary tumor-free survival [10] in normal (intact) virgin
rats compared to ovariectomized or sham-ovariectomized
virgin rats. Intact or sham-ovariectomized Hsd rats showed
age-dependent increases of mammary tumors. Late ovariectomy significantly decreased mammary tumor formation
when compared to sham-ovariectomized animals (Fig. 1,
P = 0.0124). Tumor multiplicity (number of tumors per rat)
Fig. 1. Cumulative mammary tumor incidence in Hsd rats. Female rats
were monitored for spontaneous mammary tumor development during a
period of 110 weeks. Rats were euthanized when mammary tumors reached
2 cm of diameter. The percentage of rats in each group that at any time were
surviving free of any spontaneous mammary tumors (mammary tumor
(MT)-free survival) was graphed using Kaplan–Meier plots. Mammary
tumor incidence in ovariectomized rats was significantly different from
sham-ovariectomized animals (P = 0.0124).
67
was 0.76 for non-ovariectomized Hsd rats while 0 for
ovariectomized animals. While most age-dependent mammary tumors were benign, we observed two mammary
carcinomas in 17 non-ovariectomized (intact + sham-ovariectomized) Hsd rats, an incidence comparable to the rate
(one in eight) observed in women. These results suggest that
late ovariectomy prevents development of spontaneous agedependent mammary tumors.
3.2. Hormone receptor expression of mammary
carcinomas
It is not known whether spontaneous mammary
carcinomas from outbred rats express ER. We investigated
whether the two spontaneous rat mammary carcinomas that
developed in Hsd rats expressed ER or other molecular
biomarkers found in breast cancer of older women. Fig. 2
shows the histological and molecular features of an invasive
cribriform adenocarcinoma that appeared at 583 days of age
(19 months) in an intact Hsd rat. This tumor expressed
nuclear ER, PR and the ER target cyclin D1. The second
carcinoma also expressed cyclin D1 but was ER+/PR .
Thus, spontaneous carcinomas arising in aging Hsd rats have
pathological and molecular characteristics similar to those
of breast tumors found in older women. These results
suggest that the molecular mechanisms leading to agerelated mammary carcinomas in rats are similar to those
involved in the development of hormone-dependent breast
cancer in humans.
3.3. Spontaneous mammary tumor development over
life span
Although in our first experiment no spontaneous
mammary tumors were observed in the late ovariectomized
group until 110 weeks of age, it was possible that late
ovariectomy may have increased the latency of spontaneous
mammary tumor development until after 110 weeks of age.
Therefore, another experiment was conducted to measure
mammary tumor-free survival over the life span of the rats.
As shown in Fig. 3, no tumors were observed in
ovariectomized animals over their life span. The difference
in mammary tumor incidence between sham-ovariectomized and ovariectomized animals was significant
(P = 0.0358). Tumor multiplicity (0.76 tumors/rat) and
incidence of mammary carcinoma (14.28%) obtained in
this experiment were comparable to the previous 110-week
experiment. Interestingly, maximum life span of ovariectomized animals in this experiment was 42 months while for
sham-ovariectomized it was less than 30 months. However,
as the endpoint of this experiment was mammary tumor
formation and rats were euthanized when a palpable tumor
reached 2 cm of diameter, overall survival curves were not
compared. A different experimental design was required to
determine the effect of late ovariectomy on overall survival.
Nevertheless, removal of ovaries before middle age onset
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M.D. Planas-Silva et al. / Cancer Detection and Prevention 32 (2008) 65–71
Fig. 2. Pathological features and biomarker expression in spontaneous rat mammary carcinoma. Carcinoma was stained with hematoxylin and eosin (H & E) or
immunostained with antibodies against ER, PR or cyclin D1. The light/dark brown nuclei are indicative of positive staining (200).
can successfully prevent the development of spontaneous
mammary tumors over the life span of Hsd rats.
3.4. Effect of ovariectomy on overall survival
Recently, studies on the effect of oophorectomy in
women had suggested that mortality was significantly higher
in women who had received prophylactic bilateral oophorectomy before age 45 [23] or 65 [24]. Therefore, it was
important to determine the impact of ovariectomy on
survival of Hsd rats. To evaluate overall survival, instead of
euthanizing rats upon tumor presentation, tumors were
surgically excised when possible. To compare our survival
data with a previous study on the role of ovariectomy upon
longevity [25], we also utilized rats whose ovaries were
Fig. 3. Cumulative mammary tumor incidence in Hsd rat over life span.
Female rats were monitored for spontaneous mammary tumor development
during their life span. Rats were euthanized when mammary tumors reached
2 cm of diameter. Mammary tumor (MT)-free survival was graphed as
percent (%). Mammary tumor incidence in ovariectomized rats was significantly different from sham-ovariectomized animals (P = 0.0358).
removed around 1 month of age (early ovariectomy). In this
way, our data could also be compared with the more standard
intervention protocol used in toxicological studies that have
shown the impact of hormonal therapies in development of
spontaneous mammary tumors [14,26].
In this experiment, we first compared spontaneous
mammary tumor development. Again, ovariectomy, whether
performed early or late in life, decreased the development of
spontaneous mammary tumors when compared to shamovariectomized or intact controls (Fig. 4). Only one benign
tumor was found upon necropsy in an ovariectomized rat (one
out of 12) that died at 33 months of age (142 weeks of age).
In contrast, 29 spontaneous tumors developed in 18 nonovariectomized rats (95% inhibition). While the incidence
of mammary carcinoma was still comparable to the preceding
experiment (16.67%), tumor multiplicity was significantly
increased (P = 0.002) to 1.61 tumors per rat (Table 1) due to
surgical excision of tumors. From 11 successful surgeries (i.e.,
rats survived for at least 15 days after surgery), eight rats
(73%) developed at least one additional mammary tumor. The
maximum number of tumors observed per rat was four. Thus,
surgical excision of palpable tumors can increase the total
number of spontaneous mammary tumors per rat, and
improved the ability to detect significant differences in
spontaneous mammary tumor development, when testing
novel preventive strategies.
To compare overall survival, groups whose surgical
intervention procedure (sham-ovariectomized or ovariectomized) was done early, were combined with the late
intervention groups because survival curves for the same
intervention were similar. The 110-week survival rate for nonovariectomized animals was 55.5% while for ovariectomized
M.D. Planas-Silva et al. / Cancer Detection and Prevention 32 (2008) 65–71
69
Fig. 5. Effect of ovariectomy on overall survival. Female rats were monitored for spontaneous mammary tumor development during their life span.
Tumors were surgically removed when palpable tumors reached 2 cm of
diameter. Overall survival was graphed as percent (%). Overall survival of
non-ovariectomized animals was significantly different from ovariectomized animals (P = 0.0121).
Table 2
Histopathology of spontaneous mammary tumors
Fig. 4. Cumulative mammary tumor incidence in Hsd rat in early or late
ovariectomized rats. Female rats were monitored for spontaneous mammary
tumor development during their life span. Tumors were surgically removed
when palpable tumors reached 2 cm of diameter. Mammary tumor (MT)free survival was graphed as percent (%). (a) Early ovariectomy. Mammary
tumor incidence in ovariectomized rats was significantly different from
sham-ovariectomized animals (P = 0.0014). (b) Late ovariectomy. Mammary tumor incidence in ovariectomized rats was significantly different
from sham-ovariectomized animals (P = 0.005).
Group (n)
Fibroadenoma
Adenoma
Adenocarcinoma
Intact (18)
Sham-ovariectomized
(24)
Early ovariectomy (6)
Late ovariectomy (19)
14
20
2
5
2
4
0
1
0
0
0
0
ovariectomy, can efficiently prevent development of
spontaneous mammary tumors.
4. Discussion
animals was 91.66% (P = 0.0492). Overall survival (Fig. 5)
was also significantly different (P = 0.0121). Median survival
increased from 810 days for non-ovariectomized animals to
888 for ovariectomized animals. Thus, in Hsd rats,
ovariectomy did not impair overall survival.
In summary, overall mammary tumor incidence was
73.81% for non-ovariectomized animals. Although most
tumors were benign (Table 2), 6 out of 42 (14.29%) nonovariectomized animals had spontaneous mammary carcinomas. Late ovariectomy decreased the incidence of
spontaneous mammary tumor development to 5.26%
(relative risk = 0.05, 95% CI = 0.0072–0.354, P < 0.0001)
while no mammary carcinomas were observed (Table 2).
Thus, this animal model may enable the testing of
novel prevention strategies that, as shown here for late
Table 1
Number of spontaneous mammary tumor per rat (multiplicity)
Group (n)
No. of MT
Multiplicity
Non-Ovx w/out MT Exc (24)
Non-Ovx w/MT Exc (18)
Early ovariectomy (6)
Late ovariectomy (19)
18
29
0
1
0.75
1.61*
0.0
0.05**
*P = 0.0023, **P = 0.0003, significantly different from non-ovariectomized (Ovx) w/out mammary tumor excision (MT Exc); No: number.
In this study we evaluated whether late ovariectomy
(before middle age onset) was able to prevent development
of spontaneous mammary tumors in Hsd rats. Our data
clearly indicates that late ovariectomy can efficiently
prevent development of spontaneous, benign and malignant
mammary tumors in Hsd rats. This rat model mimics the
human situation such that age-dependent ER-positive breast
carcinomas arise spontaneously, without prior treatment
with a known carcinogen, at a frequency of one in eight
women.
Currently, prevention trials for hormone-dependent
breast cancer, such as the National Surgical Adjuvant
Breast and Bowel Project P-1 (P-1) or P-2 trial needed to
enroll thousands of women for at least 5 years [2,4]. In the P1 trial, tamoxifen also decreased the risk of benign disease
and the need for biopsies [27], a considerable sociological
and socioeconomical problem today. Therefore, the use of a
Hsd rodent model, using spontaneous mammary tumor
(benign and malignant) development as surrogate for breast
cancer, could be instrumental in testing new preventive
strategies for ER-positive breast cancer before they are
tested in women. Although the incidence of mammary
carcinoma in this outbred rat is low, it is comparable to that
observed in humans. The high number of benign mammary
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M.D. Planas-Silva et al. / Cancer Detection and Prevention 32 (2008) 65–71
tumors spontaneously developing may permit efficient
testing of the effects of different preventive strategies in
blocking development of spontaneous mammary tumors.
Nevertheless, it is possible that diet or other interventions
may reproducibly increase the incidence of spontaneous
age-dependent mammary carcinomas [12]. Therefore, this
model could be used not only to test preventive strategies but
also to determine the impact of diet, hormones and other
factors in the development of spontaneous ER-positive
breast cancer.
Several studies using rats have already shown that agents,
such as tamoxifen or the aromatase inhibitor fadrozole,
when given for 2 years or less, can drastically reduce
development of spontaneous benign and malignant mammary tumors in female Sprague–Dawley rats [14,26]. In
most animal studies, hormonal interventions begin when the
animals are young (1–2 months of age). To our knowledge,
only one study has evaluated tamoxifen treatment for 40
weeks in aged animals (56 weeks old) [14]. Similar to our
results, a significant decrease in the incidence of spontaneous benign and malignant mammary tumors was observed
at the end of tamoxifen treatment and, although of less
magnitude, at the end of their life span. Thus Sprague–
Dawley rats could be used to test specific interventions
(dosage, schedule and combination strategies) in middleaged individuals or older that can modify the risk of
spontaneous hormone-dependent mammary carcinoma.
We prevented spontaneous mammary tumor formation in
female Hsd rats by removing their ovaries before 7 months
of age, when age-related changes in estrous cycles are
evident. Young adult female Sprague–Dawley rats have a 4day estrous cycle [22,28]. However, when they reach middle
age (7–9 months), 32% of Sprague–Dawley rats start
experiencing irregular cycles (4–7 days). By 15 or 16
months of age, all female rats have irregular estrous cycles
[29,30]. These age-dependent irregularities in the estrous
cycle differ from humans in having increased, rather than
decreased, blood estrogen levels (constant estrous or
pseudopregnancy). However, in the last third of their life,
when most of the mammary tumors appeared, most female
rats develop atrophic ovaries and become anestrous [30,31].
Therefore, the hormonal milieu in rats developing mammary
tumors may be comparable to that in postmenopausal
women. In women, breast cancer risk has been associated
with aberrant lobular involution [32]. Complete involution is
associated with reduced breast cancer risk [32]. Thus, it is
possible that removal of ovaries before middle age prevented
spontaneous mammary tumor development by inducing
mammary gland involution. Nevertheless, our results
suggest that the presence of estrogen during middle age
or later is crucial for spontaneous mammary development in
Hsd rats.
Interestingly, 110-week survival rates and maximum life
span were increased for ovariectomized animals as observed
in a previous study [18]. However, there is also evidence that
ovariectomy can decrease the life span of rats [25]. The
difference in outcomes may be due to the use of a different
strain of rats. Nevertheless, although development of
spontaneous mammary tumors was inhibited in ovariectomized animals, we noticed that several ovariectomized
animals died, after 110 weeks of age, due to chronic renal
failure. Chronic renal failure is a common cause of
unscheduled deaths for Hsd males but not for Hsd females
[19]. Moreover, estrogen-deficiency in rats has been shown
to accelerate progression of renal disease [33]. Thus,
although late ovariectomy can decrease spontaneous
mammary tumor formation, novel preventive strategies
are needed which can inhibit age-dependent increases in
breast cancer risk without affecting the normal function of
other organs.
In women, bilateral ovariectomy before age 45 [34] or 50
[35] can reduce breast cancer risk. However, another study,
observing an increase in mortality when bilateral oophorectomy was done before age 45 but not after 45 years of age,
found an increased risk of estrogen-related cancer [23]. As
the authors suggested, it was possible that the population
undergoing bilateral oophorectomy may have had certain
features leading to their increased mortality [23]. Nevertheless, to investigate the impact of ovariectomy at different
ages on risk of spontaneous mammary carcinoma development, other age-related processes, and longevity in an
animal model, further studies will be needed with a larger
number of rats. Nonetheless, this rat model can be used to
identify novel preventive interventions that may inhibit
spontaneous age-related mammary tumor development and,
at the same time, may increase overall survival.
Acknowledgements
We gratefully acknowledge James W. Griffith for the
pathological analyses of the mammary tumors and other
age-related pathologies, Melissa Coleman and Kecia
Hamilton for technical assistance. This work was supported
in part by a grant from the American Federation for Aging
Research. Additional support was provided by start-up funds
from the Penn State College of Medicine.
Conflict of interest
None.
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