Journal of Cancer Therapy, 2012, 3, 352-358
http://dx.doi.org/10.4236/jct.2012.324046 Published Online September 2012 (http://www.SciRP.org/journal/jct)
Estrogen Receptor β of Host Promotes the Progres sion of
Lung Cancer Brain Metastasis of an Orthotopic Mouse
Model
Lei Xu1, Guang Gao1, Jiangong Ren1, Fei Su2, Weihua Zhang1*
1Department of Biology and Biochemistry, University of Houston, Houston, USA; 2Center for Nuclear Receptors and Cell Signaling,
College of Natural Sciences and Mathematics, University of Houston, Houston, USA.
Received July 19th, 2012; revised August 23rd, 2012; accepted September 8th, 2012
ABSTRACT
Estrogen receptors (ERα and ERβ) in the brain play critical roles in maintaining brain tissue homeostasis and in tissue
repair after injury. Growth of cancer metastasis in the brain is a constant damaging process. The role of ERs of the host
in the progression of cancer brain metastasis is unknown. To determine the role of ERβ of host in the progression of
lung cancer brain metastasis, we used an isogenic murine lung cancer cell line, Lewis lung carcinoma cells (3LL), to
produce orthotopic lung cancer brain metastases in wild type and ERβ knockout (ERβ−/−) mice. In the wild type mice,
we found that ERα and ERβ appeared in the tumor associated reactive astrocytes at 24 hr after injection of tumor cells,
and ERβ remained thereafter while ERα disappeared after 1 week. The metastasis bearing ERβ−/− mice survived sig-
nificantly longer than the wild type mice. To further test the role of ERβ of reactive astrocytes in the survival of cancer
cells, we knocked down ERβ in cultured actrocytes using shRNA and performed 3D co-culture with 3LL cells in the
presence/absence of chemotherapeutic agents, oxaliplatin and 5-fluorouracil. We found that loss of ERβ in astrocytes
significantly reduced the survivability of 3LL cells co-cultured with astrocytes. It is concluded that ERβ of host, espe-
cially ERβ in reactive astrocytes, promotes the progression of lung cancer brain metastasis and ERβ might be a potential
therapeutic target for lung cancer brain metastasis.
Keywords: Lung Cancer; Brain Metastasis; Reactive Astrocytes; Estrogen Receptor β
1. Introduction
The development of brain metastasis in lung cancer pa-
tients is highly fatal. In the USA, 50% - 60% of lung
cancer patients will develop brain metastasis [1]. The
median survival of these patients is 2 - 3 months and ag-
gressive radiotherapy combined with chemotherapy may
prolong it to a median of 6 - 8 months [2]. Traditionally
the resistance of brain metastasis to chemotherapy has
been attributed to the blood-brain-barrier (BBB) [3].
However, it has been found that tumor cells in brain me-
tastasis produce VEGF/VPF which renders the BBB
permeable [4]. There are two other non-mutually exclu-
sive mechanisms that may be responsible for the chemo-
resistance of brain metastases. One is that the cancer
cells that can grow in the brain environment are a se-
lected subpopulation of cells that are chemoresistant; the
other possibility is that the brain microenvironment con-
fers drug resistance to tumor cells.
In the brain parenchyma, there are three major popula-
tions of cells, vascular cells, neuronal cells and glial cells.
The growth of brain metastasis is a process of combina-
tion of tissue destruction induced by invading tumor cells
and reactive alterations occurring around the metastases,
which results in a series of pathological microenviron-
mental changes. One of the most dramatic reactions in
the peritumoral region of brain metastasis is astrocytosis
[5], an increase in the number of reactive astrocytes due
to the destruction of nearby brain tissue. The physiologi-
cal function of reactive astrocytes is to repair/rescue da-
maged tissues by providing pro-survival inputs and scar
formation [6]. A tumor, “the wound that never heals” [7],
growing in the brain induces astrocytosis [8,9] similar to
other types of physical injury [10,11]. A pro-survival role
of reactive astrocytes to brain metastasis, as they have
with regard to the neurons, has been proposed [12].
Estrogen is neuroprotective and facilitates neuron re-
covery from injury [13]. It has been found that astrocytes
are the major mediator of estrogen’s neuroprotective ef-
fect [14]. The genomic functions of estrogen are medi-
ated by its receptors, estrogen receptor α (ERα) and
*Corresponding author.

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis
of an Orthotopic Mouse Model
353
(ERβ). Neither ERα nor ERβ can be detected by immu-
nohistochemistry in astrocytes of intact brain, however,
both receptors are significantly upregulated in the reac-
tive astrocytes following brain injuries [15,16], and both
receptors are required to carry out estrogen’s protective
effect [17].
In this study, we used an isogenic murine lung cancer
cell line, 3LL, to produce orthotopic brain metastases in
the brains wild type (ERβ+/+) and ERβ knockout (ERβ−/−)
male mice, and investigated the role of ERβ of host in the
progression of brain metastasis of lung cancer. In addi-
tion, we also determined the effects of ERβ of astrocytes
on the survival of co-cultured 3LL cells.
2. Materials and Methods
2.1. Antibodies
Goat polyclonal antibody against glial fibrillary acidic
protein (GFAP) was from Santa Cruz. Homemade chic-
ken anti-ERβ antibody, ERβ503, and rabbit anti-ERβ
antibody, ERβ-LBD, were described previously [18,19].
Rabbit polyclonal antibody against ERα was from San-
taCruz (#MC20). Horseradish peroxidase conjugated and
alkaline phosphatase conjutated secondary antibodies
were from Sigma-Aldrich.
2.2. Cells and Cell Culture
Murine cell line, 3LL (Cat. CRL-1642), and astrocytes
(Cat. CRL-2534) were purchased from American Type
of Cell Culture (ATCC). Cells were cultured in Dul-
becco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS) in an incubator at 37˚C
supplied with 5% CO2. For co-culturing 3LL cells and
astrocytes, we first stably transfected green fluorescent
protein (GFP) in to the 3LL cells, which allows us to di-
fferentiate cancer cells (GFP positive) from the co-cul-
tured astrocytes (GFP negative). The 3D cell co-culture
was performed with a seeding ratio of 3LL cells/astro-
cytes at 3:1 using the n3D cell culture system from
Nano3D Biosciences (Houston, TX) according to the
protocol provided by the manufacturer.
2.3. Knocking down ERβ by shRNA
The U6 promoter-driven shRNA expression vector
(pRNAT-U6.1/Neo) from Genscript (Piscataway, NJ)
was used to construct vectors expressing ERβ shRNA or
scrambled control shRNA. The target sequence for ERβ
was GTCCGCCTCTTGGAAAGCT, and the control se-
quence was GAACAATGTTGACCAGGTGA. Before
co-culturing with 3LL cells, asctrocytes were transfected
with either control shRNA or ERβ shRNA plasmids for
48 hr using trasfection reagent Genjuice according to the
manufacturer’s instruction (Roche). Western Blot was
used to determine the efficiency of ERβ shRNA.
2.4. Western Blot Assay
Cells were lysed for 30 minutes on ice in RIPA buffer
(Sigma Aldrich, St. Louis, MO) supplemented with pro-
tease and phsophatase inhibitors. The concentrations of
the protein samples were measured using the Qubit
flourometer (Invitrogen, Carlsbad, CA) and equal amounts
of protein samples were loaded onto a 10% SDS-PAGE
gel and transferred to a PVDF membrane. Membranes
were incubated in 5% milk to block the non specific
binding sites for 30 minutes and incubated with opti-
mized concentrations of primary antibody ERβ-LBD at
1:1000 dilutions at 4˚C overnight. After washing with 3×
PBS, membranes were incubated with horseradish per-
oxidase conjugated secondary antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA) at 1:3000 dilutions for 1
hour at room temperature. Luminescent signals were
detected using enhanced luminescence kit (Pierce Ther-
moScientific, Rockford, IL) and exposed to X-ray films
(VWR, Bridgeport, NJ).
2.5. Cell Survival Assay
Co-cultured 3LL cells and astrocytes were treated with
5-fluorouracil (5-FU, 30 µg/ml) or oxaliplatin (50 µg/ml)
the 24 hr after the establishment of 3D co-culture. Con-
trol cells were treated with an equal volume of vehicle.
The treatments lasted for 12 hr before cells were har-
vested for trypan blue uptake assay. The dead cancer
cells were identified by dual positivity of trypan blue and
GFP signals under an inverted fluorescent microscope.
Cell viability was estimated basing on the percentage of
GFP positive dead cells from triplicate samples of each
experimental group.
2.6. Animals and Intracarotid Artery Injection
of Tumor Cells
Wild type and heterozygous of ERβ knockout mice
(ERβ+/−) were purchased from the Jackson Laboratory
(Bar Harbor, Maine). ERβ−/− mice were produced by
breeding the ER+/− pairs at the animal facility of Univer-
sity of Houston. Male mice of wild type and ERβ−/− at the
age of 3 months were used for injection of 3LL cells via
the intracarotid artery into the brain according the proto-
col described previously [20]. In brief, Mice were anes-
thetized by injection (i.p.) of a mixture of ketamine (100
mg/kg)/xylazine (5 mg/kg), washed with 70% alcohol,
and restrained to a cork board in a back position and
placed under a dissecting microscope. The head of ani-
mal was stabilized with a rubber band placed between

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis
of an Orthotopic Mouse Model
354
the teeth of the upper jaw. The hair over the trachea is
shaved, the neck washed with 70% alcohol and the skin
cut by a mediolateral incision. After blunt dissection, the
trachea was exposed and the muscles were separated to
expose the carotid artery which was then separated from
the vagal nerve. The artery was prepared for injection at
a point distal to the point of division into the internal and
external carotid arteries. A ligature of 5 - 0 silk suture
was placed and tied in a position proximal to the injec-
tion site. The artery was nicked with a pair microscissors
and a plastic cannula was inserted into the blood vessel
lumen. The cells (250,000 cells in 20 µl) were injected
slowly. The cannula was removed, and the distal ligature
was tightened and the skin was closed by a tissue clamp.
2.7. Immunohistochemistry
For immunoperoxidase staining with diaminobenzidine
(DAB) labeling, tissue sections were deparaffinized in
xylene and rehydrated in a graded series of alcohol and
PBS. Antigen retrieval was performed using heated cit-
rate buffer. Endogenous peroxidase activity was blocked
with 3% hydrogen peroxide in methanol. Samples were
incubated in a blocking solution (5% donkey serum in
PBS) for 1 hour at room temperature and then overnight
at 4˚C with the primary antibodies diluted in the blocking
solution (ERα antibody was diluted at 1:200; ERβ503
antibody was diluted at 1:50; GFAP antibody was diluted
at 1:200). After three washes in PBS, samples were in-
cubated with a biotinylated goat anti-rabbit secondary
antibody (1:500) for 1 hour at room temperature and then
washed thoroughly. An ABC staining kit was used for
chromogenesis. Slides were then briefly counterstained
with hematoxylin and mounted. For double staining us-
ing horseradish peroxidase conjugated (1:500) and alka-
line phosphatase conjutated (1:500) secondary antibodies,
slides incubated with a mixture of primary antibodies of
either ERα (MC-20, rabbit origin)/GFAP (goat origin) or
ERβ503 (chicken origin)/GFAP (goat origin) were washed
with PBS and subsequently incubated with a mixture of
respective secondary antibodies. Signal of peroxidase
was first developed, then the slides were washed with
PBS before the development of alkaline phosphatase
signal. Slides were then briefly counterstained with he-
matoxylin and mounted. Staining Images were taken
using a Nikon histology imaging microscope.
2.8. Statistical Analyses
Student t-test with two tails and equal variance was used
to compare the values of two experimental groups and a
P value less than 0.05 is considered as statistically sig-
nificance.
3. Results
3.1. Characterization of Changes of ERα and
ERβ in the Brain during the Development of
Lung Cancer Brain Metastasis
The isogenic murine lung cancer cell line, 3LL (deve-
loped spontaneously in C57/BL6 mice), allows us to
study the pathogenic progression of lung cancer brain
metastasis in immnocompetent mice of the same strain.
To gain insights into the role of ERs of the brain in the
progression of lung cancer brain metastasis, by immuno-
histochemical co-staining, we profiled the expression of
ERα and ERβ in tumor associated reactive astrocytes
(identified by immnohistochemical staining of glial fi-
brillary acidic protein, GFAP) in the brain tissues of wild
type male mice collected at a series of time points after
injection of 3LL cells into the brain via intracarotid ar-
tery injection. Tissues were collected at 24 hr, 1 wk, and
3 wk after tumor injection. As shown in Figure 1, the
tumor associated reactive astrocytes are positive for ERα
and ERβ at the 24 hr time point, ERα disappeared at 1
wk while ERβ remained. These data suggest that both
ERα and ERβ are involved in the early phase of astrocyte
activation and ERβ but not ERα is involved in sustaining
the functions of reactive astrocytes in the process of tu-
mor growth.
3.2. Astrocytic ERβ Is Critical for the
Pro-Survival Function of Astrocytes in Vitro
To further determine the pro-survival role of ERβ in as-
trocytes, we performed survival analyses on 3LL cells
co-cultured with astrocytes with/without knockdown of
ERβ by shRNA. We used Western blot and immunocy-
tochemistry to determine the efficiency of ERβ shRNA.
As shown in Figure 2(a), astrocytes transfected with
Figure 1. Immunochemical double staining of ERα/GFAP
and ERβ/GFAP of tumor associated reactive astrocytes in
the brain of wild type mice. Immunoperoxidase staining
with diaminobenzidine (DAB) labeling (brown color) was
used for ERα and ERβ, and alkaline phosphatase staining
was used for GFAP (pink color). Arrows indicate nuclei of
reactive astrocytes. Arrow heads indicate tumor cells. Bar =
20 µm.

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis
of an Orthotopic Mouse Model
355
Figu re 2. Lo ss of ER β in astrocytes reduced the pro-survival function of astrocytes to tumor cells. (a) Western blot analysis of
ERβ expression in astrocytes trasnfected with control-shRNA or ERβ-shRNA. Beta actin was used as a loading control; (b)
Trypan blue uptake assay of cell viability of GFP labeled 3LL cells co-cultured with astrocytes. Image of normal light (to
visualize trypan blue staining, arrows) and image of fluorescent (to visualize GFP signal) were taken for each field of obser-
vation, and total GFP positive cells (cancer cells) and trypan blue and GFP dual positive cells (double-head arrow) were
counted. The single-head arrow indicates a dead astrocyte (trypan blue positive and GFP negative); (c) Quantification of cell
death of 3LL cells cultured alone and co-cultured with astrocytes treated with control shRNA or ERβ shRNA. Triplicate
samples were used in each group. Asterisk indicates statistically significant between the linked groups (error bars = standard
deviation, P < 0.05).
3.3. Loss of ERβ in the Host Prolonged Lives of
Mice Bearing Lung Cancer Brain Metastasis
ERβ shRNA had significantly lower levels of ERβ as
compared to the control cells. To determine the effect of
astrocytes on cancer cells’ survival, we stably expressed
green fluorescent protein (GFP) in the 3LL cells, which
allows us to distinguish 3LL cells from the co-cultured
GFP negative astrocytes. We transfected astrocytes with
either ERβ-shRNA or control-shRNA 48 hr prior to co-
culture, we then co-cultured 3LL cells with these astro-
cytes using the n3D co-culture system in the presence/
absence of 5-fluorouracil or oxalaplatin for 24 hr. After
treatments, cells were trypsinized, stained with trypan
blue, plated on tissue slides and sealed with cover slides.
Green fluorescent images of GFP signal and normal light
images of trypan blue signal were taken for each field of
observation. Dead cancer cells (positive for both GFP
and trypan blue) and live cancer cells (positive for GFP
only) (Figure 2(b)) were counted in 3 fields of each
sample (there were 3 samples in each group) under an
upright fluorescent microscope. As shown in Figure 2(c),
co-culturing with astrocytes protected cancer cells from
the toxicities of both drugs, and loss of ERβ significantly
reduced the pro-survival effects of astrocytes. These data
suggest that ERβ in the cancer cell associated astrocytes
is pro-survival to the cancer cells.
To determine the role of ERβ of host in the progression
of brain metastasis, we injected tumor cells into the
brains of age matched wild type and ERβ knockout male
(C57/BL6 background). Loss of physical balance was
used as a sign of termination of experiment. When the
survival curves of these mice were compared, it was
found that the ERβ knockout mice survived significantly
longer that the wild type, the median survival time for the
wild type mice was 22 days and for the ERβ knockout
mice was 34 days (Figure 3). These data suggest that
ERβ of the host is involved in promoting the disease
progression of lung cancer brain metastasis.
4. Discussion
Cancer brain metastasis is notoriously resistant to con-
ventional therapies. Increasing attentions have been drawn
to the contribution of brain microenvironment. Studies
have found that reactive astrocytes are pro-survival to
tumor cells [21,22]. The molecular mechanisms by which
reactive astrocytes provide tumor cells with survival
support are being revealed. It has been found that physi-

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis
of an Orthotopic Mouse Model
356
Figure 3. Brain metastases bearing ERβ knockout mice
survived longer than the wild type mice. The median sur-
vival time of wild type mice (n = 4) was 22 days, and the
median survival time of the ERβ knockout mice (n = 3) was
34 days. Asterisk indicates statistically significant between
the linked groups (error bars = standard deviation, P <
0.007).
cal contact between tumor cells and reactive astrocytes is
required for astrocytes to provide survival support to
tumor cells [21,22]. Targetable mechanisms are highly
desired. Our study, for the first time, found that ERβ, a
steroid hormone nuclear receptor, plays a key role in the
pro-survival functions of tumor associated reactive as-
trocytes.
The differential upregulation of ERα and ERβ in the
tumor associated astrocytes suggest that ERα and ERβ
plays different roles in the biology of tumor associated
reactive astrocytes. The quick up-regulation of ERα and
ERβ after the inoculation of tumor cells indicates that
both receptors are involved in the initiation process of
astrocyte activation. The disappearance of ERα and con-
tinuous expression of ERβ after 1 wk of inoculation of
tumor cells suggest that reactive astrocytes undergo a
significant genetic reprogram process in response to the
invasion of cancer cells to the brain. The continuous pat-
tern of ERβ expression in the tumor associated reactive
astrocytes indicates that ERβ is involved in maintaining
the function of astrocyte at its reactive status, which is
supported by the in vitro cell co-culture data that loss of
ERβ significantly reduced the pro-survival function of
astrocytes to tumor cells (Figure 2). The chemoprotec-
tive function of astrocytes has been demonstrated in
various melanoma cells, breast cancer cells and lung can-
cer cells [23]. Establishment of gap junctions between
cancer cells and astrocytes is required for astrocytes to
provide cancer cells with survival support [22]. Although
the exact molecular mechanisms by which astrocytes
offer tumor cells with chemoresistant strength remain to
be investigated, clues can be found from what is known
about how reactive astrocytes protect neurons from in-
sults [24,25]. Studies have found three major mecha-
nisms by which reactive astrocytes provide neurons in
damage with survival support, increased release of neu-
rotrophic factors, increased release of glutathione, and
enhanced energetic supply [10]. Reactive astrocytes se-
crete fibroblast growth factor 2 [26] and nerve growth
factor [27], and glutathione promotes cell survival by
reducing cellular oxidative stress. These secreted pro-
survival factors do not depend on cell-cell contact to exe-
cute their functions. The enhanced energetic metabolism
made in the reactive astrocytes deserves more attention
from a cancer brain metastasis point of view. Reactive
astrocytes have increased activities of fatty acid β-oxi-
dation, glycolysis, pentose phosphate pathway and Krebs
cycle [28,29]. These enhanced metabolisms generate
increased amounts of intermediate metabolites that could
be channeled to the neighboring cells in danger through
gap junctions [30], which shall also increase the meta-
bolic efficiency the neighboring cells to produce suffi-
cient amount of ATP for sustaining survival in a hostile
environment. Considering the death of cancer cells is
also determined by the intracellular ATP levels [31] and
the necessity of gap junctions between tumor cells and
astrocytes to increase tumor cells’ survival, the enhanced
energetic supply from reactive astrocytes to tumor cells
might be a major pro-survival mechanism offered by
reactive astrocytes.
The orthotopic brain metastasis model used in this
study, inoculating isogenic cancer cells into immuno-
competent gene knockout animals, is novel at two folds.
First, it overcomes the disadvantage of excluding the
involvement of immune systems of immunodeficient ani-
mal models, such as SCID (severe combined immunode-
ficiency) or nude mice; second, it allows the study of the
impact of a particular host gene on tumor progression.
The ERβ knockout mice used in this study is not tissue
specific, we can’t rule out the functions of ERβ in the
non-astrocytic cells are also involved in promoting the
progression of brain metastasis. Astrocyte specific ERβ
knockout is needed to conclusively define the role of
astrocytic ERβ in the progression of lung cancer brain
metastasis. However, one of the advantages of using the
whole body ERβ knockout mice to test the impact of host
ERβ on tumor progression is that this model is more
therapeutic relevant, i.e. if ERβ was proven to be a the-
rapeutic target for cancer brain metastasis, an ERβ spe-
cific ligand that can inhibit function of ERβ in reactive
astrocytes can be administrated systemically.
5. Acknowledgements
This study was supported by a startup grant from the
University of Houston to Z. W. Z. W. is also supported

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis
of an Orthotopic Mouse Model
357
by grants from the American Cancer Society and the De-
partment of Defense Prostate Cancer Research Program.
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