U.S. patent application number 12/174934 was filed with the patent office on 2009-02-19 for non-human animal exhibiting bone metastasis of tumor cells.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Toyokazu Miki, Saburo SONE.
Application Number | 20090049561 12/174934 |
Document ID | / |
Family ID | 18987131 |
Filed Date | 2009-02-19 |
United States Patent
Application |
20090049561 |
Kind Code |
A1 |
SONE; Saburo ; et
al. |
February 19, 2009 |
NON-HUMAN ANIMAL EXHIBITING BONE METASTASIS OF TUMOR CELLS
Abstract
The present invention provides a non-human bone metastasis model
animal in which tumor cells capable of inducing bone metastasis by
peripheral administration have been introduced, and a method for
producing the animal. The bone metastasis model animal in the
present invention may be useful for understanding the biology of
bone metastasis and developing novel therapeutic strategies for
lung cancer patient with multi-organ metastases, including bone
metastasis
Inventors: |
SONE; Saburo;
(Tokushima-shi, JP) ; Miki; Toyokazu;
(Tokushima-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
18987131 |
Appl. No.: |
12/174934 |
Filed: |
July 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11120508 |
May 2, 2005 |
7402727 |
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12174934 |
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09864364 |
May 25, 2001 |
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11120508 |
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Current U.S.
Class: |
800/10 ;
800/21 |
Current CPC
Class: |
A61P 35/04 20180101;
A01K 67/0271 20130101; A61P 19/00 20180101 |
Class at
Publication: |
800/10 ;
800/21 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/01 20060101 C12N015/01 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2001 |
JP |
2001-140538 |
Claims
1. A bone metastasis model rodent exhibiting bone metastasis of
tumor cells, wherein a suspension comprising tumor cells has been
introduced by a peripheral administration, wherein the rodent is an
immunodeficient rodent, and the metastasis occurs in the rodent's
own bone.
2. The bone metastasis model rodent according to claim 1, wherein
the animal exhibits multi-organ metastasis of tumor cells.
3. The bone metastasis model rodent according to claim 2, wherein
the multi-organ metastases include metastases to one or more organs
selected from lung, liver, kidney, and lymph node.
4. A method for producing a rodent exhibiting bone metastasis of
tumor cells, comprising: (i) providing an immunodeficient rodent;
and (ii) introducing a suspension comprising tumor cells into the
rodent by a peripheral administration, wherein the metastasis
occurs in the rodent's own bone.
5. The method according to claim 4, wherein the immunodeficient
rodent is produced by inactivating NK cells in the rodent.
6. The method according to claim 4, wherein the immunodeficient
rodent is produced by reducing the number of NK cells in the
rodent.
7. The method according to claim 4, wherein the immunodeficient
rodent is produced by depleting NK cells in the rodent.
8. The method according to claim 4, wherein the immunodeficient
rodent is produced by administering an anti-IL-2 receptor antibody
to the rodent.
9. The method according to claim 8, wherein the antibody is an
anti-IL-2 receptor .beta.-chain antibody.
10. The method according to claim 8, wherein the antibody is
derived from a rodent.
11. The method according to claim 4, wherein the immunodeficient
rodent is produced by administering an asialo-GM1 antibody.
12. The method according to claim 4, wherein the immunodeficient
rodent is produced by irradiating with an X-ray the whole body of
the rodent.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a non-human animal
exhibiting bone metastasis of tumor cells and a method for
producing the animal. Specifically, the present invention relates
to the animal in which tumor cells capable of inducing bone
metastasis by peripheral administration have been introduced, and a
method for producing the animal.
BACKGROUND OP THE INVENTION
[0002] Lung cancer is an increasingly common cause of
cancer-related death in the world, and over 90% of deaths from lung
cancer can be attributed to metastasis (Yano, S. et al., Int. J.
Cancer 67:211-217, 1996). Lung cancer is histologically classified
into two groups, small cell carcinoma (SCLC) and non-small cell
carcinoma (NSCLC), based on different clinical behavior (the rate
of tumor growth, the pattern of tumor progression, the sensitivity
to chemo- and radiotherapy). In both type, however, metastasis to
the multiple organs are frequently observed at the time of the
diagnosis. Bone, following the liver and brain, is the third most
common organs of metastasis in lung cancer patients. In particular,
bone metastasis causes various severe symptoms by inducing
pathological fracture, compression of spinal cord, and
hypercalcemia, and reduces the quality of life of the patients
(Iguchi, H. et al., Cancer Res. 56:4040-4043, 1996). Though
palliative radiotherapy is given to reduce the symptoms for
patients with bone metastasis, novel therapeutic modality is
necessary for these patients to improve their prognosis.
[0003] Recently much attention has been paid to develop bone
metastasis model with human cancer cells in order to understand the
molecular mechanism and to develop therapeutic modality. When
cancer cells were injected through the intracardiac route in
immunodeficient mice, melanoma (Arguello, F. et al., Cancer Res.
48:6876-6881, 1988; Nakai, M. et al., Cancer Res. 52:5395-5399,
1988), breast cancer (Hall, D. G. and Stocia, G., J. Bone Miner.
Res. 9:221-230, 1994; Sasaki, A. et al., Cancer Res. 55:3551-3557,
1995), prostate cancer (Shevrin, D. H. et al., Prostate 19:149-154,
1991), and lung cancer (Iguchi, H. et al., Cancer Res.
56:4040-4043, 1996) have been reported to produce bone
metastasis.
[0004] In these models, tumor cells were inoculated into left
ventricle of the heart and bone metastasis was developed in 75%
recipient mice. However, intracardiac inoculation of tumor cells
does not seem to be technically easy, and is associated with sudden
death to some extent. Therefore, intracardiac injection of tumor
cells imposes a lot of burden on animals, causing an ethical and
economic problems.
SUMMARY OF THE INVENTION
[0005] We have succeeded in producing a bone metastasis model
animal, which can be used in order to elucidate a mechanism of bone
metastasis of tumor cells and develop a useful agents for treating
and preventing bone metastasis, by peripheral administration of
tumor cells.
[0006] We examined the abilities of 8 human lung cancer cell lines
injected intravenously into natural killer (NK)-cell depleted SCID
(severe combined immunodeficient) mice to generate metastatic
nodules in bone and multi-organs, and explored the correlation of
parathyroid hormone related protein (PTHrP) to the bone metastasis.
Though all the cell lines of small cell carcinoma (SBC-5, SBC-3,
SBC-3/ADM, H69, H69/VP) formed metastatic nodules in multiple
organs (liver, kidney, and lymph nodes), only SBC-5 cells
reproducibly developed bone metastases. Squamous cell carcinoma
(RERF-LC-AI) cells metastasized mainly into the liver and kidneys,
whereas adenocarcinoma (PC14, A549) produced colonies mainly in the
lungs. As assessed by X-ray photography, osteolytic bone metastases
produced by SBC-5 cells were detected as early as on day 28, and
all recipient mice developed bone metastasis by day 35. The
expression of PTHrP in 8 cell lines directly correlated with the
formation of bone metastasis. No correlation was observed between
the formation of bone metastasis and expression Other
metastasis-related cytokines (IL-1, IL-6, IL-8, IL-10, IL-11,
TNF-.alpha., VEGF, M-CSF). Consistent with the formation of bone
metastasis by SBC-5 cells, the levels of PTHrP and calcium in the
mouse serum were increased in a time-dependent manner, suggesting
that PTHrP produced by human lung cancer may play a crucial role in
the formation of bone metastasis and hypercalcemia. These results
indicate that bone metastasis model of SBC-5 cells may be useful
for clarifying the molecular aspects of metastatic process under
the different organ microenvironments and developing therapeutic
modalities for lung cancer patients with bone metastases.
[0007] Therefore, the present invention provides the following (1)
to (25):
(1) A non-human bone metastasis model animal exhibiting bone
metastasis of tumor cells, in which tumor cells capable of inducing
bone metastasis have been introduced by peripheral administration.
(2) The non-human bone metastasis model animal according to claim
1, wherein the tumor cells are human lung cancer or breast cancer
derived cells highly expressing PTHrP. (3) The non-human bone
metastasis model animal according to claim 1, wherein the tumor
cells are cells from human lung small cell carcinoma. (4) The
non-human bone metastasis model animal according to claim 1, which
exhibits multi-organ metastasis of tumor cells. (5) The non-human
bone metastasis model animal according to claim 4, wherein the
multi-organ metastases include metastases to one or more organs
selected from the group consisting of lung, liver, kidney, and
lymph node. (6) The non-human bone metastasis model animal
according to claim 1, wherein the animal belongs to rodents. (7)
The non-human bone metastasis model animal according to claim 6,
wherein the animal is mouse. (8) The non-human bone metastasis
model animal according to claim 7, wherein the animal is
immunodeficient mouse. (9) The non-human bone metastasis model
animal according to claim 8, wherein the animal is SCID mouse. (10)
A method for producing a non-human animal exhibiting bone
metastasis of tumor cells, comprising the steps of:
[0008] (i) providing a non-human animal having reduced immunity;
and
[0009] (ii) introducing tumor cells capable of inducing bone
metastasis into the animal by peripheral administration.
(11) The method according to claim 10, wherein the tumor cells are
human lung cancer- or breast cancer-derived cells highly expressing
PTHrP. (12) The method according to claim 10, wherein the tumor
cells are cells from human lung small cell carcinoma. (13) The
method according to claim 10, wherein the step of providing a
non-human animal having reduced immunity includes a step of
inactivating NK cells in the animal. (14) The method according to
claim 10, wherein the step of providing a non-human animal having
reduced immunity includes a step of reducing the number of NK cells
in the animal. (15) The method according to claim 10, wherein the
step of providing a non-human animal having reduced immunity
includes a step of depleting NK cells in the animal. (16) The
method according to claim 10, wherein the step of providing a
non-human animal having reduced immunity includes a step of
administering anti-IL-2 receptor antibody to the animal. (17) The
method according to claim 16, wherein the antibody is anti-IL-2
receptor .beta.-chain antibody. (18) The method according to claim
16, wherein the antibody is mouse antibody. (19) The method
according to claim 10, wherein the step of introducing tumor cells
capable of inducing bone metastasis to the animal by peripheral
administration includes a step of injecting the tumor cells into
the animal intravenously. (20) The method according to claim 10,
wherein the animal belongs to rodents. (21) The method according to
claim 10, wherein the animal is mouse. (22) The method according to
claim 21, wherein the animal is an immunodeficient mouse. (23) The
method according to claim 21, wherein the animal is SCID mouse.
(24) A method for evaluating efficiencies of treatment against bone
metastasis of tumor cells, comprising the step of:
[0010] (i) applying a treatment to the non-human bone metastasis
model animal according to any one of claims 1 to 9; and
[0011] (ii) comparing the size and/or extent of bone metastasis,
and/or symptoms resulted from bone metastasis, with control
animal.
(25) Use of the non-human bone metastasis model animal according to
claim to any one of claims 1 to 9 for determining the effect of a
test substance on bone metastasis, comprising the steps of:
[0012] (i) administering the test substance to the animal; and
[0013] (ii) comparing the size and/or extent of bone metastasis,
and/or symptoms resulted from bone metastasis, with control
animal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows X-ray photograph of bone metastasis model mouse
of the present invention. 1A indicates osteolytic bone metastases
induced mainly in the spine (1B) and bone of extremities (1C).
[0015] FIG. 2 shows histological analysis of bone metastasis. Bone
metastatic lesions consist of tumor cells with multi-nucleated
cells (2A and B).
[0016] FIG. 3 shows mRNA expression of PTHrP and IL-11 in various
tumor cells.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Formation of bone metastasis has been reported to involve
various soluble mediators such as cytokines and parathyroid hormone
related protein (PTHrP), which was originally identified as a 17
kDa PTH-like adenylate cyclase-stimulating protein from a tumor
associated with humoral hypercalcemia of malignancy (HHM)(Suva, L.
J. et al., Science 237:893-896, 1987; Broadus, A. E. et al, N.
Engl. J. Med. 319:556-63, 1988). Interestingly, a variety of tumor
cells, including breast cancer, prostate cancer and lung cancer
overexpress PTHrP (Burtis, W. J. et al., N. Engl. J. Med.
322:1106-12, 1990). Recently, a highly bone metastatic lung cancer
cell line was found to overexpress PTHrP, and that treatment of
nude mice with anti-PTHrP antibody inhibited the formation of bone
metastasis (Iguchi, H. et al., Cancer Res. 56:4040-4043, 1996),
suggesting that PTHrP plays a critical role in the formation of
bone metastasis from human lung cancer.
[0018] Recently we showed that depletion of NK cells by anti-IL-2
receptor .beta.-chain antibody (TM .beta.1) in SCID mice resulted
in formation of multi-organ metastasis (lung, liver, kidney and
lymph nodes) induced by intravenous injections of various human
lung cancer cell lines (Yano S., 2 papers). Since there is no
reports about evidences showing ability of cancer cells injected
intravenously into tail to form bone metastasis in solid tumor
other than those of haemal system, we examined whether bone
metastasis could be produced by 8 different cell lines of human
lung cancer inoculated intravenously in NK-cell depleted SCID mice.
Moreover, we also evaluated the correlation of the expression of
PTHrP and cytokines with the formation of bone metastasis in human
lung cancer cell lines.
[0019] The bone metastasis model animal of the present invention
refers to a non-human animal exhibiting bone metastasis of tumor
cells. In the present invention, the phrase "exhibiting bone
metastasis of tumor cells" means the animal carries one or more
bone metastasis originated from tumor cells which has been
introduced by peripheral administration. Animals which carries
metastasis to other organs such as lung, liver, kidney, lymph node
together with bone metastasis are also included. In the case of
such multi-organ metastasis, effectiveness of the same agents for
bone metastasis and metastasis to other organs can be examined
simultaneously. Although the existence and degrees of bone
metastasis in non-human animals can be detected as a image of bone
absorption (punch-out image) with X-ray photography and/or identify
by means of histological analysis, the means of identification of
bone metastasis is not particularly restricted.
[0020] Tumor cells which can be used in the present invention are
capable of inducing bone metastasis by peripheral administration
and include, for example, lung cancer such as squamous cell
carcinoma, small cell carcinoma, adenocarcinoma, large cell
carcinoma, adenosquamous carcinoma, carcinoid tumor, breast cancer
such as noninfiltrating carcinoma, invasive carcinoma (invasive
galactophore carcinoma, mucus carcinoma, invasive lobular
carcinoma, squamous cell carcinoma and the like); and other cancers
such as kidney cancer, prostate cancer, stomach cancer, liver
cancer, intestinal cancer, pancreas cancer. Tumor cells are
preferably those highly expressing PTHrP. The term "highly
expressing" means herein that PTHrP can be detected at a certain
concentration or higher in cell culture supernatant, in particular,
in the case of measuring PTHrP C-terminus, that PTHrP can be
detected in the concentration higher than that detected in normal
individuals. The above term also means that PTHrP can be detected
at a certain concentration or higher in blood of non-human animals
such as a nude mouse which is implanted with the tumor. For
example, the cases where 1.1 pmol/L or higher PTHrP is detected in
measuring PTHrP-N terminus (for example, Mitsubishi kagaku
Bio-chemical Laboratory Inc.), and where 55.3 pmol/L or higher
PTHrP is detected in measuring PTHrP-C terminus (for example, SRL,
Inc.) are included in the situation. Further, the term includes the
case where 55.3 pmol/L or higher, preferably 100 pmol/L or higher,
more preferably 300 pmol/L or higher, yet more preferably 499
pmol/L or higher PTHrP is detected when measuring PTHrP
(C-terminus) in cell culture supernatant according to the method
described in the Examples below. In the present invention, human
SCLC cell line can be used as particularly suitable cells. Although
the human SCLC cell lines SBC-3 and SBC-5 cells which are
preferably used were kindly provided by Dr. K-Hiraki (Okayama
University, Okayama, Japan), they can also be commercially
available from Human Science Research Resources Bank (Osaka,
Japan). The multi-drug resistant SCLC cell line SBC-3/ADM was
established as previously reported (Nakamura, K. et al., Cancer
Res. 59:5323-5330, 1999). The human lung squamous cell carcinoma
RERF-LC-AI cells were kindly provided by Dr. Akiyama (Radiation
Effects Research Foundation, Hiroshima, Japan), but they can also
be commercially available from RIKEN Cell Bank (Ibaragi, Japan).
The human lung adenocarcinoma PC-14 cells, human SCLC cells H69 and
its etoposide-resistant variant, H69/VP, were kindly provided by
Dr. N. Saijo (National Cancer Center, Tokyo, Japan). The human lung
adenocarcinoma PC-14 cells can also be commercially available from
RIKEN Cell Bank (Ibaragi, Japan), and human SCLC cells H69 are
commercially available from ATCC (American Type Culture
Collection).
[0021] The method for culturing tumor cells may be any methods
commonly used in the art, and is not particularly limited. For
example, SBC-3, SBC-3/ADM and SBC-5 cells can be maintained in
Eagle MEM supplemented with 10% heat-inactivated fetal bovine serum
(GIBCO, Grand Island, N.Y.) and gentamicin (Schering-Plough, Osaka,
Japan) and 4 mM HEPES. The RERF-LC-AI, PC-14, H69 and H69/VP cells
can be maintained in RPMI 1640 supplemented with 10%
heat-inactivated fetal bovine scrum (CRPMI 1640) and gentamicin.
All cell lines are preferably incubated at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 in air.
[0022] Cells for implanting into animals can be prepared as
follows. Tumor cells harvested are washed, for example, with
Ca.sup.2+- and Mg.sup.2+-free phosphate buffered saline (CMF-PBS).
Cell viability can be determined by trypan blue exclusion test, and
only single cell suspensions of > 90% viability are preferably
used.
[0023] In the present invention, non-human animals include
vertebrate, specifically mammals such as mouse, rat, hamster,
guinea pig, rabbit, pig, miniature pig, sheep, cat, and dog, birds
such as chicken, and fishes, but any animals other than human are
included without any restriction. Animals which belong to rodents,
for example, mouse, rat, hamster, and the like, are preferably used
in the present invention, and mouse is most preferably used, due to
the ease of breeding and manipulation. Any kinds of mouse which are
used in the art can be suitably used. For example, Male
CB-17/Icr-scid mice, ages 6-8 weeks, can be obtained from Charles
River Japan, Inc. (Yokohama, Japan) and maintained under specific
pathogen-free conditions throughout the experiment.
[0024] The bone metastasis model animal of the present invention
can be produced from normal animals. However, more suitable results
can be obtained where immunodeficient animals are used.
Immunodeficient animals may be obtained or produced by way of known
means in the art. For example, SCID mouse can be commercially
available from CLEA Japan Inc. (Tokyo, Japan) or Charles River
Japan Inc. (Yokohama, Japan).
[0025] The term "peripheral administration" means herein parenteral
administration route by which agents are administered directly into
the body, and includes intravenous, intramuscle, intracutaneous,
subcutaneous, and intraperitoneal administrations, and does not
include percutaneous, inhalation, intracerebral, and ophthalmic
administrations.
[0026] The bone metastasis model animal of the present invention
can be produced, for example, according a process comprising
following steps:
(i) The Step of Providing a Non-Human Animal having Reduced
Immunity
[0027] When implanting heterologous (tumor) cells into animals, it
is necessary to reduce host immunity. Generally, nude mouse which
is deficient of T cell function due to lack of thymus, or SCID
mouse which is deficient of B cell function as well as T cell
function, is used as a immunodeficient model for implanting tumor.
However, NK cell functions in these models, and NK cell
inactivation is required in order to assure a good take of tumor.
In that case, asialo-GM1 antibody or TM .beta. 1 treatment employed
herein is preferably used. Another method may be that of producing
immune-suppressed mouse by irradiating X-ray to the whole body of a
mouse.
[0028] Immunodeficient animal of the present invention includes
commonly available nude mouse, SCID mouse, a mouse obtained by
administration of asialo-GM1 antibody or TM .beta. 1 into nude
mouse or SCID mouse, and X-ray irradiated mouse, and the like.
[0029] In order to facilitate the metastasis of human lung cancer
cell lines, natural killer (NK) cells may preferably be depleted in
SCID mice. The depletion of NK cells may be carried out, for
example, by treatment of SCID mice with anti-IL-2 receptor
antibody, especially anti-IL-2 receptor .beta.-chain antibody (TM
.beta. 1). Antibody can be derived from any kinds of animals
including human, and mouse antibody is particularly preferable.
Although anti-mouse IL-2 receptor .beta. chain monoclonal Ab,
TM-.beta. 1 (IgG2b), was kindly supplied by Drs. M. Miyasaka and T.
Tanaka (Osaka University, Osaka, Japan), it can be produced
according to the method described in Japanese Patent No. 3040451
(Tanaka, T. et al., J. Exp. Med. 178 1103-1107, 1993). For example,
TM-.beta. 1 Ab (1 mg/1 ml PBS/mouse) may be injected i.p. into SCID
mice 2 days before tumor inoculation (Yano, S. et al., Int. J.
Cancer 67:211-217, 1996).
(ii) The Step of Introducing Tumor Cells Capable of Inducing Bone
Metastasis into the Non-Human Animal by Peripheral
Administration
[0030] Introduction of tumor cells by administration can be carried
out, preferably by injection, more preferably by intravenous
injection. Injection may be performed with 0.3 ml of tumor cells
(approximately 1-5.times.10.sup.6) into the lateral tail vein of
unanesthetized SCID mice pretreated with TM-.beta. 1 Ab. After the
indicated periods, bone metastasis can be evaluated by X-ray
photography (Fuji Film, Tokyo, Japan). To determine the visceral
metastases, the mice are sacrificed and the number of metastatic
foci on the lungs, liver, kidneys and lymph nodes are counted
macroscopically.
[0031] For histo-pathological study, the major organs of the animal
harvested may be fixed in 10% phosphate-buffered formalin,
sectioned and stained with hematoxylin and eosin by standard
procedure.
[0032] The present invention also provides a method for evaluating
efficiencies of treatment against bone metastasis of tumor cells,
comprising the step of:
[0033] (i) applying a treatment to the non-human bone metastasis
model animal according to any one of claims 1 to 9; and
[0034] (ii) comparing the size and/or extent of bone metastasis,
and/or symptoms resulted from bone metastasis, with control
animal.
[0035] The term "treatment" includes treatment by some agents and
radiotherapy, and also includes prevention (inhibition of
enlargement) of bone metastasis. When a treatment is effective,
contraction or disappearance, or inhibition of enlargement of bone
metastasis can be observed, and relief of some symptoms resulted
from bone metastasis, such as paralysis, can also be observed. The
term "control animal" means an animal under the same condition as
the animal applied the treatment, except for without the treatment.
Alternatively, the control animal may be the same animal before the
treatment.
[0036] Further, the present invention provides use of the non-human
bone metastasis model animal according to claim to any one of
claims 1 to 9 for determining the effect of a test substance on
bone metastasis, comprising the steps of:
[0037] (i) administering the test substance to the animal; and
[0038] (ii) comparing the size and/or extent of bone metastasis,
and/or symptoms resulted from bone metastasis, with control
animal.
[0039] In the present specification, test substance is not
particularly limited, and include, for example, peptides, proteins,
non-peptide compounds, synthetic compounds, fermentation products,
cell extract, various factors in cells. The administration of test
substance can be carried out by oral or parenteral administration
which are known in the art such as intravenous injection,
subcutaneous injection, and the like, and the route of
administration can be appropriately selected depending on animals
used, the degrees of bone metastasis, and the like. Alternatively,
normal condition can be the condition of administration of a
specified factor in cells, by removing the factor from the cells or
reducing the function of the factor. The term "control animal"
means herein an animal under the same condition as the animal
administered with test substance, except for without the
administration. Alternatively, the control animal may be the same
animal before the administration. The use of the bone metastasis
model animal according to the present invention allows a simple
evaluation of whether the test substance is effective or not.
[0040] As described above, we established a novel bone metastasis
model animal by iv injection of tumor cells. Mice with multiple
bone metastases showed neurological disorders (paraplesia or
monoplesia of hind leg) and hypercalcemia, which are frequently
observed in lung cancer patients with bone metastases, suggesting
that our bone metastasis model seems to reproduce the
characteristics of bone metastasis of lung cancer in humans.
[0041] The advantage of our model over prior art are 1) the
incidence of bone metastases is 100%, 2) the procedure involved is
easy, though immunodeficient animal is preferable to facilitate
metastasis formation, 3) since visceral metastasis is also
reproducibly developed, antimetastatic effect of drugs in various
organs can be evaluated in one model.
[0042] The formation of bone metastasis consists of several steps,
including tumor-cell arrest at capillaries in bone marrow,
extravasation, tumor-cell invasion with bone resorption, and tumor
growth. Therefore, various cytokines that modulate these steps can
facilitate bone metastasis formation. Pro-inflammatory cytokines
(IL-1, IL-6, TNF-.alpha.) up-regulate bone resorption and
expression of adhesion molecules expressed on the capillaries, and
pro-angiogenic cytokines (VEGF and IL-8) can stimulate
angiogenesis. In addition, M-CSF and IL-10 can activate macrophages
and lymphocytes, respectively, can inhibit tumor growth (28-29).
Nevertheless, there was no correlation between the expression of
these cytokines in human lung cancer cells used and the formation
of bone metastasis.
[0043] Recently, it was reported that PTHrP--expression in primary
tumors directly correlated with the incidence of bone metastasis in
breast cancer patients (Powell, G. J. et al. Cancer Res.
51:3059-3061, 1991). In addition, the high level of PTHrP
expression was detected in the bone metastases in the patients with
breast or prostate cancer (Bundred, N. J. et al., Eur. J. Cancer
28:690-692, 1992; Bouizar, Z. et al. Cancer Res. 53:5076-5078,
1993). Consistent with these reports, highly PTHrP expressing cells
had highest potential to produce bone metastasis in NK-cell
depleted SCID mice. Therefore. PTHrP, which stimulates bone
resorption, may play a crucial role on developing bone metastasis
in our model.
[0044] Hypercalcemia is frequently observed in lung cancer
patients. It can be caused by two different mechanisms, namely HHM
and LOH (local osteolytic hypercalcemia) (Burtis, W. J. et al., N.
Engl. J. Med. 322:1106-12, 1990). HHM is mediated via PTHrP
produced by tumor cells. Since PTHrP indirectly stimulates bone
resorption by osteoclasts and augments resorption of calcium in the
kidneys, the calcium level in the serum is elevated. In LOH,
locally produced cytokines (IL-1, IL-6, TNF-.alpha.) by tumor cells
promote proliferation and differentiation of osteoclast-linage
cells, induced bone resorption, and hence cause hypercalcemia
(Mundy, G. R., Bone, 12(supple) S1-S6, 1991; Yoneda, T., M. Noda
(ed.), Cellular and molecular biology of bone, 375-412, New York,
Academic Press, 1993; Jilka, R. L. et al., Science 257:88-91, 1992;
Roodman, G. D., Calcif. Tissue Int. 53, S94-S98, 1993; Tamura, T.
et al., Proc. Natl. Acad. Sci. USA 90:11924-11928, 1993; Kurihara,
N. et al., J. Immunology 144:4226-4230, 1990; Tashjian, Jr A. H. et
al., Proc. Natl. Acad. Sci USA 82:4535-4538, 1985; Oreffo, R. O. et
al., Biochem. Biophys. Res. Commun. 158:817-823, 1989). In the
present study, hypercalcemia was observed in SBC-5-cell inoculated
SCID mice in a time-dependent manner. Tumor cells highly expressed
PTHrP at both protein and mRNA levels, whereas no IL-1, IL-6, or
TNF-.alpha. was detected in the culture supernatants of the cells,
suggesting that hypercalcemia observed in mice inoculated with
these cells may be due to PTHrP produced by these cells (HHM
mechanism).
[0045] Interesting was the present finding that paralysis, a sign
caused by bone metastasis, happened in all the mice inoculated with
SBC-5 cells. Nevertheless, the paralysis was also observed at lower
incidence (10-60%) in mice with bearing SBC-3, SBC-3/ADM, H69, or
H69/VP, but the radiolucent lesions (a hallmark of osteolytic bone
metastasis) could not be detected on X-ray photograph, suggesting
that metastases to the brain or spinal cord might be involved in
these mice.
[0046] The formation of multi-organ metastasis is critical problem
in the management of lung cancer patients. In this study, all 8
human lung cancer cell lines tested developed multiple organ
metastases in NK-cell depleted SCID mice. Interestingly, two
adenocarcinoma cell lines produced metastases mainly in the lungs,
whereas five small cell carcinoma cell lines formed lesions mainly
in the liver, kidneys and systemic lymph nodes, resembling their
clinical behavior. Although we examined the expression of various
cytokines in these cell lines to identify key molecules responsible
for organ specific metastasis, no correlation was observed between
cytokine expression and metastatic potential. Of 8 cell lines
tested, SBC-5, overexpressing PTHrP, most aggressively produced
metastases into multiple visceral organs (such as the lungs, liver,
kidneys and systemic lymph nodes) in addition to bone metastasis.
Recent report showed that PTHrP-gene transfection to human prostate
cancer cells resulted in resistant to apoptotic stimuli and
facilitating tumor growth in vivo (Dougherty, M. K. et al., Cancer
Res. 59:6015-6022, 1999). Therefore, PTHrP might facilitate the
metastasis formation of SBC-5 cells not only to the bone but also
to visceral organs.
[0047] In summary, we established the model for bone metastasis
with dissemination by intravenous injection with tumor cells. Our
model resembles characteristics of small cell lung cancer in
humans, because tumor cells produced multi-organ metastasis with
osteolytic bone leasions causing paralysis and induced
hypercalcemia in recipient mice. In addition, our data suggest that
PTHrP secreted by tumor cells plays a crucial role in the process
of bone metastasis and hypercalcemia. Therefore, our bone
metastasis model may be useful for understanding the biology of
bone metastasis and developing novel therapeutic strategies for
lung cancer patient with multi-organ metastases, including bone
metastasis.
EXAMPLES
[0048] The present invention is further described in detail
hereafter referring to Examples. However, it is intended that the
present invention should not limited in these Examples. Moreover,
none of materials used in the experiments contained endotoxins, as
judged by the limulus amebocyte assay (Seikagaku Kogyo, Tokyo,
Japan: minimum detection level, 0.1 ng/ml).
Example 1
Pattern of Metastasis Produced by Human Lung Cancer Cell Lines in
NK-Cell Depleted SCID Mice
[0049] We examined the pattern of multi-organ metastasis produced
by 8 different human lung cancer cell lines in NK-cell depleted
SCID mice. NK-cell depleted SCID mice were inoculated i.v. through
tail vein into the mice with 1-5.times.10.sup.6 tumor cells, and
were sacrificed on the day after the indicated periods, and the
number of metastatic colonies into the lungs, livers, kidneys and
lymph nodes were counted All recipient mice developed tumor lesion
and many of the mice became morbid by the time of sacrifice (Table
1).
TABLE-US-00001 TABLE 1 Pattern of Metastasis Produced by Human Lung
Cancer Cell Lines in NK-Cell Depleted SCID Mice Day of Number of
Metastasis; median (Range) Cell Line Sacrifice Bone Lungs Liver
Kidneys Lymph Nodes Squamous cell carcinoma RERF-LC-AI.sup.a day 42
All 0 All 0 67(38->100) 19(15-30) 4(0-18) Adenocarcinoma
PC-14.sup.a day 28 All 0 >100 3(1-7) 5(3-13) 1(0-3) A549.sup.a
day 56 All 0 >100 1(0-2) All 0 10(4-14) small cell carcinoma
SBC-5.sup.a day 35 6(4-9) 31(9-38) 58(32-84) 6(3-20) 3(1-4)
SBC-3.sup.a day 42 All 0 All 0 12(7-21) 12(10-17) 13(6-22)
SBC-3/ADM.sup.a day 35 All 0 All 0 54(41-58) 1(0-2) 4(2-7)
H69.sup.b day 56 All 0 All 0 2(1-3) 6(2-12) 32(23-43) H69/VP.sup.b
day 56 All 0 All 0 15(9-16) 19(17-25) 21(10-28) .sup.a1 .times.
10.sup.6 cells were injected. .sup.b5 .times. 10.sup.6 cells were
injected.
[0050] Squamous cell carcinoma (RERF-LC-AI) cells produced
metastatic colonies mainly in the liver and kidneys. Adenocarcinoma
PC-14 and A549 cells formed metastases mainly in the lungs. Four
out of 5 small cell carcinoma cell lines (SBC-3, SBC-3/ADM, H69 and
H69/VP) developed metastases mainly in the liver, kidneys and
systemic lymph nodes. Among these cell lines, only one cell line of
small cell lung cancer (SBC-5) developed metastatic colonies in
bones as well as other organs such as lungs, liver, kidneys and
systemic lymph nodes. Paralysis (paraplesia and monoplesia in the
hind leg) was occasionally observed in the recipient mice bearing
bone metastasis of SBC-5.
Example 2
X-Ray and Histological Analysis of Bone Metastasis Produced by
SBC-5 Cells
[0051] Bone metastases obtained in Example 1 were determined by
X-ray photography. Multiple bone metastases were reproducibly
developed in the mice injected iv with SBC-5 cells and bone
metastasis lesions were detected as radiolucent lesions on X-ray
photograph (FIG. 1A), indicating osteolytic bone metastases mainly
in the spine (FIG. 1B) and bone of extremities (FIG. 1C).
Histological analysis show that these lesions consist of tumor
cells with multi-nucleated cells (FIGS. 2A and B). The mice with
these lesions had paralysis of hind legs and urinary retention with
enlarged bladder, probably due to pathological fracture and/or
compression of spinal cord caused by bone metastasis.
Example 3
Effect of Tumor-Cell Number on Bone Metastasis in NK-Cell Depleted
SCID Mice
[0052] To determine the optimal experimental conditions for bone
metastasis, we injected various numbers of SBC-5 cells into NK-cell
depleted SCID mice. When the mice became moribund, the mice were
sacrificed and the number of bone metastasis was determined by
X-ray photograph. The number of visceral organs was determined
macroscopically. The number of bone metastases, as well as visceral
metastases, depended on the number of cells injected (Table 2).
Based on these results, 1.times.10.sup.6 SBC-5 cells were injected
in subsequent experiments.
TABLE-US-00002 TABLE 2 Pattern of Multiple Organ Metastases
Produced by SBC-5 Cells in NK-Cell Depleted SCID Mice Number of
Days of Bone Lungs Liver Kidneys Lymph Nodes Injection Sacrifice
Inc..sup.a Med.sup.b. Range Inc. Med.sup.b Range Inc. Med.sup.b
Range Inc. Med.sup.b Range Inc. Med.sup.b Range 1 .times. 10.sup.5
121 3/5 3 0-6 0/5 0 All 0 5/5 30 7-45 0/5 0 All 0 2/5 3 0-5 5
.times. 10.sup.5 49 5/5 6 1-8 2/5 3 0-10 5/5 25 14-50 3/5 2 0-4 3/5
2 0-3 1 .times. 10.sup.6 35 5/5 4 2-5 5/5 5 3-18 5/5 47 25-58 5/5 2
1-3 5/5 2 1-3 2 .times. 10.sup.6 35 5/5 4 3-6 5/5 11 9-24 5/5 49
33-84 5/5 9 7-10 5/5 3 2-5 .sup.aInc., incidence; .sup.bMed.,
median.
Example 4
Time Kinetics of Bone and Visceral Metastases Produced by Cells
[0053] We next explored the kinetics of bone metastasis formation
by SBC-5 cells. Twenty-five SCID mice depleted of NK cells were
intravenously injected with 1.times.10.sup.6 SBC-5 cells, and the
formation of bone metastasis was examined by X-ray photography on
days 10, 14, 21, 28 and 35. Four to five mice were sacrificed at
each point to determined visceral organ metastasis. Bone metastasis
was detected as early as on day 28 and was observed in all
recipient mice on day 35 (Table 3). Consistent with bone metastasis
formation, loss of body weight was observed as early as on day 28
(Table 4). On the other hand, metastases to the lung and liver were
developed by day 21, and metastases to the kidneys and lymph nodes
were produced by day 28. On day 35, all mice developed metastatic
lesions into multiple organs, such as the lungs, liver, kidneys and
lymph nodes.
TABLE-US-00003 TABLE 3 Time Kinetics of Multiple Organ Metastases
Produced by SBC-5 Cells in NK-Cell Depleted SCID Mice Days of Bone
Lungs Liver Kidneys Lymph Nodes Sacrifice Inc..sup.a Med.sup.b.
Range Inc. Med Range Inc. Med Range Inc. Med Range Inc. Med Range
10 0/25 0 All 0 0/4 0 All 0 0/4 0 All 0 0/4 0 All 0 0/4 0 All 0 14
0/21 0 All 0 0/4 0 All 0 0/4 0 All 0 0/4 0 All 0 0/4 0 All 0 21
0/17 0 All 0 4/4 18 1-21 4/4 6 5-8 0/4 0 All 0 0/4 0 All 0 28 8/13
2 0-4 5/5 17 8-34 5/5 57 34-87 5/5 5 3-13 2/5 1 0-2 35 8/8 6 4-9
8/8 31 9-38 8/8 58 32-84 8/8 6 3-20 8/8 3 1-4 .sup.aInc.,
incidence; .sup.bMed., median.
TABLE-US-00004 TABLE 4 The Kinetics of Body Weight and Levels of
Calcium and PTHrP of Nk-Cell Depleted SCID Mice Injected with SBC-5
Cells Days of Body Weight(g) Ca (mg/dl) PTHrP (pmol/l) Sacrifice
Med. Range Med. Range Med. Range 10 21.57 20.1-23.8 8.0 8.0-8.2
24.1 <10.0-63.2 14 22.50 21.2-23.8 9.2 9.0-10.4 88.2 26.3-143 21
21.92 21.7-26.0 9.6 9.2-10.5 95.3 <10.0-115 28 18.64 16.6-21.3
9.9 9.0-10.2 299.0 146-527 35 16.00 13.3-18.4 12.0 9.5-15.0 544.3
317-1020
The level of calcium in the mouse serum was determined using
methylxylenol blue as a substrate for luminescence (Ohtsuka
Pharmaceutical Co, Tokushima, Japan).
Example 5
Expression of PTHrP and Metastasis-Related Molecules in Human Lung
Cancer Cell Lines
[0054] PTHrP and IL-11 are reported to play a crucial role in bone
resorption and bone metastasis (20-27). We determined whether
expression of PTHrP and IL-11 in the human lung cancer cell lines
correlated with their potential to produce bone metastasis. Tumor
cells (5.times.10.sup.5) cells were incubated in CRPMI1640 for 24
hr, and the supernatants were harvested. The level of PTHrP In
culture supernatants and mouse serum can be determined using a
radioimmuno assay (Ohtsuka Pharmaceutical Co, Tokushima, Japan).
The levels of cytokines (VEGF, IL-6, IL-8, IL-10, M-CSF) in the
culture supernatants were measured by ELISA (Ohtsuka Pharmaceutical
Co. Tokushima, Japan).
[0055] Polymerase chain reaction (PCR) analysis was carried out as
follows. First-strand cDNA was synthesized from 1 .mu.g total RNA
using a RNA LA PCR Kit (Takara, Tokyo, Japan) in 20 .mu.l of
reaction mixture, according to the manufacture's instructions. The
synthesized first-strand cDNA (20 .mu.l) was amplified by
polymerase chain reaction (PCR) in a final volume of 100 .mu.l
containing 1.times.LA PCR Buffer II, 2.5 mM MgCl.sub.2, 20 pmol of
each primer, and 2.5 U of TaKaRa LA Taq polymerase RNA LA PCR Kit
(Takara, Tokyo, Japan). Sequences of PTHrP primers used were sense
5'-ATG CAG CGG AGA GAC TGG TTC AG-3' (SEQ ID NO: 1) and antisense
5'-TCA ATG CCT CCG TGA ATC GAG CTC CAG AGC CGT-3' (SEQ ID NO: 2)
(Walsh, C. A. et al. Bone and Mineral 27 43-50, 1994). PCR
amplification of PTHrP cDNA was performed under the following
conditions: 30 cycles, 30 sec at 94.degree. C., 30 sec at
58.degree. C., 30 sec at 72.degree. C. Before the first cycle, a
denaturation step for 2 min at 94.degree. C. was included, and
after 30 cycles the extension was prolonged for 7 min at 72.degree.
C. (Walsh, C. A. et al., Bone and Mineral 27 43-50, 1994).
Sequences of IL-11 primers used were sense 5'-ACT GCT GCT GCT GAA
GAC TCG GCT GTG A-3' (SEQ ID NO: 3) and antisense 5'-ATG GGG AAG
AGC CAG GGC AGA AGT CTG T-3' (SEQ ID NO: 4) (Auernhammer, C. J. and
Melmed, S., Endocrinology 140:1559-1566, 1999). PCR amplification
of IL-11 cDNA was performed under the following conditions: 40
cycles, 30 sec at 94.degree. C., 30 sec at 58.degree. C., 45 sec at
72.degree. C. Before the first cycle, a denaturation step for 2 min
at 94.degree. C. was included, and after 30 cycles the extension
was prolonged for 3 min at 72.degree. C. (Auernhammer, C. J. and
Melmed, S., Endocrinology 140:1559-1566, 1999). PCR products were
analyzed by 1.5% agarose gel electrophoresis and visualized by
ethidium bromide staining with UV light.
[0056] As a result, all the eight cell lines tested expressed IL-11
mRNA, but there was no discernible differences in IL-11 mRNA
expression among 8 cell lines (FIG. 3). On the other hand, 6 out of
8 cell lines expressed PTHrP mRNA and the strongest expression was
observed in SBC-5 cells. Consistent with mRNA expression, SBC-5
cells secreted more than 10 times higher levels of PTHrP protein
compared to other cell lines (Table 5), indicating direct
association of the expression of PTHrP and bone metastasis in human
lung cancer cells.
TABLE-US-00005 TABLE 5 Cytokine Production by Human Lung Cancer
Cell Lines PTHrP.sup.a VEGF.sup.b IL-6.sup.b IL-8.sup.b M-CSF.sup.b
IL-10.sup.b RERF-LC-AI <10 1142 67 1025 454 <20 PC-14 15.1
11429 106 236 911 <20 A549 30.8 743 <20 576 89 <20 SBC-5
499 2040 <20 <20 <20 <20 SBC-3 19.6 1106 <20 13061
297 <20 SBC-3/ADM 14.4 1591 <20 16502 310 <20 H69 <10
3344 <20 <20 <20 <20 H69/VP <10 3273 <20 <20
<20 <20 .sup.a10 pmol/L .sup.bpg
Data shown are the representation of three sets of independent
experiments.
Example 6
Levels of PTHrP and Calcium in the Serum of SBC-5 Cell-Bearing
Mice
[0057] PTHrP has been reported to be responsible for hypercalcemia
in HHM. We evaluated the levels of PTHrP and calcium in the serum
of SBC-5 cell-bearing mice. The levels of serum calcium and PTHrP
were increased in a time-dependent manner, indicating direct
correlation of bone metastasis (or tumor burden) and levels of
these markers and loss of body weight (Table 4). These phenomena
reflect clinical patients of human lung cancer with bone
metastasis.
Example 7
Abilities of Lung Cancer Cells to Produce Cytokines Related to Bone
Metastasis
[0058] We also examined abilities of lung cancer cells to produce
bone metastasis-related cytokine such as IL-1.alpha., IL-6,
TNF-.alpha., XL-10, M-CSF, VEGF and IL-8. Although 8 cell lines
secreted various levels of these cytokines (Table 5), there was no
correlation between the expression level of these cytokines and
formation of bone metastasis.
[0059] All publication, patents and patent application cited herein
are incorporated herein by reference in their entirety.
Sequence CWU 1
1
4123DNAArtificial SequenceSynthetic primer PTHrP 1atgcagcgga
gagactggtt cag 23233DNAArtificial SequenceSynthetic primer PTHrP
2tcaatgcctc cgtgaatcga gctccagagc cgt 33328DNAArtificial
SequenceSynthetic primer IL-11 3actgctgctg ctgaagactc ggctgtga
28428DNAArtificial SequenceSynthetic primer IL-11 4atggggaaga
gccagggcag aagtctgt 28
* * * * *