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Progesterone Is Essential for Maintenance and

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CANCER-ONCOGENES

Progesterone Is Essential for Maintenance

and Growth of Uterine Leiomyoma
Hiroshi Ishikawa, Kazutomo Ishi, Vanida Ann Serna, Rafael Kakazu,
Serdar E. Bulun, and Takeshi Kurita
Division of Reproductive Biology Research, Feinberg School of Medicine at Northwestern University,
Chicago, Illinois 60611

Uterine leiomyomata (ULs) represent the most common tumor in women and can cause abnormal
uterine bleeding, large pelvic masses, and recurrent pregnancy loss. Although the dependency of UL
growth on ovarian steroids is well established, the relative contributions of 17-estradiol and progesterone are yet to be clarified. Conventionally, estradiol has been considered the primary stimulus for
UL growth, and studies with cell culture and animal models support this concept. In contrast, no
research model has clearly demonstrated a requirement of progesterone in UL growth despite accumulating clinical evidence for the essential role of progesterone in this tumor. To elucidate the functions of ovarian steroids in UL, we established a xenograft model reflecting characteristics of these
tumors by grafting human UL tissue beneath the renal capsule of immunodeficient mice. Leiomyoma
xenografts increased in size in response to estradiol plus progesterone through cell proliferation and
volume increase in cellular and extracellular components. The xenograft growth induced by estradiol
plus progesterone was blocked by the antiprogestin RU486. Furthermore, the volume of established
UL xenografts decreased significantly after progesterone withdrawal. Surprisingly, treatment with
estradiol alone neither increased nor maintained the tumor size. Although not mitogenic by itself,
estradiol induced expression of progesterone receptor and supported progesterone action on leiomyoma xenografts. Taken together, our findings define that volume maintenance and growth of human
UL are progesterone dependent. (Endocrinology 151: 24332442, 2010)

terine leiomyomata (ULs) or fibroids are benign

smooth muscle tumors originating from the myometrium. The estimated cumulative incidence of tumors by
age 50 yr is greater than 80% for African American
women and nearly 70% for Caucasian women (1). Patients with UL can experience symptoms such as pelvic
pressure, abnormal uterine bleeding, and recurrent pregnancy loss that require medical intervention (2). Surgical
removal is the primary treatment option for symptomatic
ULs, and more than 1 million hysterectomies were performed for the treatment of UL between 2001 and 2005 in
the United States alone (3). With the high number of hysterectomies for this tumor, management of ULs has placed
an enormous burden on the health care system. Therefore,
development of efficacious preventative and therapeutic

modalities that will allow avoidance of surgery not only

improves quality of life but also carries significant public
health implications.
Importance of 17-estradiol (E2) and progesterone (P4)
in the etiology of UL is well established. However, the
relative contributions of estrogen vs. progestin and their
functions in the pathogenesis of UL are still controversial.
Conventionally, estrogen has been considered to be the
primary growth promoter of ULs, and experimental evidence in both animal and cell culture models support this
concept (4 14). In contrast, the effect of P4 on UL has not
been well elucidated in these research models. For example, growth of spontaneous UL was stimulated by E2 but
not P4 in the Eker rat model (7). Within a guinea pig model,
UL formation induced by E2 treatment was inhibited by

ISSN Print 0013-7227 ISSN Online 1945-7170

Printed in U.S.A.
Copyright 2010 by The Endocrine Society
doi: 10.1210/en.2009-1225 Received October 14, 2009. Accepted March 15, 2010.
First Published Online April 7, 2010

Abbreviations: E2, 17-Estradiol; ECM, extracellular matrix; ER, estrogen receptor; H&E,
hematoxylin and eosin; IHC, immunohistochemistry; OVX, ovariectomy; P4, progesterone;
PR, progesterone receptor; SCID, severe combined immunodeficiency; UL, uterine
leiomyoma.

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Ishikawa et al.

Uterine Leiomyoma Xenograft Study

coadministration of P4 (15). In vitro, progestin can show

similar growth-promoting effects as estrogen on primary
culture of human UL cells by inhibiting apoptosis and
stimulating proliferation (10, 16, 17). However, progestin
can also inhibit UL growth under certain culture conditions (18, 19).
In contrast to these research models, clinical studies
have suggested that P4 plays an equally important role as
E2 in the growth of ULs. Proliferation markers such as
Ki67 and proliferating cell nuclear antigen (PCNA) are
highest in UL in the luteal/secretory phase (20 22). Moreover, quantitative proliferation indices of ULs in postmenopausal women increased significantly with combined estrogen plus progestin replacement but not with
estrogen replacement alone (20). These authors have even
suggested that P4 may be the primary hormone driving the
growth of UL. However, systemic hormone-levels in patients cannot be manipulated experimentally, and thus,
the effects of estrogen vs. progestin on UL growth cannot
be delineated solely by analysis of clinical specimens.
In total, there remains significant conflict regarding the
role of P4 on human UL. Within human subjects, manipulation of systemic hormone levels beyond routine administration of estrogen/progesterone-containing medications is unethical, and thus, study findings are hindered by
factors such as dosing and patient compliance. Furthermore, most UL-related procedures are scheduled within
the first half of the menstrual cycle to avoid the possibility
of pregnancy, thereby limiting the number of UL specimens obtained within the luteal/secretory phase. Thus, to
advance research on UL, development of a novel model
system that incorporates characteristics of original human
tumors is essential.
In human ULs, cells are embedded in extracellular matrix (ECM), which is largely composed of collagens and
glycosaminoglycans. ECM is known to influence several
critical cellular functions, including apoptosis and cell
proliferation (23, 24). Indeed, myometrial and UL cells
alter their gene expression pattern significantly in the organotypic three-dimensional environment in vivo vs. an
artificial, two-dimensional culture within plastic dishes
(25, 26). In addition, accumulation of ECM contributes
significantly to the growth of the tumor (27). These observations raise additional questions regarding the correlation between in vitro studies to human in situ tumors.
We report a method of xenografting and growing human ULs in immunodeficient mice. Xenografting into an
immunodeficient mouse host is a standard approach in
studying human tissues in vivo. Previously UL tissues with
ECM were implanted under the skin of severe combined
immunodeficiency mice (28). However, the UL xenografts
were maintained only when their original phenotypes

Endocrinology, June 2010, 151(6):24332442

were altered by transduction of proangiogenic genes,

PTGS2 (COX2) and VEGFA (28). These findings imply
that the limiting factor for UL xenograft survival within
subcutaneous (sc) tissue with low intrinsic vascular density was angiogenesis. Thus, we chose to use a subrenal
capsule graft site because of its superior blood supply (29).
On the kidney of nonobese diabetic (NOD)-scid IL2Rnull
mice (30), the survival rate of UL and normal myometrial
tissue xenografts was nearly 100%, and the xenografts
retained the histological characteristics of original tissue.
Using this model, the objective of this study was to address
the effects of E2 vs. P4 on UL growth.

Materials and Methods

Subrenal grafting of myometrial and UL tissues
All procedures involving animals in this study were approved
by Northwestern Universitys Animal Care and Use Committee.
Protocol for the acquisition of surgical specimens was approved
by Northwestern Universitys Institutional Review Board. Surgically removed UL and normal myometrial tissue from the same
patient were cut into small pieces (1 2 2 mm) and grafted
onto opposing kidneys of adult female nonobese diabetic-scid
IL2Rnull mouse hosts (Jackson Laboratory, Bar Harbor, ME).
The tissue pieces were high in water content, and thus, they
became smaller under the pressure of subrenal capsule. The estimated starting volume of tissue grafts under the renal capsule
was approximately 1 mm3. The growth of UL in intact female
hosts without hormone treatment was minimum and inconsistent. Therefore, all hosts were ovariectomized (OVX) and supplemented with sc implantation of 50 mg P4 plus 50 g E2 60-d
slow-release pellets (Innovative Research of America Inc., Sarasota, Fl). Stable dosages of E2 (50 g per 60 d) and P4 (50 mg per
60 d) were used throughout the study. These dosages were chosen because previous studies demonstrated that they were able to
sustain systemic E2 and P4 levels within cycling women (3135).
Additionally, if required by the experimental design of the specific study, RU486, a progesterone receptor (PR) antagonist, was
also given as sc implant (slow release pellet containing 25 mg per
60 d; Innovative Research of America). The effects of ovariectomy and hormone treatments were then confirmed by gross
appearance and histology of host female reproductive tracts. The
presence of hormone pellets was also confirmed at the time of
termination of the host.

Preparation of cell graft

Fresh surgical specimens of human myometrial/UL tissues
were digested into single cells by type I collagenase (Sigma, St.
Louis, MO) and cultured for 23 d as previously described (36).
Cells were collected from the culture plates by trypsin digestion
and suspended into rat-tail collagen (type I) solution (BD Bioscience, San Jose, CA) at 106 cells per 10 l. The method has been
successfully used to study hormonal response of human endometrial tissue in our previous report (37). The low-density collagen gel consisted mostly of water, and thus, the pellet volume
(10 l) did not reflect the starting volume of tumor. When cell
pellets were incubated at 37 C overnight as floating-culture, they
became smaller than 1 mm in diameter by contraction of collagen

Endocrinology, June 2010, 151(6):24332442

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2435

by UL cells. Therefore, the estimated starting volume of cell graft

was smaller than 0.6 mm3.

Data analysis
The tumor volume was estimated using the formula: volume
(cubic millimeters) 0.52 (derived from /6) length
width height (millimeters) (38). When tumor volume of the
E2P4 group was less than 1.0 mm3 at 8 wk after grafting, the
UL was categorized as growth negative. In the statistical analysis,
average value (e.g. tumor volume, cell density, labeling index) of
three to six xenografts per group in each experiment was considered as a single measurement. Only the experiments with the
complete set of hormone treatment groups (e.g. OVX, E2, P4,
E2P4, and E2P4RU486) were included in the analyses of
tumor volume, Ki67 labeling, cell size index, and cell density.

Histological analysis
The following histological analyses were performed routinely
for all xenografts and the original tissues: hematoxylin and eosin
(H&E) staining, immunohistochemistry (IHC) for estrogen receptor (ER)- (LabVision, Fremont, CA), PR (Dako, Carpinteria, CA), Ki67 (proliferation marker; Novacastra Laboratories,
Burlingame, CA) and active caspase-3 (apoptosis marker; Cell
Signaling, Danvers, MA) (37, 39). Terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assay
was performed using Apo-Brd UTM DNA fragmentation assay
kit (BioVision, Mountain View, CA). The cellular component
was detected by IHC for -actin (Abcam, Cambridge, MA). The
cell density was determined on H&E sections by counting the
number of nuclei per optical field with a 20 objective lens.
Morphometric analysis of immunohistochemical staining has
been described previously (39). Images of -actin-stained tissues
were captured with a Leica microscope imaging system (Leica
Microsystems Inc., Bannockburn, IL). Total and -actin-positive
tissue areas were measured with ImageJ (National Institutes of
Health, http://rsbweb.nih.gov/ij/index.html). The area with OD
higher than 180 [from 255 (white) to 0 (black)] in blue channel
of RGB mode was considered negative. Relative cell size was
calculated by -actin-positive area per cell number and expressed
as relative value to that of 2 wk after grafting.

Results
Growth of UL tissue in mouse hosts
All myometrial grafts (74 grafts) survived for 8 wk but
did not show detectable growth with any hormone treatments (Fig. 1), indicating the presence of an intrinsic
growth-suppressive mechanism within normal adult myometrium. Due to small tissue size, further analysis was
difficult for myometrial xenografts. Thus, we focused on
UL in the following analyses. In contrast, UL xenografts
increased their size in response to E2P4 but not to either
E2 or P4 alone. Among a total of 29 UL cases, xenografts
of 14 cases (48.2%) increased in size in response to E2P4
treatment (growth-positive UL), whereas there was no detectable growth (tumor size 1 mm3) in the other 15 cases
(growth-negative UL). The most noticeable difference be-

FIG. 1. Gross appearance and histology of myometrial xenografts.

Gross appearance of myometrial tissue and cell xenografts (indicated
by an arrow) on the host kidney (treated with E2P4) at 8 wk after
grafting. Although the myometrial xenografts did not increase in size
with any hormone treatment, both tissue and cell xenografts (with E2
treatment) showed typical histology of myometrium comparable with
the original tissue (top panels) in H&E and ER-IHC staining.

tween growth-positive and -negative ULs was the cell density

in the original patient tissue. Specifically, growth-positive
xenografts arose from patient tissue in which the original cell density was significantly higher (831 113
cells/area, n 12), whereas growth-negative UL cases
demonstrated fewer cells within the original patient tissue (425 155 cells/area, n 12) (Students t test, P
0.05). Thus, the number of cells/xenograft significantly
affected growth of UL xenografts. This conclusion was
further confirmed by cell-grafting experiments in the
following section. Because the absence of growth was
simply due to the quality of the original tissue, we excluded the growth-negative UL cases from the following
analyses.
Tumor volume was significantly higher in the E2P4
group than the other four groups (Fig. 2, A and B). The
growth-promoting effect of E2P4 was completely
blocked by coadministration of RU486, a PR antagonist
(Fig. 2). Therefore, P4 induced tumor growth via PR.
There was no significant difference in the tumor volume
(P 0.05) between E2P4RU486, E2 alone, P4 alone,
and untreated control groups, confirming the requirement
of both E2 and P4 for human UL growth. In fact, E2 alone
failed to increase tumor size, even when a supraphysiological dose was administered (10 g/d) (n 4).
The Ki67 index was significantly higher (Fig. 2C) in the
E2P4 group than the other four groups, indicating that
E2P4-stimulated tumor growth via proliferation of UL
cells. At the same time, cell density was significantly lower
in the E2P4-treated group (Fig. 2D), suggesting that an
increase in ECM volume and/or cell size also contributed
to the enlargement of tumor. This issue was further assessed in the following section.

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Endocrinology, June 2010, 151(6):24332442

this possibility, xenografts were established

first in the hosts with the optimum hormone
treatment (E2P4) and then subjected to different hormone treatments. All hosts were
ovariectomized and supplemented with E2 and
P4 at the time of grafting. Two weeks later, to
allow establishment of vascularization, the
hosts were divided into four groups, and hormone pellets were changed as indicated (Fig.
4A). After the hormone pellet change, established UL xenografts increased their size further only with E2P4 treatment (Fig. 4B). In
contrast, E2 or P4 alone failed to maintain tumor size. All xenografts of groups in which E2
and/or P4 was withdrawn exhibited significant
reduction in tumor volume and Ki67 labeling
index (Figs. 4 and 5). These results confirm the
essential role of E2 and P4 in growth and maintenance of ULs. All xenografts were negative
for apoptotic cells as assessed by active
caspase-3 IHC and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Absence of apoptotic
FIG. 2. Effect of E2 and P4 on the growth of UL xenografts. OVX, OVX control (no
cells at 8 wk after hormone withdrawal (data
hormone); E, E2 treated; P, P4 treated; EP, E2P4 treated; RU, E2P4RU486
not shown) does not necessarily mean that UL
treated groups. UL tissues were grown as subrenal grafts in OVX mouse host treated
cells do not die via apoptosis during regression
with E2 (E), P4 (P), E2P4 (EP), or E2P4RU486 (RU) for 8 wk. Gross appearance
of xenografts on host kidney (A; graft is indicated by an arrow), tumor volume (B),
of tumor. Nevertheless, UL cells appeared
Ki67 labeling index (C), and cell density (D). The bar indicates average value SEM.
healthy and showed no signs of tissue degraStatistical differences were assessed by using the Kruskal-Wallis test (n 6, P
dation as shown in Fig. 5. Thus, loss of cells via
0.05). UL tissues enlarged in response to E2 plus P4 treatment (A and B). E2P4 also
increased ki67 labeling index (C) and decreased cell density (D). This growthapoptosis does not appear to play a major role
promoting effect of E2 plus P4 treatment was blocked by RU486. E2 and P4 alone
in tumor regression. Instead, UL cells reduced
had no effect on the growth of UL tissues.
their size significantly after withdrawal of E2
and/or P4 (Fig. 4D). Accordingly, cell density
As assessed by IHC, ER was expressed in the xeno- increased significantly in these groups (Figs. 4E and 5).
grafts of all five groups (Fig. 3). In contrast, expression
This result indicates that cell size reduction is one of the
of PR in UL xenografts was totally dependent on E2. The
mechanisms via which UL shrinks.
level of PR was very low to undetectable in the untreatedIn the E2P4-treated group, cell size remained the same
control (OVX) and P4 groups but high in the E2,
(Fig. 4D), but the cell density significantly decreased from
E2P4 and E2P4RU486 groups (Fig. 3). These re2 to 10 wk after grafting (Fig. 4E), suggesting an enlargesults indicate that E2 is essential for P4 to act on ULs
ment of ECM. Indeed, -actin-negative areas significantly
in vivo.
increased from 2 to 10 wk (Fig. 4F). Therefore, E2 and P4
stimulate UL growth via increase in cell number and ECM
Growth of established tumors
In our xenograft model, tissue grafts must first establish volume. Ki67 was coexpressed with ER and PR (Supa blood vessel network to survive and subsequently grow. plemental Fig. 1 published on The Endocrine Societys
Although the kidney is highly vascular and thus capable of Journals Online web site at http://endo.endojournals.org),
supporting survival of tissues grafted onto it, efficiency in suggesting that Ki67-positive cells were the direct target of
blood vessel recruitment can be a limiting factor for sub- E2 and P4. As tumor volume increased, the Ki67 labeling
sequent growth of xenograft (29). Therefore, lack of index decreased significantly from 9.4 3.4% at 2 wk to
growth within xenografts treated with E2 alone may be 3.1 1.6% at 10 wk (mean SD) (Fig. 4C). This result
explained by a requirement of P4 for UL xenografts to indicates that the peak for cell proliferation is an early
sufficiently establish an adequate vascular network. Thus, event in the UL growth, whereas increase of ECM appears
E2 may still be a mitogen for established ULs. To examine to occur at a constant rate.

Endocrinology, June 2010, 151(6):24332442

FIG. 3. Histology of UL xenografts and original tumor. OVX, No

hormone control. UL xenografts survived for 8 wk and retained
histology typical for leiomyoma in all hormone treatment groups.
Whereas ER was expressed in all xenografts irrespective of hormone
treatment, expression of PR was dependent on E2. Thus, PR was
undetectable in OVX and P4 groups. All histology images were taken
under a 20 objective lens and in the same magnification.

Although it was not statistically significant, cell size

appeared smaller (Fig. 4D) and ECM volume appeared
higher (Fig. 4E) in the P4-alone group than control and
E2-alone groups (Fig. 5). It suggests that P4 may have some
effects on UL cell, even in the absence of E2.
Reconstruction of UL tissues from cultured UL cells
Because of the significant heterogeneity that exists
within human tissue, comparison between subjects is difficult. Factors including cell density of the original surgical
specimen and the distribution of UL cells within the sampled portion of the tumor can affect growth of the xenograft. To address this issue, tumor tissue was digested into
single cells, and UL tissue was reconstructed by suspending the isolated UL cells in type I collagen. After 8 wk of
in vivo growth with E2P4 treatment, UL cell grafts containing 106 cells formed uniform tissues with histology
comparable with the original tumor (Fig. 6). For efficient
tumor growth, a concentration of 106 cells/graft was the
optimum. With lower cell concentrations (5.0 105 cell/
graft and lower), growth of xenografts was inconsistent.

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2437

FIG. 4. Effect of hormone withdrawal on UL xenograft. A, Time line.

UL xenografts were grown for 2 wk with E2P4 (EP 2wks), and then
hormone pellets were replaced with E2P4 (EP), E2 (E), P4 (P), or no
hormone (OVX). Eight weeks after pellet replacement (10 wk after
grafting), xenografts were harvested and analyzed. Tumor volume (B),
Ki67 labeling index (C), relative cell size (D; relative value to average
cell size at 2 wk after grafting), cell density (E), and extracellular area
(-actin negative area; F). Bars indicate average value SEM. Statistical
differences were assessed by using the Kruskal-Wallis test (n 6, P
0.05). Tumor volume increased only with E2P4 treatment (B). In
response to withdrawal of E2 and/or P4, tumor volume (B), Ki67
labeling index (C), and cell size (D) significantly declined. Accordingly,
cell density increased in OVX, E, and P groups (E). In the EP group,
cell size remained the same; however, the cell density decreased
significantly (E) with increase of extracellular component (F) from 2 to
10 wk after grafting, indicating tumor volume increased by both cell
number and ECM volume.

With higher cell concentration (5.0 106 UL cells/graft),

the collagen gel became too soft to graft. To test whether
normal myometrium was able to grow as cell grafts, the
same cell-graft protocol was applied to the normal human
myometrium. Xenografts of reconstructed myometrium
(106 cells/graft) formed a tissue with typical histology of
myometrium, but graft volume did not increase with E2,
P4, or E2P4 treatment for 8 wk (Fig. 1, n 12).
Previous studies demonstrated that UL cells alter gene
expression profile in vitro and lose expression of ER and
PR rapidly (26). Therefore, expression of these receptors
in cell grafts was assessed by IHC. As in the tissue xeno-

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Uterine Leiomyoma Xenograft Study

Endocrinology, June 2010, 151(6):24332442

the low cell number and/or thick

ECM layer in the original tissues from
which the tissue xenografts were generated. The growth-positive and -negative ULs do not represent two different
entities with different hormone requirement. Our study suggests that all human ULs require E2 and P4 for growth
and volume maintenance.

Discussion
There has been conflicting evidence for
the role of P4 in the regulation of UL
growth. This is the first study to definitively demonstrate that both growth
and maintenance of human UL in vivo
are dependent on P4 action via PR. Our
conclusion agrees with the results of recent clinical trials of selective progesterone receptor modulators for ULs
(40 44). This study also defined for the
first time that estrogen by itself is not a
mitogen for human UL in vivo. NoneFIG. 5. Histology of UL xenografts subjected to hormone withdrawal. Proliferation activity
theless, our findings do not discount the
significantly declined in response to withdrawal of E2 and/or P4 as assessed by Ki67
importance of estrogen in UL. PR is
expression. PR was also down-regulated in response to E2 withdrawal. However, UL
widely recognized as a marker for esxenografts did not show sign of tissue degradation or cell death, and expression of ER and
trogen action, and regulation of the PR
-actin was maintained without E2 and/or P4, even after 8 wk. All histology images were
taken under a 20 objective lens and in the same magnification.
gene (PGR) by estrogen/ER has been
well defined (45). Because the PR prografts, ER was expressed in all xenografts of UL cells, tein level is thought to be a critical determinant of sensiwhereas PR was expressed only in the presence of E2 (Fig. tivity to P , almost all hormone treatment protocols de4
6). The growth response of UL cell grafts to E2 and P4 was signed to elicit effects of P involve priming with estrogen.
4
identical with that of tissue grafts. The growth of UL cell This was also true for human UL in vivo, and the expresgrafts was stimulated by E2P4 and inhibited by RU486 sion of PR was dependent on E
2 in UL xenografts. Based
(Fig. 7). Moreover, E2P4 treatment increased Ki67 laon the results of the current study, we propose the followbeling index (Fig. 7B) and reduced cell density significantly
ing model of UL growth control. P4 action via PR increases
(Fig. 7C).
tumor volume through cell proliferation and ECM accuThe efficacy of the cell graft method was experimenmulation. E2 is not a mitogen but is required for the growth
tally tested by comparing growth of the UL cell graft vs.
and maintenance of ULs to sensitize cells to P4 by inducing
that of the UL tissue xenograft in tissue/cells obtained from
the same subject. Among three UL cases, two cases with PR. This model is currently being tested by whether PR
relatively low cell density (cases 128 and 135) did not overexpression replaces E2 action in P4-dependent UL
show detectable growth in response to E2P4 as tissue growth.
Because coadministration of E2 with P4 was essential
xenografts (Table 1). However, when cell grafts were genfor
growth and maintenance, inhibition of ER should also
erated from these same UL tissues, the volume of all three
cases increased significantly in response to E2P4 treat- be an effective treatment for UL. However, efficacy of
ment (Table 1 and Fig. 7). The overall rate of growth- selective ER modulators in clinical trials for UL has been
positive tumors in the entire experiment improved from inconsistent (46). This may be due to the technical diffi48.2% (14 of 29) in tissue grafts to 76.4% (13 of 17) in culty in blockade of ER signaling in vivo (47). In addition,
cell grafts. This result further confirmed that the ab- our study suggests that E2 is required only for up-regulasence of growth as tissue xenograft was mostly due to tion of PR, which should be achieved with a relatively low

Endocrinology, June 2010, 151(6):24332442

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2439

systemic inhibition of E2/ER by antiestrogens may not be a practical option

for treatment of symptomatic ULs.
Previously clinical observations associated with pregnancy have raised
questions about the growth-promoting
effect of P4 on human UL. The major
criticism is a lack of conclusive evidence
for the enlargement of UL during pregnancy despite the elevated systemic P4
level (48 50). Furthermore, epidemiological evidence indicates that parity is
protective, rather than promoting, for
UL development (51, 52). However,
there are several reasons these findings
may not necessarily reflect the role of P4
on ULs. First, the majority of longitudinal studies examining the natural history of UL in pregnancy enrolled patients that were found to have UL on
their obstetrical ultrasounds after pregnancy was well established. It is possible that early exposure to elevated P4
levels induced UL growth that was not
detected given the timing of enrollment of the subjects. This is supported
FIG. 6. Gross appearance and the histology of UL cell xenografts. Xenografts constructed
by the fact that when ULs do increase
with 106 dissociated leiomyoma cells formed tumors with typical histology of UL. Expression
in size during pregnancy, the majority
of ER and PR was also comparable with that of tissue xenografts (Fig. 3). All histology
images were taken under a 20 objective lens and in the same magnification.
of growth occurs by the 10th week of
gestation (49). Furthermore, pregnancy-associated stretching and hypertrolevel of E2. In reality, effective blockade of E2/ER signaling
phy of myometrium may affect tumor volume. Lastly,
is difficult to achieve and should cause severe adverse side
pregnancy physiology affects multiple systems including
effects such as hot flashes and osteoporosis. Hence, the
immunological and vascular systems within the uterus
(53). In total, multiple factors likely
contribute to UL size in pregnancy, and
the phenotype of UL in pregnancy is not
necessarily representative of increased
systemic E2 and P4. Indeed, data on miscarriage or induced abortion suggest
that the protective effects of parity for
UL may be associated with an event at
delivery or during the postpartum process (52, 54 56). Therefore, the absence of detectable growth in ULs durFIG. 7. Growth of UL cell xenografts. OVX, OVX control (no hormone); E, E2 treated; P, P4
treated; EP, E2P4 treated; RU, E2P4RU486 treated groups. UL cell xenografts were
ing pregnancy does not necessarily
grown in OVX mouse host treated with E2 (E), P4 (P), E2P4 (EP), or E2P4RU486 (RU) for
contradict with our findings of UL P4
8 wk. As assessed by tumor volume (A), Ki67 labeling index (B), and cell density (C), the
dependency.
response of UL cell xenografts to E2 and P4 was essentially identical with that of tissue
xenografts (Fig. 2). Xenografts increased their size in response to E2 plus P4 treatment (A).
Our study also clearly demonstrated
E2P4 also increased ki67 labeling index (B) and decreased cell density (C). This growththat E2 was not mitogenic for human
promoting effect of E2 plus P4 treatment was blocked by RU486. E2, P4 alone had no effect
ULs in vivo. In contrast, studies with
on the growth of UL cell grafts. The bars indicate average value SEM. Statistical differences
cell culture and animal models repeatwere assessed by using the Kruskal-Wallis test (n 6, P 0.05).

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TABLE 1. Efficacy comparison between tissue and cell grafts

Donor

Xenograft size (mm3)

Original UL

Case no.

Age (yr)

Phase

Nodule size (cm)

Cell density

Tissue graft

Cell graft

128
130
135

31
34
37

Follicular
Follicular
Luteal

12 12 10
866
886

513
901
373

Growth negative (0.5)

4.3
Growth negative (0.5)

2.4
9.6
1.5

edly demonstrated mitogenic effects of estrogens on UL

cells (4 14). Therefore, estrogen/ER signal transduction
in cell culture and animal models should significantly differ from that in human UL in vivo. In the case of UL cell
culture, the reduced levels of ER and PR may be the
reason for the aberrant actions of E2 and P4 in vitro (26).
The difference in hormone responsiveness between human
and rodent leiomyoma may reflect differences in the intrinsic growth control mechanism of human and rodent
myometrial cells. Previously we demonstrated fundamental differences of human vs. mouse endometrial epithelial
cells in the estrogen-regulated proliferation by xenograft
experiments (37). Considering fundamental differences in
cellular kinetics of menstrual vs. estrous cycles and the
duration of cycles, growth control of uterine cells should
be significantly different in human vs. rodent. Rodent UL
models may have some limitation in the studies of growth
control of human UL.
Within the clinical realm, tumor growth can be assessed
only by gross volume change. However, the volume of UL
reflects multiple factors such as cell number/size, ECM,
and water content. Therefore, tumor growth is not necessarily due to the proliferation of UL cells. To envision
therapeutic strategies, it is essential to understand how
ovarian steroids regulate the tumor volume. For example,
if tumors grow solely by accumulation of ECM, inhibition
of cell proliferation should not be an effective treatment.
The xenograft model of human UL serves as an ideal tool
to dissect the mechanism of tumor growth. Our results
indicate that the enlargement of UL xenografts involved
cell proliferation, hypertrophy of UL cells, and volume
increase in extracellular component, all of which were
stimulated by E2P4. Whereas Ki67 labeling index of UL
xenografts significantly declined, the rate of tumor growth
by volume appeared to increase as the tumor grew. It is
quite possible that efficiency in the exchange of oxygen,
nutrients, and waste products between UL cells and blood
reduces as the ECM thickens, and in turn ECM negatively
affects proliferation activity of UL cells. In such a case,
inhibition of ECM accumulation may stimulate proliferation of UL cells by improving the molecular exchange
efficiency between UL cells and blood. Parallel to the
growth, shrinkage of UL also appeared to be primarily via
the reduction in the cell size and extracellular components

but not the loss of leiomyoma cells. Therefore, inhibition

of PR is only a temporary suppression of tumor and not a
cure for UL. It agrees with the clinical observation that
regrowth of shrunken UL occurs slowly after cessation of
RU486 treatment for premenopausal woman (57).
To improve the efficiency of UL xenograft growth, we
developed the cell graft system in which UL tissues were
reconstructed with dissociated single UL cells. The phenotype and hormone responsiveness of cell grafts were
identical with those of tissue xenografts. The advantages
of cell graft over tissue graft were reproducibility of cell
number in each xenograft and improved success rates in
xenograft growth. In addition, transgenic human ULs can
be generated from UL cells transduced with genes of interest. In total, the cell graft model is a novel and relevant
tool for basic research on UL growth. The knowledge
gained from these studies is critical if effective treatments
are to be discovered.

Acknowledgments
The authors thank Dr. Emily Su (Northwestern University) for
critical reading of the paper.
Address all correspondence and requests for reprints to:
Takeshi Kurita, Ph.D., Division of Reproductive Biology Research, Northwestern University Feinberg School of Medicine,
Department of Obstetrics and Gynecology, 4th Floor, Suite
4-127, 303 East Superior Street, Chicago, Illinois 60611. E-mail:
t-kurita@northwestern.edu.
This work was supported by Friends of Prentice and The
National Institutes of Health Grant HD057877.
Disclosure Summary: H.I., K.I., V.A.S., R.K., and T.K. have
nothing to declare. S.E.B. has served on the advisory board for
Repros Inc., Meditrina Inc., and Novartis Inc.

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