U.S. patent application number 15/837905 was filed with the patent office on 2018-04-12 for reduction of tgf beta signaling in myeloid cells in the treatment of cancer.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Li Yang.
Application Number | 20180100157 15/837905 |
Document ID | / |
Family ID | 47712810 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180100157 |
Kind Code |
A1 |
Yang; Li |
April 12, 2018 |
REDUCTION OF TGF BETA SIGNALING IN MYELOID CELLS IN THE TREATMENT
OF CANCER
Abstract
Methods of inhibiting metastasis in cancer patients are
provided, wherein the methods comprise reducing TGF.beta.
signaling, for example, by reducing TGF.beta. receptor II
expression in myeloid cells. Vectors comprising a TGF.beta.
receptor II RNAi nucleic acid sequence operably linked to a myeloid
specific promoter also are provided. A method of diagnosing cancer
in an individual by determining TGF.beta. receptor II expression in
myeloid cells in the individual is provided. Additionally, a method
of modulating TGF.beta. activity in myeloid cells in a cancer
patient comprising administering a regulator of at least one of the
GSK3 and PI3K pathways to the patient is provided.
Inventors: |
Yang; Li; (Potomac,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
47712810 |
Appl. No.: |
15/837905 |
Filed: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15402865 |
Jan 10, 2017 |
9868954 |
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15837905 |
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14820697 |
Aug 7, 2015 |
9562237 |
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15402865 |
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13589769 |
Aug 20, 2012 |
9115371 |
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14820697 |
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61525025 |
Aug 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/14 20130101;
G01N 33/57492 20130101; C12Q 2600/158 20130101; A61K 31/7088
20130101; A01K 2227/105 20130101; C12N 15/86 20130101; A61K 31/4025
20130101; C07K 14/71 20130101; A01K 67/0276 20130101; A61K 35/28
20130101; A61P 35/04 20180101; C12N 2320/30 20130101; C12Q 1/6886
20130101; G01N 2333/71 20130101; A01K 2217/075 20130101; A01K
2267/0331 20130101; G01N 33/5091 20130101; C12N 15/1138 20130101;
A61K 31/5377 20130101; A61K 35/15 20130101; C12N 15/85 20130101;
A01K 2217/206 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/86 20060101 C12N015/86; G01N 33/574 20060101
G01N033/574; A61K 31/4025 20060101 A61K031/4025; A61K 31/5377
20060101 A61K031/5377 |
Claims
1.-14. (canceled)
15. A vector comprising a TGF.beta. receptor II RNAi nucleic acid
sequence operably linked to a myeloid-specific promoter.
16. The vector of claim 15, wherein the vector is a viral vector, a
plasmid, a yeast, or a nanoparticle.
17. (canceled)
18. A method of modulating TGF.beta. activity in myeloid cells in a
cancer patient comprising administering a regulator of at least one
of the GSK3 and PI3K pathways to the patient.
19. The method of claim 18, wherein TGF.beta. activity in myeloid
cells is reduced by administering an inhibitor of PI3K or an
enhancer of GSK3.
20. The method of claim 18, wherein the PI3K inhibitor is LY294002.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/525,025 filed Aug. 18, 2011,
which is incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Conventional cancer therapeutic approaches, including
radiation and chemotherapy, are nonselective and damage normal
cells. Gene therapies have exhibited limited success. This likely
is because the vector or virus inefficiently reaches the targeted
tumor and/or the agents used in gene therapy interact with normal
cells and to yield adverse effects (McCormick, Nat. Rev. Cancer, 1:
130-141 (2001), and Scanlon, Anticancer Res., 24: 501-504
(2004)).
[0003] TGF.beta. targeted therapies, such as neutralizing
antibodies, small molecular inhibitors, and adenoviruses have been
used in preclinical and clinical settings (Dumont et al., Cancer
Cell, 3: 531-536 (2003)). However, TGF.beta. is well known to work
as a tumor suppressor in early stage tumorigenesis and as a tumor
promoter in later stages of tumor progression (Yang et al., Cancer
Res., 68: 9107-9111 (2008), and Yang et al, Trends Immunol., 31:
220-227 (2010). The underlying mechanisms for this switch in
function are not clear and pose a great challenge for TGF.beta.
targeted therapies. This challenge in TGF.beta. targeted therapy
represents a general problem of cancer biology, as many cancer
related molecules demonstrate a dual role of pro- and anti-cancer
properties.
[0004] Thus, there remains a need for effective and specific
treatment of cancer for both animals and humans.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides a method of inhibiting metastasis in
a cancer patient comprising reducing TGF.beta. receptor II
expression in myeloid cells in the cancer patient.
[0006] The invention also provides a method of inhibiting
metastasis in a cancer patient comprising (a) removing bone marrow
comprising myeloid cells from the cancer patient, (b) reducing
TGF.beta. signaling in the myeloid cells of the bone marrow ex vivo
to yield TGF.beta. signaling-deficient bone marrow, and (c)
administering the TGF.beta. signaling-deficient bone marrow to the
cancer patient, so as to inhibit metastasis in the cancer
patient.
[0007] Additionally, the invention provides a method of inhibiting
metastasis in a cancer patient comprising transplanting TGF.beta.
receptor II (Tgfbr2)-deficient myeloid cells to the cancer
patient.
[0008] The invention further provides a vector comprising a Tgfbr2
RNAi nucleic acid sequence operably linked to a myeloid specific
promoter.
[0009] The invention provides a method of diagnosing cancer in an
individual comprising (a) obtaining a sample comprising myeloid
cells from the individual; and (b) determining TGF.beta. receptor
II expression in the myeloid cells, wherein increased TGF.beta.
receptor II expression in the myeloid cells relative to a control
indicates a diagnosis of cancer in the individual.
[0010] The invention also provides a method of modulating TGF.beta.
activity in myeloid cells in a cancer patient comprising
administering a regulator of at least one of the GSK3 and PI3K
pathways to the patient.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0011] FIGS. 1A and 1B are graphs with percent expression (y-axis)
of immune cells from spleen (sp), thymus (thy), bone marrow (bm),
and lymph node (ln) (x-axis) for floxed control
Tgfbr2.sup.flox/flox (white bar) and Tgfbr2.sup.MyeKo (black bar)
mice. Expression of CD4+ T cells, CD8+ T cells, NKT cells, NK
cells, B cells, MDSCs (Gr-1+CD11b+), and macrophages are
represented.
[0012] FIG. 2 is a graph with the number of 4T1 lung metastases
(y-axis) from floxed control Tgfbr2.sup.flox/flox (white bar) and
Tgfbr2.sup.MyeKo (black bar) mice that received injection of 4Ti
mammary tumor cells into the #2 mammary fat pad (MFP) (x-axis).
[0013] FIG. 3 is a graph with the number of 4T1 lung metastases
(y-axis) for floxed control Tgfbr2.sup.flox/flox (white bar) and
Tgfbr2.sup.MyeKo (black bar) mice that received a tail vein
injection of 5.times.10.sup.5 4T1 tumor cells (x-axis)
[0014] FIG. 4 is a graph with the number of 3LL Lewis lung
metastases on the y-axis for floxed control Tgfbr2.sup.flox/flox
(white bar) and Tgfbr2.sup.MyeKo (black bar) mice that received
tail vein injection of 3LL Lewis lung cancer cells (x-axis).
[0015] FIG. 5 is a schematic showing the experimental design for
adoptive transfer of bone marrow to wild-type tumor-bearing mice.
4T1 cells (5.times.10.sup.5) were injected into mammary fat pad of
wild-type mice on day 0 (D0). The tumors were surgically removed on
day 15 (D15), and the mice were left to recover until day 34 (D34)
after tumor injection (allowing development of tumor invasion and
metastasis). On D34, mice received a bone marrow transplant from
floxed control Tgfbr2.sup.flox/flox or Tgfbr2.sup.MyeKO mice. Lung
metastasis was examined on day 63 (D63).
[0016] FIGS. 6A and 6B are graphs demonstrating the differences in
survival and the number of lung metastases between floxed control
Tgfbr2.sup.flox/flox (.circle-solid.) (n=9) or Tgfbr2.sup.MyeKO
mice (.box-solid.) (n=8). FIG. 6A has percent survival on the
y-axis and days after tumor injection on the x-axis. FIG. 6B has
the number of lung metastases on the y-axis for floxed control
Tgfbr2.sup.flox/flox (white bar) or Tgfbr2.sup.MyeKO (black bar)
mice.
[0017] FIG. 7 is a graph showing the fold changes in Gr-1+CD11b+
myeloid cell (MDSC) production (y-axis) in spleen, bone marrow, and
peripheral blood for normal (white bar) and 4T1 tumor bearing (D35
tu) (black bar) mice.
[0018] FIG. 8 is a graph showing the percentage of CD33+CD34+CD15+
cells (y-axis) for healthy donors (.circle-solid.) and lung cancer
patients (.box-solid.). Flow cytometry analysis of immature myeloid
cells was performed with anti-CD33, CD34, and CD15 antibodies.
[0019] FIGS. 9A and 9B are graphs showing the density of PF4
expression (y-axis) in myeloid cells (9A) and in the premetastatic
lungs (9B) of floxed control Tgfbr2.sup.flox/flox (white bar) and
Tgfbr2.sup.MyeKo (black bar) mice.
[0020] FIGS. 9C and 9D are graphs demonstrating the differences
between lung metastases and tumor weight (g) between wild-type
(.circle-solid.) and CXCR3 knockout (.box-solid.) mice that
received injection of 4Ti mammary tumor cells into the #2 mammary
fat pad. FIG. 9C has the number of lung metastasis on the y-axis.
FIG. 9D has tumor weight (g) on the y-axis.
[0021] FIGS. 10A and 10B are graphs showing relative expression of
cytokines in Gr-1+D11b+ cells of floxed control
Tgfbr2.sup.flox/flox (white bar) or Tgfbr2.sup.MyeKO (black bar)
mice. FIG. 10A is a graph with relative expression (y-axis) for
each of arginase, INOS, VEGF, TNF.alpha., MMP9, IL4, IL-10, IL12,
and IFN.gamma. (x-axis). FIG. 10B is a graph with relative
expression (y-axis) for each of IL4, IL5, IL6, IL-10, IL13,
IFN.gamma., and IL2 (x-axis) bar.
[0022] FIG. 11A is a graph showing the percentage of IFN.gamma.
positive CD8+ T cells in the spleen of tumor-bearing
Tgfbr2.sup.MyeKO (black) mice compared to Tgfbr2.sup.flox/flox
(white bar) mice.
[0023] FIG. 11B is a graph showing the number of
IFN.gamma.-producing cells detected by ELISPOT in the spleens of
Tgfbr2.sup.MyeKO (black bar) and Tgfbr2.sup.flox/flox (white bar)
mice.
[0024] FIGS. 11C and 11D are graphs demonstrating that IFN.gamma.
neutralization increased lung metastases (11C) and tumor size (11D)
in both Tgfbr2.sup.MyeKO and Tgfbr2.sup.flox/flox mice. Mice were
inoculated with 5.times.10.sup.4 4T1 cells in the #2 MFP. The mice
were treated with IFN.gamma. neutralizing antibody (1 mg on day 1,
3, and 6) or IgG control (0.5 mg on day 9, 12, 15, 18, 21, 24, and
27) through intraperiotoneal injection. For evaluation of lung
metastases, mice were euthanized on day 28 after tumor
injection.
[0025] FIG. 11E is a graph demonstrating TGF.beta.1 level (pg/mL)
in myeloid cells (Gr-1+CD11b+) of wild-type (Nor), tumor-bearing
Tgfbr2.sup.MyeKO, and Tgfbr2.sup.flox/flox mice on the y-axis.
Sorted Gr-1+CD11b+ cells were cultured overnight and supernatants
were collected for TGF.beta.1 ELISA.
[0026] FIG. 12A depicts fluorescence analysis and magnetic cell
sorting (MACS) of human immature myeloid cells (CD33+CD34+CD15+)
before (left panel) and after (right panel) sorting.
[0027] FIG. 12B depicts the results of Western blot analysis of
T.beta.RII expression in CD33+CD34+CD15+ myeloid cells, wherein
.beta.-actin served as a positive control. Samples from two normal
individuals and six later stage lung cancer patients were
examined.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The inventors discovered that myeloid TGF.beta. signaling is
an essential part of the tumor promoting role of TGF.beta..
Specifically, the inventors discovered that the expression of
TGF.beta. receptor II in myeloid cells of tumor hosts plays an
essential role in metastasis. Increased TGF.beta. signaling in
myeloid cells constitutes a critical part of the tumor-promoting
role of TGF.beta.. When TGF.beta. receptor II is deleted in myeloid
cells, there is a significant decrease in tumor metastasis.
[0029] Accordingly, the invention provides a method of inhibiting
metastasis in a cancer patient comprising reducing TGF.beta.
signaling in the cancer patient. TGF.beta. signaling can be reduced
by any suitable method known in the art, but is preferably reduced
by reducing TGF.beta. receptor II expression in myeloid cells of
the cancer patient.
[0030] The cancer patient can be any suitable patient, such as an
animal (e.g., mouse, rat, guinea pig, rabbit, hamster, cat, dog,
horse, cow, pig, simian, or human) with cancer or who is at risk
for cancer.
[0031] Non-limiting examples of specific types of cancers include
cancer of the head and neck, eye, skin, mouth, throat, esophagus,
chest, bone, lung, colon, sigmoid, rectum, stomach, prostate,
breast, ovaries, kidney, liver, pancreas, brain, intestine, heart
or adrenals. More particularly, cancers include solid tumor,
sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor, acute lymphoblastic leukemia,
acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell
leukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acutenonlymphocyctic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia, chronic lymphocytic
leukemia, hairy cell leukemia, or multiple myeloma. See, e.g.,
Harrison's Principles of Internal Medicine, Eugene Braunwald et
al., eds., pp. 491 762 (15th ed. 2001).
[0032] In one aspect of the invention, metastasis in a cancer
patient is inhibited by (a) removing bone marrow comprising myeloid
cells from the cancer patient, (b) reducing TGF.beta. signaling in
the myeloid cells of the bone marrow ex vivo to yield TGF.beta.
signaling-deficient bone marrow, and (c) administering the
TGF.beta. signaling-deficient bone marrow to the cancer patient, so
as to inhibit metastasis in the cancer patient. The TGF.beta.
signaling deficiency preferably is a result of decreased TGF.beta.
receptor II expression.
[0033] TGF.beta. receptor II is a member of the Ser/Thr protein
kinase family and the TGF.beta. receptor subfamily. The encoded
protein is a transmembrane protein that has a protein kinase
domain, forms a heterodimeric complex with another receptor
protein, and binds TGF.beta.. This receptor/ligand complex
phosphorylates proteins, which then enter the nucleus and regulate
the transcription of a subset of genes related to cell
proliferation. Alternatively spliced transcript variants encoding
different isoforms have been characterized (GenBank Accession Nos.
NM_001024847 and NM_003242).
[0034] TGF.beta. receptor II expression can be reduced by any
suitable method, such as RNA interference (RNAi), mutation of the
nucleic acid sequence encoding TGF.beta. receptor II (to produce a
non-functional TGF.beta. receptor II), or knockout (deletion of the
native gene or portion thereof) of the nucleic acid sequence
encoding TGF.beta. receptor II. Alternatively, TGF.beta. receptor
II activity can be reduced by inhibition of the polypeptide, for
example, by administration of TGF.beta. receptor II-specific
antibodies, peptidomimetics, or small molecules.
[0035] In another embodiment, TGF.beta. and/or TGF.beta. receptor
II activity can be mediated (e.g., reduced) by administration of a
modifier (e.g., inhibitor or enhancer) of a member of a related
signaling pathway (e.g., the GSK3 and PI3K signaling pathways). For
example, Example 7 describes the use of a PI3K inhibitor
(LY294002), which can be used to target or modify myeloid cells in
a cancer patient.
[0036] Accordingly, the invention also provides a method of
modulating TGF.beta. and/or TGF.beta. receptor II activity in
myeloid cells in a cancer patient comprising administering a
regulator of at least one of the GSK3 and PI3K pathways to the
patient. For example, the myeloid cells of the cancer patient can
be removed from the cancer patient, contacted with a PI3K inhibitor
or GSK3 enhancer, which results in a reduction in TGF.beta. and/or
TGF.beta. receptor II activity in the myeloid cells, and then
reintroduced into the cancer patient. TGF.beta. and/or TGF.beta.
receptor II activity in the myeloid cells includes, but is not
limited to, TGF.beta. regulation of type 2 cytokines (e.g., IL-10
and IL-4) and PF4 in the myeloid cells.
[0037] In one aspect, the invention provides a vector comprising or
consisting of a nucleotide sequence that encodes an RNAi agent,
which is an RNA molecule that is capable of RNA interference. Such
RNA molecules are referred to as siRNA (short interfering RNA that
is a short-length double-stranded RNA, including, for example, a
short hairpin RNA). The nucleotide sequence that encodes the RNAi
agent preferably has sufficient complementarity with a cellular
nucleotide sequence of TGF.beta. receptor II to be capable of
inhibiting the expression of TGF.beta. receptor II.
[0038] The siRNA can comprise an antisense code DNA coding for the
antisense RNA directed against a region of the TGF.beta. receptor
II gene mRNA and/or a sense code DNA coding for the sense RNA
directed against the same region of the TGF.beta. receptor II gene
mRNA. The siRNA can be any suitable length, such as 15-50 (e.g.,
20, 25, 30, 35, 40, or 45) nucleotides.
[0039] Alternatively, the RNAi agent can be an antisense RNA, which
is an RNA strand having a sequence complementary to the TGF.beta.
receptor II gene mRNA. Antisense RNA induces RNAi by binding to the
TGF.beta. receptor II gene mRNA. The antisense RNA can be any
suitable length, such as 15-50 (e.g., 20, 25, 30, 35, 40, or 45)
nucleotides.
[0040] The vector comprising or consisting of a nucleotide sequence
that encodes an RNAi agent can be any suitable vector, such as a
viral vector, a plasmid, a yeast, a nanoparticle, or naked DNA.
Suitable viral vectors, included poxviruses (e.g., orthopox
viruses, such as vaccinia viruses, and avian poxviruses, such as
fowlpox virus and canarypox virus), adenoviruses, adeno-associated
viruses, and retroviruses.
[0041] The nucleotide sequence that encodes an RNAi agent can be
operably linked to a promoter (in the vector). The promoter
preferably is a myeloid-specific promoter so that expression of the
RNAi agent is specific to myeloid cells. The CD11b promoter and the
c-fes promoter are examples of myeloid-specific promoters.
[0042] The RNAi agent, vector, or regulator of at least one of the
GSK3 and PI3K pathways can be administered alone or in a
composition (e.g., pharmaceutical composition) that can comprise at
least one carrier (e.g., a pharmaceutically acceptable carrier), as
well as other therapeutic agents. The RNAi agent, vector, or
regulator or the respective composition can be administered by any
suitable route, including parenteral, topical, oral, or local
administration.
[0043] The composition (e.g., pharmaceutical composition) can
comprise more than one compound or composition of the invention.
Alternatively, or in addition, the composition (e.g.,
pharmaceutical composition) can comprise one or more other
pharmaceutically active agents or drugs. Examples of such other
pharmaceutically active agents or drugs that may be suitable for
use in the composition (e.g., pharmaceutical composition) include
anticancer agents. Suitable anticancer agents include, without
limitation, alkylating agents; nitrogen mustards; folate
antagonists; purine antagonists; pyrimidine antagonists; spindle
poisons; topoisomerase inhibitors; apoptosis inducing agents;
angiogenesis inhibitors; podophyllotoxins; nitrosoureas; cisplatin;
carboplatin; interferon; asparginase; tamoxifen; leuprolide;
flutamide; megestrol; mitomycin; bleomycin; doxorubicin;
irinotecan; and taxol, geldanamycin (e.g., 17-AAG), and various
anti-cancer peptides and antibodies.
[0044] The carrier can be any of those conventionally used and is
limited only by physio-chemical considerations, such as solubility
and lack of reactivity with the active compound(s), and by the
route of administration. The pharmaceutically acceptable carriers
described herein, for example, vehicles, adjuvants, excipients, and
diluents, are well-known to those skilled in the art and are
readily available to the public. It is preferred that the
pharmaceutically acceptable carrier be one which is chemically
inert to the active agent(s) and one which has no detrimental side
effects or toxicity (other than that desired by the active
compounds) under the conditions of use.
[0045] The choice of carrier will be determined in part by the
particular compound or composition of the invention and other
active agents or drugs used, as well as by the particular method
used to administer the compound and/or composition. Accordingly,
there are a variety of suitable formulations of the composition
(e.g., pharmaceutical composition) of the inventive methods. The
following formulations for oral, aerosol, parenteral, subcutaneous,
intravenous, intramuscular, interperitoneal, rectal, and vaginal
administration are exemplary and are in no way limiting. One
skilled in the art will appreciate that these routes of
administering the compound of the invention are known, and,
although more than one route can be used to administer a particular
compound, a particular route can provide a more immediate and more
effective response than another route.
[0046] Injectable formulations are among those formulations that
are preferred in accordance with the invention. The requirements
for effective pharmaceutical carriers for injectable compositions
are well-known to those of ordinary skill in the art (See, e.g.,
Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982),
and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages
622-630 (1986)).
[0047] Topical formulations are well-known to those of skill in the
art. Such formulations are particularly suitable in the context of
the invention for application to the skin.
[0048] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the compound
or composition dissolved in diluents, such as water, saline, or
orange juice; (b) capsules, sachets, tablets, lozenges, and
troches, each containing a predetermined amount of the active
ingredient, as solids or granules; (c) powders; (d) suspensions in
an appropriate liquid; and (e) suitable emulsions. Liquid
formulations may include diluents, such as water and alcohols, for
example, ethanol, benzyl alcohol, and the polyethylene alcohols,
either with or without the addition of a pharmaceutically
acceptable surfactant. Capsule forms can be of the ordinary hard-
or soft-shelled gelatin type containing, for example, surfactants,
lubricants, and inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one or more of
lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline cellulose, acacia, gelatin, guar gum,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium
stearate, calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible excipients. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to
the active ingredient, such excipients as are known in the art.
[0049] The compounds and compositions of the invention, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an atomizer.
Such spray formulations also may be used to spray mucosa.
[0050] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The compounds and
compositions of the invention can be administered in a
physiologically acceptable diluent in a pharmaceutical carrier,
such as a sterile liquid or mixture of liquids, including water,
saline, aqueous dextrose and related sugar solutions, an alcohol,
such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, dimethylsulfoxide,
glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol,
ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a
fatty acid ester or glyceride, or an acetylated fatty acid
glyceride with or without the addition of a pharmaceutically
acceptable surfactant, such as a soap or a detergent, suspending
agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or
emulsifying agents and other pharmaceutical adjuvants.
[0051] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0052] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-b-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0053] Preservatives and buffers may be used. In order to minimize
or eliminate irritation at the site of injection, such compositions
may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5% to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0054] Additionally, the compounds of the invention, or
compositions comprising such compounds, can be made into
suppositories by mixing with a variety of bases, such as
emulsifying bases or water-soluble bases. Formulations suitable for
vaginal administration can be presented as pessaries, tampons,
creams, gels, pastes, foams, or spray formulas containing, in
addition to the active ingredient, such carriers as are known in
the art to be appropriate.
[0055] The invention also provides a method of diagnosing cancer in
an individual comprising (a) obtaining a sample comprising myeloid
cells from the individual; and (b) determining TGF.beta. receptor
II expression in the myeloid cells, wherein increased TGF.beta.
receptor II expression in the myeloid cells relative to a control
indicates a diagnosis of cancer in the individual.
[0056] Expression of TGF.beta. receptor II mRNA and/or protein can
be determined by any suitable method including, but not limited to,
PCR (RT-PCR, quantitative RT-PCR), microarrays, Northern blotting,
and Western blotting.
[0057] The control can be any suitable control, such as an
expression level of TGF.beta. receptor II mRNA and/or protein from
myeloid cells of a normal (healthy) individual or group of normal
(healthy) individuals.
[0058] The individual can be any suitable individual, such as a
mammal including a mouse, rat, hamster, guinea pig, rabbit, cat,
dog, pig, horse, cow, or primate (e.g., human).
[0059] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0060] This example demonstrates the generation of a
myeloid-specific TGF.beta. receptor II knockout mouse.
[0061] Myeloid cells play an important role in tumor progression.
They suppress host immune surveillance and influence the tumor
microenvironment (see, e.g., Gabrilovich et al., Nat. Rev.
Immunol., 9: 162-174 (2009); Pollard, Nat. Rev. Immunol., 9:
259-270 (2009); Mantovani, Nature, 457: 36-37 (2009), Fridlender et
al., Cancer Cell, 16: 183-194 (2009); Balkwill et al., Nature, 431:
405-406 (2004), Yang et al., Cancer Cell, 6: 409-421 (2004); and
Yang et al., Cancer Cell, 13: 23-35 (2008)). Myeloid cells are also
present in the lungs prior to tumor cell arrival and contribute to
pre-metastatic niche formation and environment alteration (see
Kaplan et al., Nature, 438: 820-827 (2005); and Yan et al., Cancer
Res., 70: 6139-6149 (2010)). These include tumor-associated
macrophages (TAM, Mac-1+ or F4/80+ cells), Gr-1+CD11b+ cells or
myeloid derived suppressor cells (MDSCs), and tumor associated
neutrophils (TAN, CD11b+ Ly6G+ cells). One of the most important
properties of these cells is the increased TGF.beta. production and
increased Th2 polarization (see, e.g., Yang, et al., Cancer Cell,
13: 23-35 (2008); and Flavell et al., Nat. Rev. Immunol. 10:
554-567 (2010)). However, there are no reports as to how TGF.beta.
signaling in myeloid cells affects tumor phenotype.
[0062] To investigate the effect of myeloid specific abrogation of
TGF.beta. signaling on tumor phenotype, knock out mice with a
specific deletion of the TGF.beta. receptor II in myeloid cells
(Tgfbr2.sup.MyeKO) were generated. Mice with targeted deletion of
Tgfbr2 in myeloid cells (Tgfbr2.sup.MyeKO) were generated through
the cross breeding of floxed Tgfbr2 (Tgfbr2.sup.flox/flox) (see,
e.g., Chytil et al., Genesis, 32: 73-75 (2002); and Forrester et
al., Cancer Res., 65: 2296-2302 (2005)) mice with LysM-Cre
transgenic mice (in C57BL/6 and 129 background). Mice heterozygous
for LysM-Cre and heterozygous for the floxed Tgfbr2 allele were
further bred with wild-type Balb/c or C57BL/6 for 10 generations to
generate a Balb/c or C57BL/6 background. LysM-Cre transgenic mice
have been well characterized and used in many other studies to
delete floxed genes specifically in myeloid cells, both neutrophils
and monocytes/macrophages (see, e.g., Sinha et al., J. Immunol.,
173: 1763-1771 (2004); Sun et al., Blood, 104: 3758-3765 (2004);
and Hazenbos et al., Blood, 104: 2825-2831 (2004)). The
Tgfbr2.sup.MyeKO mice appeared to be normal with no alteration in
hematopoiesis (see FIGS. 1A and 1B). This includes macrophages
(CD11b+F4/80+), MDCSs (Gr-1+CD11b+), B cells (B220), CD4+ T cells,
CD8+ T cells, as well as NK cells (NK 1.1), and NKT cells (NK1.1
and TCR.beta.) derived from spleen, thymus, bone marrow and lymph
nodes (FIGS. 1A and 1B).
EXAMPLE 2
[0063] This example demonstrates that reduced expression of
TGF.beta. signaling reduces lung metastasis.
[0064] The 4T1 mammary tumor model shares many characteristics with
human breast cancer, particularly its ability to spontaneously
metastasize to the lungs. In an orthotopic metastasis design,
5.times.10.sup.4 4T1 cells in 50 .mu.L PBS were injected into the
#2 mammary fat pad (MFP) of Tgfbr2.sup.MyeKO and floxed control
Tgfbr2.sup.flox/flox mice. Mice were sacrificed 28 days later, and
the number and size of lung metastasis was evaluated.
Tgfbr2.sup.MyeKO mice showed a decreased ability to support tumor
metastasis after injection of 4T1 mammary tumor cells (see FIG. 2),
with no difference in primary tumor size.
[0065] In an experimental metastasis design, 2.times.10.sup.5 4T1
cells were injected into the tail vein of Tgfbr2.sup.MyeKO and
floxed control Tgfbr2.sup.flox/flox mice. The size of tumors was
determined by direct measurement of tumor dimensions at 2-3 day
intervals using calipers. The number of lung metastasis was
evaluated when the mice were euthanized at day 25 after injection.
There was a significant reduction in metastasis in Tgfbr2.sup.MyeKO
mice (see FIG. 3). This result was recapitulated in the LLC
experimental metastasis model in which Tgfbr2.sup.MyeKO mice in a
C57BL/6 background were injected in the tail vein with or
2.5.times.10.sup.5 LLC cells and sacrificed at day 21 after
injection (see FIG. 4).
[0066] These data support that decreased expression of TGF.beta.
signaling results in a decrease of lung metastasis in a mouse
model.
EXAMPLE 3
[0067] This example demonstrates that the inventive methods inhibit
metastasis.
[0068] To further confirm the inhibitory effect of myeloid-specific
Tgfbr2 deletion on tumor metastasis, Tgfbr2.sup.MyeKO bone marrow
(B.M.) was transplanted into wild-type mice bearing 4T1 tumors.
5.times.10.sup.5 4T1 cells were injected into the #4 MFP on day 0.
To better model clinical metastatic disease the primary tumor was
surgically removed on day 15 and metastasis allowed to continue
until day 34 allowing the mice to develop tumor invasion and
metastasis. On day 34, the mice were irradiated and subjected to
B.M. transplantation from either Tgfbr2.sup.MyeKO mice or
Tgfbr2.sup.flox/flox control mice. Lung metastases were examined on
day 63 and thereafter (see FIG. 5).
[0069] 100% survival of the mice that received B.M. from the
Tgfbr2.sup.MyeKO mice was observed, whereas approximately 55% of
the mice that received B.M. from Tgfbr2.sup.flox/flox control mice
exhibited decreased survival (see FIG. 6A). In addition, a
significantly reduced number of lung metastases were observed in
mice that received Tgfbr2.sup.MyeKO B.M. relative to those that
received control B.M. (see FIG. 6B).
[0070] These data suggest that myeloid-specific TGF.beta. signaling
constitutes an essential part of the metastasis-promoting role of
TGF.beta.. When Tgfbr2 is ablated in myeloid cells, tumor
metastasis is significantly decreased. Notably, this experimental
model uncovers mechanisms different from those observed in mice
with Tgfbr2 deletion in FSP+ fibroblasts, which develop invasive
squamous cell carcinoma in the fore-stomach, and intraepithelial
neoplasia in the prostate (see, e.g., Bhowmick et al., Science,
303: 848-851 (2004)). The consequences of myeloid-specific Tgfbr2
ablation are also different from deletion of Smad4 signaling in T
cells, which induces gastrointestinal cancer development (see,
e.g., Kim et al., Nature, 441: 1015-1019 (2006)).
[0071] Instead, these data are reminiscent of those observed
following blockade of TGF.beta. signaling in T cells using
CD4dnTGF-RII mice, which confers resistance to an EL-4 lymphoma or
a B16-F10 melanoma tumor challenge (see, e.g., Gorelik et al., Nat.
Med., 7: 1118-1122 (2001)). However, those mice developed an
autoimmune pathology that is not seen in the current mouse model.
The lack of pathology in the current mouse model is likely due to
the fact that myeloid cells are massively expanded under tumor
conditions (see FIG. 7). In particular, Gr-1+CD11b+ cells, which
produce large quantities of TGF.beta., constitute the majority of
the tumor-associated myeloid cells in the 4T1 mammary tumor and
Lewis lung carcinoma (LLC) models. Splenic Gr-1+CD11b+ cells from
tumor-bearing mice expressed significantly higher levels of
TGF.beta. receptor II compared with their non-tumor-bearing
counterparts.
[0072] These data support that the specific deletion of myeloid
Tgfbr2 produces a pronounced antitumor effect with very few adverse
effects.
EXAMPLE 4
[0073] This example demonstrates the correlation between TGF.beta.
receptor II overexpression and cancer.
[0074] Immature myeloid cells are overproduced in tumor hosts
including patients with a variety of cancers (see, e.g., Yang et
al., Cancer Cell, 6: 409-421 (2004); and Almand et al., J.
Immunol., 166: 678-689 (2001)). In humans, these cells have been
identified as myeloid linage marker CD33+CD34+CD15+ cells (Yang et
al., Cancer Cell, 6: 409-421 (2004); Zea et al., Cancer Res., 65:
3044-3048 (2005); Srivastava et al., Cancer Immunol. Immunother., 5
7: , 1493-1504 (2008); Hoechst et al., Hepatology, 50: 799-807
(2009); and Chalmin et al., J. Clin. Invest., 120: 457-471
(2010)).
[0075] To investigate whether TGF.beta. receptor II was
over-expressed in these human myeloid cells, peripheral blood from
16 patients with metastatic non-small cell lung cancers was
collected. CD33+CD34+CD15+ myeloid cells accounted for
approximately 85% of the total leukocytes in these patients,
clearly higher than that of healthy individuals (28%, n=4) (see
FIG. 8). Sorted immature myeloid cells from cancer patients also
showed overtly increased TGF.beta. receptor II expression compared
to those from healthy individuals (see FIGS. 12A-B), suggesting a
strong clinical relevance for TGF.beta. receptor II over-expression
in myeloid cells.
[0076] Furthermore, the expression of Tgf.beta.1 and TGF.beta.
receptor II mRNA in human peripheral blood mononuclear cells in
cohorts of lung cancer (GSE20189) and breast cancer (GSE27567) was
determined. The lung cancer and breast cancer datasets were
analyzed by Genespring GX 10.0 software. TGF.beta. receptor II
expression correlated with the degree of malignancy in the lung and
breast cancer datasets. These results indicate that TGF.beta.
receptor II expression in monocytes can be used for diagnosis
and/or prognosis of cancer.
[0077] The over-expression of TGF.beta. receptor II in both human
and mouse myeloid cells from cancer hosts strongly supports that
TGF.beta. receptor II signaling in myeloid cells affects tumor
progression and metastasis.
EXAMPLE 5
[0078] This example demonstrates the characterization of reduced
TGF.beta. signaling.
[0079] Gr-1+CD11b+ cells are present in the premetastatic lung
(prior to tumor cell arrival). They change the lung into an
inflammatory and proliferative environment, diminish immune
protection, and promote metastasis through aberrant vasculature
formation. The premetastatic lung is characterized by increased
growth factors, inflammatory cytokines, and chemokines, such as the
chemokine PF4. PF4, also known as CXCL4, was significantly
increased in lungs of 4T1 tumor bearing mice at day 10 and 14 when
compared with non-tumor control mice.
[0080] PF4 belongs to a CXCL chemokine family that includes CXCL9,
CXCL10, and CXCL11. These chemokines signal through CXCR3, a G
protein coupled receptor. Interestingly, other than PF4, there was
no change in the expression of other members of this chemokine
family (i.e., CXCL9, CXCL10, and CXCL11). Notably, the deletion of
Tgfbr2 decreased PF4 expression in myeloid cells (see FIG. 9A) and
in the premetastatic lungs of Tgfbr2.sup.MyeKO mice (see FIG.
9B).
[0081] To determine the functional significance of decreased
PF4/CXR3 signaling in tumor metastasis, CXCR3 knockout mice (see,
e.g., Pan et al., J. Immunol., 176: 1456-1464 (2006)) were used.
Deletion of CXCR3 dramatically decreased the number of lung
metastasis in the mice that received 4T1 tumor injection in the #2
MFP (see FIG. 9C) with no effect on primary tumor size or weight
(see FIG. 9D).
[0082] These data support that PF4/CXCR3 chemokine axis plays a
specific and critical role in 4T1 tumor lung metastasis.
EXAMPLE 6
[0083] This example demonstrates the further characterization of
reduced TGF.beta. signaling.
[0084] TGF.beta. signaling is a critical mediator of polarization
of myeloid cells. Therefore, Th1/Th2 cytokine expression of
Gr-1+CD11b+ cells was examined. Interestingly, the expression of
Th2 type cytokines, including IL-10 and IL4, was reduced in myeloid
cells with the Tgfbr2 deletion compared to controls, with no
difference in Th1 type cytokine production (e.g., IL-12 and
IFN.gamma.) (see FIG. 10A). There also was a reduction in arginase
and iNOS levels (see FIG. 10A), which are implicated in the immune
suppression effects of Gr-1+CD11b+ cells. These results were
further confirmed with a cytokine protein array assay (see FIG.
10B).
[0085] Since tumor associated myeloid cells exert immune
suppression through inhibiting multiple immune cell function in
tumor hosts, whether myeloid-specific Tgfbr2 deletion resulted in
an improved function of CD4, CD8, B, NK, or macrophage cells was
examined. Single cell suspensions from spleen were made, and
IFN.gamma. ELISPOT and intracellular cytokine staining of IL2,
IL-10, IL4 (CD4 T cells), IFN.gamma., IL2 (CD8 T cells), CD69, 41BB
(B cell function), IFN.gamma. (NK cell function), as well as IL12
and IL-10 (macrophage function) were performed between
Tgfbr2.sup.MyeKO and the control littermates.
[0086] An increased percentage of IFN-.gamma. positive CD8+ T cells
was observed in the spleen of tumor-bearing Tgfbr2.sup.MyeKO mice
compared to Tgfbr2.sup.flox/flox mice (see FIG. 11A). No difference
was found in other cytokines or cell types. This was consistent
with the increased number of IFN-.gamma. producing cells detected
by ELISPOT in the spleens of Tgfbr2.sup.MyeKO mice (see FIG. 11B).
Importantly, systemic neutralization of IFN-.gamma. diminished the
inhibitory effect of myeloid Tgfbr2 deletion on metastasis (see
FIG. 11C) with no effect on tumor size (see FIG. 11D).
[0087] This data suggests that genetic inactivation of TGF.beta.
receptor II in myeloid cells likely decreases immune suppression in
tumor bearing hosts through improved Th1/Th2balance and elevated
IFN.gamma. production in CD8 T cells, which likely contributes to
reduced tumor metastasis in Tgfbr2.sup.MyeKO mice.
[0088] Gr-1+CD11b+ cells are one of the major sources of TGF.beta.
in the tumor bearing host. Deletion of Tgfbr2 decreased TGF.beta.1
production in Gr-1+CD11b+ cells (see FIG. 11E), suggesting possible
autocrine and/or paracrine loops that enhance TGF.beta. production
and signaling in myeloid cells. It is not clear whether TGF.beta.
production directly converts myeloid cells from an M1 to M2
phenotype or is the result of M2 TAM polarization. The data show
that deletion of myeloid Tgfbr2 induced a decrease of both Th2
cytokines and TGF.beta.1 production, which was associated with
increased IFN-.gamma. expression in CD8 T cells. This likely
improves the host immune surveillance in the Tgfbr2.sup.MyeKO mice.
Taken together, these studies demonstrate that myeloid-specific
TGF.beta. signaling is a significant part of the tumor-promoting
effects of TGF.beta., and provides a therapeutic opportunity for
new approaches to cancer therapy.
EXAMPLE 7
[0089] This example demonstrates the mechanism of reduced TGF.beta.
signaling.
[0090] To determine the mechanisms underlying decreased Th2
cytokine or PF4 expression in myeloid cells of Tgfbr2.sup.MyeKO
mice, the expression of potentially relevant genes was determined.
Increased expression of glycogen synthase kinase 3 (GSK3) and
NF.kappa.B was observed in Gr-1+CD11b+ cells sorted from
Tgfbr2.sup.MyeKO mice. GSK3 is a serine/threonine protein kinase
that mediates the addition of phosphate molecules into serine and
threonine amino acid residues. GKS3 has been implicated in the
production of inflammation-associated cytokines (see, e.g., Park et
al., Nat. Immunol., 12: 607-615 (2011); and Woodgett et al., Nat.
Immunol., 6: 751-752 (2005)).
[0091] Gr-1+CD11b+ myeloid cells were sorted with MACS from
Tgfbr2.sup.MyeKO and cultured for 6 hours with an inhibitor of GSK3
(SB216763, Sigma) or PI3K (LY294002, Calbiochem) at different doses
(2, 5, and 10 .mu.M for the GSK3 inhibitor; 5, 10, and 20 .mu.M for
the PI3K inhibitor). SB216763 is a potent and selective
ATP-competitive inhibitor of the serine/threonine protein kinas GSK
.alpha. and .beta.. LY294002 competitively inhibits ATP binding of
the catalytic subunit of P13 kinases, consequently enhancing GSK3
activity. GSK3 Western blotting was performed to detect inhibiting
activity. The expression of IL-10, IL-4, and PF4 was examined using
Western blotting and quantitative PCR.
[0092] The GSK3-specific inhibitor (SB216763) reversed the
down-regulation of IL-10, IL-4, and PF4 in the myeloid cells
lacking Tgfbr2 at both the mRNA level and protein level. The
inhibitor of PI3K (LY294002), the upstream mediator of GSK3,
resulted in the inverse effect of GSK3 inhibition (i.e., decreased
IL-10, IL4, and P4). This is consistent with a negative regulatory
role of PI3K on GSK3 that promotes inhibitory phosphorylation of
GSK3. Inhibition of NFK.kappa. with a specific inhibitor
(BMS-345541) did not show a significant effect. These data indicate
that TGF.beta. regulation of Th2 and PF4 likely is mediated by the
PI3K and GSK3 signaling pathways. Therefore, P13K inhibitors (e.g.,
LY294002) and GSK3 enhancers can be used to target or modify
myeloid cells in cancer patients to inhibit cancer and/or
metastasis.
[0093] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0094] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0095] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *