U.S. patent application number 15/666472 was filed with the patent office on 2017-11-30 for cd11 b[low] macrophages and conditioned media thereof for treating cancer and/or fibrosis.
The applicant listed for this patent is CARMEL-HAIFA UNIVERSITY ECONOMIC CORPORATION LTD. Invention is credited to Amiram Ariel, Dalit Barkan, Odelya Gilon, Sagie Schif-Zuck, Aroesti-Assi Simaan.
Application Number | 20170340670 15/666472 |
Document ID | / |
Family ID | 51019998 |
Filed Date | 2017-11-30 |
United States Patent
Application |
20170340670 |
Kind Code |
A1 |
Barkan; Dalit ; et
al. |
November 30, 2017 |
CD11 B[low] MACROPHAGES AND CONDITIONED MEDIA THEREOF FOR TREATING
CANCER AND/OR FIBROSIS
Abstract
A conditioned cell culture medium of CD11b.sup.low human
macrophages or a biologically active fraction thereof can be
prepared by a method that includes (i) culturing a population of
human mononuclear cells of the monocyte/macrophage lineage for 5-7
days, so as to induce differentiation of the mononuclear cells to
macrophages; (ii) incubating the macrophages obtained in (i) with
apoptotic cells or in the presence of a pro-resolving lipid
mediator to reduce the CD11b expression, thus obtaining a culture
of CD11b.sup.low macrophages; and (iii) collecting the conditioned
cell culture medium of CD11b.sup.low macrophages. Pharmaceutical
compositions containing the CD11b.sup.low macrophages conditioned
medium or a culture of CD11b.sup.low macrophages can be used in the
treatment of cancer or fibrosis.
Inventors: |
Barkan; Dalit; (Zichron
Yaakov, IL) ; Gilon; Odelya; (Kiryat Bialik, IL)
; Ariel; Amiram; (Kiryat Motzkin, IL) ;
Schif-Zuck; Sagie; (Haifa, IL) ; Simaan;
Aroesti-Assi; (Maale Adumim, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARMEL-HAIFA UNIVERSITY ECONOMIC CORPORATION LTD |
Haifa |
|
IL |
|
|
Family ID: |
51019998 |
Appl. No.: |
15/666472 |
Filed: |
August 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14758569 |
Jun 30, 2015 |
9724366 |
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PCT/IL2013/051084 |
Dec 30, 2013 |
|
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15666472 |
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61747312 |
Dec 30, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2502/1157 20130101;
C12N 2501/15 20130101; A61K 35/12 20130101; C12N 5/0645 20130101;
A61K 45/06 20130101; C12N 2500/70 20130101; C12N 2502/00 20130101;
C12N 2501/58 20130101; C12N 2506/115 20130101; C12N 2506/1307
20130101; C12N 5/0656 20130101; A61K 35/15 20130101; C12N 2506/11
20130101 |
International
Class: |
A61K 35/15 20060101
A61K035/15; A61K 35/12 20060101 A61K035/12; C12N 5/077 20100101
C12N005/077; A61K 45/06 20060101 A61K045/06; C12N 5/0786 20100101
C12N005/0786 |
Claims
1. A conditioned cell culture medium of CD11b.sup.low human
macrophages or a biologically active fraction thereof.
2. A method for preparing the conditioned cell culture medium of
CD11b.sup.low human macrophages of claim 1, the method comprising:
(i) culturing a population of human mononuclear cells of the
monocyte/macrophage lineage for 5-7 days, so as to induce
differentiation of said mononuclear cells to macrophages; (ii)
incubating the macrophages obtained in said (i) with apoptotic
cells or in presence of a pro-resolving lipid mediator to reduce a
CD11b expression, thus obtaining a culture of CD11b.sup.low
macrophages; and (iii) collecting the conditioned cell culture
medium of CD11b.sup.low macrophages.
3. The method according to claim 2, wherein said human mononuclear
cells of the monocyte/macrophage lineage are human monocytes.
4. The method according to claim 3, wherein the human monocytes are
derived from peripheral blood mononuclear cells of an
individual.
5. The method according to claim 2, wherein the incubation of the
macrophages in said (ii) is carried out in presence of apoptotic
cells.
6. The method according to claim 5, wherein a ratio of said
macrophages to apoptotic cells is between 1:1 and 1:1.5.
7. The method according to claim 2, wherein the incubation of the
macrophages in said (ii) is carried out in presence of a
pro-resolving lipid mediator selected from the group consisting of
a lipoxin (LX), a resolvin (Rv), a protectin (PD), a maresin (MaR),
and a glucorticoid.
8. The method according to claim 7, wherein said lipoxin is
LXB.sub.4 or 15-epi-LXA.sub.4, said resolvin is RvD1 or RvD2, said
protectin is PD1, and said glucorticoid is dexamethasone.
9. A conditioned cell culture medium of CD11b.sup.low macrophages
produced according to claim 2.
10. An active agent selected from the group consisting of (i) a
conditioned cell culture medium of CD11b.sup.low macrophages; (ii)
a biologically active fraction thereof; (iii) a pharmaceutical
composition comprising said (i) or (ii) and a pharmaceutically
active carrier, an excipient or a diluent; (iv) a culture of human
CD11b.sup.low macrophages; or (v) a pharmaceutical composition
comprising said (iv) and a pharmaceutically active carrier, an
excipient or a diluent, for use in treatment of a cancer or a
fibrosis.
11. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 10, for use in treatment of the cancer.
12. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 11, wherein said treatment of the cancer
comprises preventing or delaying a cancer recurrence or an
occurrence of metastasis or inhibiting a cancer metastasis.
13. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 10, wherein said cancer is selected from the
group consisting of breast, prostate, esophageal, skin, lung, head
and neck, colon and liver cancer.
14. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 10, wherein said treatment of fibrosis comprises
preventing or inhibiting the fibrosis.
15. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 14, wherein said fibrosis is associated with
cancer.
16. The conditioned cell culture medium of CD11b.sup.low
macrophages or the pharmaceutical composition comprising it
according to claim 14, wherein said fibrosis is associated with a
disease or a condition selected from the group consisting of acute
lung injury, Alzheimer's disease, chronic diabetic wounds, chronic
granulomatous disease (CGD), chronic obstructive pulmonary disease
(COPD), constrictive pericarditis, Dupuytren's disease, emphysema,
hypertrophic burn scars, keloid, liver cirrhosis, plantar
fibromatosis, retinal detachment inflammation, scleroderma, cystic
fibrosis, endometrial fibrosis, idiopathic pulmonary fibrosis,
myocardial fibrosis, nephrogenic fibrosis, pancreatic fibrosis,
perineural fibrosis, renal interstitial fibrosis, secondary
fibrosis in the gastrointestinal tract or fibrosis resulting after
surgery.
17. A pharmaceutical composition comprising an active agent
selected from the group consisting of (i) a conditioned cell
culture medium of CD11b.sup.low macrophages; (ii) a biologically
active fraction thereof; (iii) a pharmaceutical composition
comprising (i) or (ii) and a pharmaceutically active carrier, an
excipient or a diluent; (iv) a culture of human CD11b.sup.low
macrophages; or (v) a pharmaceutical composition comprising (iv)
and a pharmaceutically active carrier, an excipient or a
diluent.
18. A method for preparing a culture of CD11blow macrophages, the
method comprising: (i) culturing a population of mononuclear cells
of the monocyte/macrophage lineage for 5-7 days, so as to induce
differentiation of said mononuclear cells to macrophages; and (ii)
incubating the macrophages obtained in said (i) with apoptotic
cells or in presence of a pro-resolving lipid mediator to reduce
the CD11b expression, thus obtaining a culture of CD11b.sup.low
macrophages.
19. A method for treating a cancer comprising administering to a
subject in need an effective amount of an active agent selected
from the group consisting of: (i) a conditioned cell culture medium
of CD11b.sup.low macrophages; (ii) a biologically active fraction
thereof; (iii) a pharmaceutical composition comprising (i) or (ii)
and a pharmaceutically active carrier, an excipient or a diluent;
(iv) a culture of human CD11b.sup.low macrophages; and (v) a
pharmaceutical composition comprising (iv) and a pharmaceutically
active carrier, an excipient or a diluent, wherein said treatment
comprises preventing or delaying a cancer recurrence or an
occurrence of metastasis or inhibiting a cancer metastasis.
20. The method according to claim 19, wherein said treating cancer,
preventing or delaying the cancer recurrence or occurrence of
metastasis, or inhibiting cancer metastasis comprises preventing or
inhibiting an outbreak of dormant cancer cells.
21. The method according to claim 19, wherein said subject in need
is in cancer remission.
22. The method according to claim 19, wherein said administering is
performed in combination with additional one or more anti-cancer
agents or treatments.
23. A method for treating a fibrosis comprising administering to a
subject in need an effective amount of an active agent selected
from the group consisting of: (i) a conditioned cell culture medium
of CD11b.sup.low macrophages; (ii) a biologically active fraction
thereof; (iii) a pharmaceutical composition comprising (i) or (ii)
and a pharmaceutically active carrier, an excipient or a diluent;
(iv) a culture of human CD11b.sup.low macrophages; and (v) a
pharmaceutical composition comprising (iv) and a pharmaceutically
active carrier, an excipient or a diluent, wherein said treatment
fibrosis comprises preventing or inhibiting fibrosis.
Description
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
[0001] The present application is a continuation application of
application Ser. No. 14/758,569 filed Jun. 30, 2015, which is the
US national phase entry of International Patent Application No.
PCT/IL2013/051084, filed Dec. 30, 2013, and which claims priority
from U.S. Provisional Patent Application No. 61/747,312, filed Dec.
30, 2012. Any and all applications for which a foreign or domestic
priority claim is identified in the Application Data Sheet as filed
with the present application are hereby incorporated by reference
under 37 CFR 1.57.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates, generally, to cell
conditioned media and, more specifically, to cell conditioned media
of specific types of macrophages and methods for preparation
thereof. The invention further relates to compositions comprising
said specific types of macrophages or a conditioned medium thereof
and their uses in the treatment of cancer or fibrosis.
Description of the Related Art
Metastasis and Tumor Dormancy
[0003] Early diagnosis of neoplastic disorders such as breast
cancer, melanoma and renal cancer, can increase the chances of
disease-free survival of patients. However, these disorders can
recur as metastatic disease many years after primary tumor
resection and adjuvant therapy. This metastatic disease appears to
arise from tumor cells that disseminated early in the course of the
disease but did not develop into clinically apparent lesions, and
is the major cause of mortality of breast cancer patients.
[0004] These long-term surviving, disseminated tumor cells maintain
a state of dormancy and are resistant to conventional therapies
that target actively-dividing cells, but may be triggered to
proliferate through largely unknown mechanisms. Therefore,
understanding the mechanisms that regulate tumor dormancy or the
switch to a proliferative state is critical to discovering novel
targets and interventions to prevent disease recurrence.
[0005] Chemotherapy is the main treatment for disseminated,
malignant cancers. However, chemotherapeutic agents are limited in
their effectiveness for treating many cancer types, including many
common solid tumors. This failure is in part due to the intrinsic
or acquired drug resistance of many tumor cells. Another drawback
to the use of chemotherapeutic agents is their severe side effects.
These include bone marrow suppression, nausea, vomiting, hair loss,
and ulcerations in the mouth.
[0006] In addition, currently available cancer therapy commonly
targets actively proliferating tumor cells, and fails to eradicate
quiescent, non-proliferating tumor cells (dormant tumor cells)
which are the source for the recurrent disease and metastasis.
[0007] Two states of tumor dormancy have been described in the
literature based on experimental and clinical evidence. Dormant
tumor cells may exist in a quiescent state for many years as
solitary quiescent tumor cells in the bone narrow, lymph nodes and
blood circulation of breast cancer patients. Their quiescence was
demonstrated by their negative staining for the proliferation
marker Ki67 and their negative staining for apoptosis. These cells
are resistant to conventional therapies that target actively
dividing cells, leading to possible disease recurrence following
adjuvant therapy.
[0008] Alternatively, tumor dormancy may exist as micrometastases
where cellular proliferation is balanced by apoptosis.
Consequently, in this balanced state, there is no net increase in
tumor mass over time. These micrometastases remain dormant because
of lack of recruitment of the vasculature needed to nourish the
tumor, known as the angiogenic switch and/or involvement of the
adaptive immune system such as cytotoxic CD8.sup.+ T cells.
Macrophage Types
[0009] Macrophages are highly plastic monocyte-derived cells that
acquire different molecular and functional phenotypes following
exposure to different bioactive molecules and environments. The
early studies on the interactions of macrophages and lymphocytes in
battling bacterial infections revealed the T helper type 1 (Th1)
secreted cytokine IFN.hoarfrost. to be involved in the classical
activation of macrophages. However, seminal studies by the groups
of Gordon and Mantovani have extensively characterized additional
macrophage subtypes activated in alternative manners (reviewed in
Mantovani et al., 2004, Martinez et al., 2009).
[0010] Since the major polarizing cytokines initially found to be
involved in classical and alternative activation were derived from
Th1 (IFN.lamda.) and Th2 (IL-4 and IL-13) lymphocytes these
activated macrophages were named M1 and M2, respectively. Later
studies revealed that, in addition to IL-4, alternative activation
can also be induced by immune complexes and glucocorticoids, and
accordingly the subdivision of alternatively-activated macrophages
to M2a-c was instilled. M1 macrophages are important inducers and
effectors in the Th1 response. They are instrumental in immune
responses against intracellular microbes and tumors. M2 macrophages
are more heterogeneous, but generally play a role in Th2 responses,
such as killing and encapsulation of extracellular parasites,
stopping inflammation and promoting tissue repair and remodeling.
M2 macrophages also play a role in immune regulation and promote
tumor progression (Mantovani et al., 2005, Martinez et al., 2009).
M1 and M2 macrophages are not only distinct in function, but also
express different receptors and enzymes required for their
activities. M1 macrophages express high levels of inflammatory
cytokines (IL-12, IL-23, TNF.alpha., IL-1.beta., and IL-6) and
chemokines (CXCL9, 10, and 11, CCL2, 3, 4, and 5, and CXCL2), as
well as enzymes involved in the generation of reactive oxygen
species (ROS) and nitric oxide (NO). M2 macrophages express lower
levels of inflammatory mediators, but high levels of IL-10,
scavenger, mannose, and galactose receptors.
[0011] The prototypic Th2 cytokines IL-4, IL-13 and IL-10, as well
as immune responses to parasites were found to promote many of the
outcomes of efferocytosis (the process by which dying or dead cells
are removed by phagocytic cells) in macrophages. These cytokines
are well appreciated antagonists of the M1 response and macrophage
pro-inflammatory properties while IL-4 and IL-13 can also promote
fibrosis through TGF.beta. production. IL-4 and IL-13 also activate
PPAR-.lamda. and PPAR-.delta. to promote monocyte/macrophage
alternative activation. Liver X receptor (LXR) was recently found
to synergize with IL-4 in the induction of arginase 1 expression
and promotion of an M2 phenotype in regressive atherosclerotic
lesions. Thus, efferocytosis induces phenotypic and molecular
switches and activates signaling pathways in macrophages that
resemble M2 polarization. In addition, M2 polarization promotes
efferocytosis through induction of different molecular modules,
whereas M1 macrophages exert reduced uptake of apoptotic cells.
Along these lines, recent studies also found that efferocytosis is
a self-promoting process, and that M2 pathways play key roles in
mediating this feature of macrophage function.
Role of Macrophages in Inflammation
[0012] When inflammation occurs, polymorphonuclear neutrophils
(PMNs) are among the first leukocyte responders to accumulate in
the inflamed site. These cells are crucial as the first line of
defense of the innate immune system because of their phagocytotic
and microbicidal functions. The initial accumulation of neutrophils
is followed by a second wave of cellular infiltration, of
mononuclear phagocytes (monocytes). Differentiation of monocytes
into macrophages promotes the removal of apoptotic neutrophils and
debris by nonphlogistic phagocytosis. Notably, the apoptotic
neutrophil uptake blocks the release of pro-inflammatory mediators
(including chemokines, cytokines and lipid mediators) from M1
macrophages, in a phenomenon termed reprogramming/"immune
silencing", and promotes production and release of
anti-inflammatory and reparative cytokines (M2 macrophages). These
M2 macrophages promote myofibroblast proliferation, support matrix
deposition, and express inhibitors of metalloproteinase that
impairs the remodeling of deposited ECM (Ariel et al., 2012).
Therefore, the milieu that governs macrophage differentiation along
the M1-M2 axis may dictate the magnitude of myofibroblast
activation and deposition of ECM.
Extra Cellular Matrix (ECM) as a Regulator of Tumor Dormancy
[0013] In recent studies, the inventors have clarified potential
mechanisms by which the ECM, which is part of the microenvironment
milieu surrounding the dormant tumor cells, may regulate tumor
dormancy and its outbreak to growing metastases (Barkan et al.,
2008, 2010 and 2010a). Specifically, it was shown that ECM
composition may play a critical role in determining whether
solitary dormant tumor cells remain quiescent or begin to actively
proliferate, using the well-characterized D2.0R/D2A1 mammary cell
line model system to study dormant vs. metastatic proliferative
growth. These cell lines were derived from tumors arising from
implants of the same D2 hyperplastic alveolar nodule line, but
display distinct metastatic properties. Whereas disseminated D2A1
cells in mice transition from dormancy to metastatic growth after
few weeks, disseminated D2.0R cells remain dormant for months with
occasional formation of metastatic lesions. Using a modified 3D
culture system constituted from growth factor reduced basement
membrane (BME; a specialized form of ECM), the inventors
demonstrated for the first time that the quiescent or proliferative
behavior of these cells could be recapitulated in vitro (Barkan et
al., 2010a; Barkan et al., 2011). Thus D2.0R cells persist in a
quiescent state for at least 14 days in the 3D BME system and only
few cells occasionally emerge from dormancy after this period. D2A1
cells, on the other hand, remain quiescent for only 4-6 days in the
3D BME system and then begin to proliferate extensively.
Importantly, the quiescent state is characterized by growth arrest
associated with expression of the negative cell-cycle regulators
p27 and p16, and is not due to a balance between cell proliferation
and apoptosis. Notably, this 3D model system was validated with
additional cell lines demonstrating dormant and metastatic behavior
in vivo. Hence, the model system provides the first in vitro method
to model tumor dormancy and study the transition to proliferative
growth induced by the microenvironment.
Characterization of Fibrotic Tissue
[0014] Fibrosis is one of the pathological features of metastatic
outbreak and is a major pathological feature of many other
diseases. Fibrosis can lead to permanent scaring, organ malfunction
and, ultimately, death, as seen in end-stage liver disease, kidney
disease, idiopathic pulmonary fibrosis and heart failure. There are
some drugs aimed to treat fibrotic disease, such as corticosteroids
(to decrease Col-I synthesis) and angiotensin blocking agents (for
inhibition of collagen synthesis and TGF.beta.1). Unfortunately,
none of them has the ability to cure fibrosis.
[0015] Excessive deposition of matrix proteins such as Col-I and
fibronectin and .alpha.-smooth muscle actin (.alpha.-SMA)
expression in differentiated fibroblasts are the hallmarks of
fibrotic processes. Fibrosis is a complex and tightly orchestrated
phenomenon; it involves multiple signals mediated by various
stromal cells, such as myofibroblasts (which differentiate from
fibroblasts), endothelial cells and macrophages.
[0016] Fibroblasts are considered to be the primary source of the
reparative matrix in all tissues. In response to tissue injury,
they proliferate, migrate to the site of injury, and differentiate
into their activated form, myofibroblasts. Following
differentiation, myofibroblasts acquire an increased contractile
ability and are characterized by the expression of .alpha.-SMA
positive phenotype. In wound healing, these myofibroblasts mediate
wound contraction and the formation of a collagen-rich
extracellular matrix. Increased activation and proliferation of
resident fibroblasts at the wound edge is therefore an important
early step that is central to the wound healing process.
[0017] The cytokine transforming growth factor-.beta.1 (TGF.beta.1)
is a mediator of tissue repair and wound healing and is also
implicated in progressive tissue fibrosis. In addition to its
effect on extracellular matrix turnover, TGF.beta.1 is known to
have direct effects on cell phenotype, including the induction of a
contractile phenotype and the up-regulation of .alpha.-SMA both in
vitro and in vivo.
[0018] The myofibroblast, by virtue of its ability to express high
levels of cytokines, extracellular matrix and .alpha.-SMA, is
expected to have key roles in inflammation, connective tissue
deposition, and lung tissue mechanics, respectively, but that can
severely impair organ function when contraction and ECM protein
secretion become excessive, such as in fibrosis.
[0019] Fibrosis occurs upon deregulated and exaggerated tissue
repair that fails to subside and resolve. During active resolution
of inflammation, specific signals down-regulate macrophage
activation and promote the clearance of activated macrophages,
either by apoptosis or by migration through the lymphatic drainage.
Disruption of any of these processes can lead to chronic persistent
inflammation and fibrosis.
Fibrotic-Like Microenvironment Promotes Dormant Tumor Cells
Proliferation
[0020] A recent report by Barkan and colleagues has demonstrated
for the first time that remodeling of the ECM can regulate the
switch of dormant tumor cells to their metastatic outgrowth.
Specifically, it has been shown that metastatic D2A1 cell line
transitioned from a quiescent state to proliferation upon
production of fibronectin in vitro (Barkan et al., 2008).
Furthermore, metastatic lesions arising from D2A1 cells in vivo
were associated with significant deposition of fibronectin and type
I Collagen (Col-I), whereas a related dormant D2.0R cell line did
not express fibronectin or Col-I. However, supplementing the 3D BME
system with fibronectin and or Col-I induced the transition of
D2.0R cells from quiescence to growth. Furthermore, it has been
recently demonstrated by the inventors for the first time that the
induction of fibrosis, with deposition of Col-I in the in vivo
metastatic microenvironment, induced dormant D2.0R cells to form
proliferative metastatic lesions (Barkan et al., 2010a). Hence,
D2A1 cells were able to escape tumor dormancy by inducing a stromal
fibrotic-like response in vivo, whereas D2.0R cells required an
exogenous fibrotic stimulus to initiate their proliferative
response.
[0021] Importantly, these findings are consistent with several
clinical observations demonstrating a correlation between fibrosis
and breast cancer recurrence (Hasebe et al., 2002). Therefore, a
fibrotic-like microenvironment, which can either be induced
externally or be associated with the pathology induced by the
residing tumor cells, may provide the permissive microenvironment
triggering the transition from dormancy to metastatic growth.
[0022] Immunomodulating therapies designed to affect various immune
cells which may positively or negatively affect tumor progression
are also being considered as potential modulators in the treatment
of cancer and other pathologies.
[0023] WO 1999/029345 discloses a method for the inhibition of
angiogenesis in a cell population in a mammal by inhibiting a host
cell angiogenic effect in said mammal, optionally by creating for
said cell population an environment substantially free of activated
macrophages. The method is said to be useful for the treatment of
cancer. Other publications based on inhibiting or eliminating
macrophages are disclosed in US 2009/258025 and WO 2010/091206.
[0024] EP 0211684 discloses a method of promoting tumoricidal
activity of cells of macrophage lineage, comprising the step of
subjecting the cells of macrophage lineage to an effective amount
of a granulocyte-macrophage colony stimulating factor.
[0025] US 2003/0108534 discloses a method of treating cancer,
comprising administering to a patient in need an effective amount
of macrophages produced by culturing monocytes in vitro, said
macrophages having at least one of the following properties: their
cytotoxic activity without IFN-.gamma. is increased with respect to
standard macrophages; their cytotoxic activity is increased with
IFN-.gamma. with respect to standard macrophages; and deactivation
of the cytotoxic activity following activation of IFN-.gamma. is
defined by residual cytotoxic activity compared to the maximum
cytotoxic activity presented by the macrophages due to IFN-.gamma.
activation.
[0026] Macrophages have also been reported to be involved in
anti-cancer effects induced by Mycobacterium bovis bacillus
Calmette-Guerin immunotherapy of bladder cancer (Luo and Knudson,
2010).
[0027] Thus, a number of distinct macrophages phenotypes may exist
in the mammalian body during different stages of inflammation and
pathology and during steady state homeostasis. Different macrophage
populations have been suggested to exert opposing effects on tumor
progression and inflammation. In addition, interactions between the
macrophages and their environment, such as the inflammatory site,
may also influence the phenotype and hence the function of the
macrophages. It is therefore considered difficult to determine
appropriate parameters for using macrophages for controlled,
predictable therapy, and indeed their clinical use is currently
limited.
[0028] None of the prior art discloses or fairly suggests that
isolated macrophage populations may be used for preventing the
recurrence of cancer and metastatic progression in patients having
cancer remission. There remains an unmet medical need for providing
effective and safe treatments for the treatment of cancer, and
especially for treating patients in cancer remission against tumor
recurrence. Identification of isolated macrophages and secretory
products thereof for inducing cell death in dormant,
non-proliferating cells would be highly advantageous.
SUMMARY OF THE INVENTION
[0029] It has now been unexpectedly found in accordance with the
present invention that certain types of macrophages, particularly
macrophages, herein designated CD11b.sup.low macrophages,
characterized by CD11b expression that is significantly lower than
surface expression of this marker on activated inflammatory
macrophages, or a conditioned medium of said CD11b.sup.low
macrophages, can be useful for inducing cell death in malignant
cells, including dormant, non-proliferating tumor cells. These
cells and the conditioned medium thereof were also unexpectedly
found to inhibit fibrosis as manifested by inhibition of the
transition of fibroblasts into myofibroblasts, including inhibition
of Type I collagen (Col-I) secretion and of .alpha.-SMA
expression.
[0030] Thus, in one aspect, the invention is directed to a
conditioned cell culture medium of CD11b.sup.low macrophages or a
biologically active fraction thereof. In preferred embodiments,
said conditioned medium is of CD11b.sup.low human macrophages.
[0031] In another aspect, the invention is directed to a method for
preparing said conditioned cell culture medium of CD11b.sup.low
macrophages, the method comprising: [0032] (i) culturing a
population of mononuclear cells of the monocyte/macrophage lineage
for 5-7 days, so as to induce differentiation of said mononuclear
cells to macrophages; [0033] (ii) incubating the macrophages
obtained in (i) with apoptotic cells or in the presence of a
pro-resolving lipid mediator to reduce the CD11b expression, thus
obtaining a culture of CD11b.sup.low macrophages; and [0034] (iii)
collecting the conditioned cell culture medium of CD11b.sup.low
macrophages.
[0035] In some embodiments, the mononuclear cells of the
monocyte/macrophage lineage used in the method above are monocytes.
In some embodiments, the monocytes are human monocytes. In
preferred embodiments, the monocytes are autologous, namely, they
are obtained from the patient to whom the CD11b.sup.low macrophages
or a conditioned medium thereof will be administered.
[0036] In some embodiments, the mononuclear cells of the
monocyte/macrophage lineage are human monocytes isolated from a
blood sample of an individual by any conventional method followed
by suspending the cells in a suitable medium. In some embodiments,
the human monocytes are derived from peripheral blood mononuclear
cells (PBMC) of an individual, preferably autologous PBMC.
[0037] In some embodiments, the human monocytes are cultured in
step (i) in autologous serum or in a suitable medium to which
granulocyte-macrophage colony stimulating factor (GM-CSF) or
macrophage colony stimulating factor (M-CSF) is added, and are then
differentiated into a population of human macrophages.
[0038] In some embodiments, for reduction of the CD11b marker
expression, the macrophages obtained in step (ii) are incubated in
the presence of apoptotic cells. In some preferred embodiments, the
ratio of said macrophages to the apoptotic cells is between 1:1 and
1:1.5.
[0039] In some other embodiments, for reduction of the CD11b marker
expression, the incubation of the macrophages in step (ii) is
carried out in the presence of a pro-resolving lipid mediator,
which may be selected from a lipoxin (LX) such as LXB.sub.4 or
15-epi-LXA.sub.4, a resolvin (Rv) such as RvD1 or RvD2, a protectin
(PD) such as protectin PD1, a maresin (MaR), or a glucorticoid such
as dexamethasone.
[0040] In some embodiments, the invention is further directed to a
conditioned cell culture medium of CD11b.sup.low human macrophages
produced according to the method of the invention described
hereinabove.
[0041] In a further aspect, the present invention relates to a
pharmaceutical composition comprising the conditioned cell culture
medium of CD11b.sup.low macrophages of the invention, or a
biologically active fraction thereof, and a pharmaceutically active
carrier, excipient or diluent.
[0042] In some embodiments, the invention relates to the
conditioned cell culture medium of CD11b.sup.low macrophages, a
biologically active fraction thereof or the pharmaceutical
composition comprising said conditioned medium or said active
fraction, for use in the treatment of cancer or fibrosis.
[0043] When the invention is directed to treatment of cancer, said
treatment may comprise preventing or delaying cancer recurrence or
occurrence of metastasis. In some embodiments, said treatment of
cancer comprises inhibiting cancer metastasis. The cancer may be
selected from breast, prostate, esophageal, skin, lung, head and
neck, colon or liver cancer. In some embodiments, the cancer is
breast cancer.
[0044] When the invention is directed to treatment of fibrosis,
said treatment may comprise preventing or inhibiting fibrosis. In
some embodiments, the fibrosis is associated with cancer. In some
other embodiments, the fibrosis is associated with a disease or a
condition selected from acute lung injury, Alzheimer's disease,
chronic diabetic wounds, chronic granulomatous disease (CGD),
chronic obstructive pulmonary disease (COPD), constrictive
pericarditis, Dupuytren's disease, emphysema, hypertrophic burn
scars, keloid, liver cirrhosis, plantar fibromatosis, retinal
detachment inflammation, scleroderma, cystic fibrosis, endometrial
fibrosis, idiopathic pulmonary fibrosis, myocardial fibrosis,
nephrogenic fibrosis, pancreatic fibrosis, perineural fibrosis,
renal interstitial fibrosis, secondary fibrosis in the
gastrointestinal tract or fibrosis resulting after surgery.
[0045] In a further aspect, the present invention is directed to a
culture of isolated CD11b.sup.low human macrophages or a a
pharmaceutical composition comprising said culture and a
pharmaceutically active carrier, excipient or diluent, for use in
the treatment of cancer or fibrosis.
[0046] According to this aspect of the invention, a method is
provided for preparing a culture of said human CD11b.sup.low
macrophages, the method comprising: [0047] (i) culturing a
population of human mononuclear cells of the monocyte/macrophage
lineage for 5-7 days, so as to induce differentiation of said
mononuclear cells to macrophages; and [0048] (ii) incubating the
macrophages obtained in (i) with apoptotic cells or in the presence
of a pro-resolving lipid mediator to reduce the CD11b expression,
thus obtaining a culture of CD11b.sup.low human macrophages.
[0049] All the features described above for the preparation and
uses of the conditioned medium of CD11b.sup.low human macrophages
apply mutatis mutandis to the preparation and uses of the culture
of human CD11b.sup.low macrophages and the pharmaceutical
composition comprising said culture.
[0050] In yet a further aspect, the present invention provides a
method for treating cancer comprising administering to a subject in
need an effective amount of an active agent selected from: (i) a
conditioned cell culture medium of CD11b.sup.low macrophages; (ii)
a biologically active fraction thereof; (iii) a pharmaceutical
composition comprising (i) or (ii) and a pharmaceutically active
carrier, excipient or diluent; (iv) a culture of human
CD11b.sup.low macrophages; or (v) a pharmaceutical composition
comprising (iv) and a pharmaceutically active carrier, excipient or
diluent, wherein said treatment comprises preventing or delaying
cancer recurrence or occurrence of metastasis or inhibiting cancer
metastasis.
[0051] In some embodiments, the above method of preventing or
delaying cancer recurrence or occurrence of metastasis, or
inhibiting cancer metastasis, comprises preventing or inhibiting an
outbreak of dormant cancer cells, particularly wherein said subject
in need is in cancer remission.
[0052] In still a further aspect, the present invention provides a
method for treating fibrosis comprising administering to a subject
in need an effective amount of an active agent selected from: (i) a
conditioned cell culture medium of CD11b.sup.low macrophages; (ii)
a biologically active fraction thereof; (iii) a pharmaceutical
composition comprising (i) or (ii) and a pharmaceutically active
carrier, excipient or diluent; (iv) a culture of human
CD11b.sup.low macrophages; or (v) a pharmaceutical composition
comprising (iv) and a pharmaceutically active carrier, excipient or
diluent, wherein said treatment comprises preventing or inhibiting
fibrosis.
[0053] In some embodiments, the above method of preventing or
inhibiting an outbreak of dormant cancer cells is achieved by
preventing or treating fibrosis associated with said dormant cancer
cells.
[0054] In some embodiments, the method for treating cancer may be
performed in combination with one or more anti-cancer agents or
treatments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIGS. 1A-1B show that CD11b.sup.low macrophages differ in
their protein expression signature from CD11b.sup.high macrophages.
Resolving peritoneal exudates were recovered 66 hours after zymosan
A (1 mg) injection into mice, the cells were immunostained for
Ly-6G, F4/80, and CD11b, and the Ly-6G.sup.-/F4/80.sup.+
macrophages were sorted to CD11b.sup.high and CD11b.sup.low
populations. The recovered macrophages were lysed and their protein
content was analyzed by SDS-PAGE and Western blotting for CD11b,
iNOS, arginase-1, COX-2, 12/15-LO, MMP-9, and .beta.-actin. Tubulin
was used a loading control. Results are shown as representative
blots (A) and mean.+-.SE of three independent experiments (B). Fold
difference in expression was calculated according to the following
formula: Densitometric units (DU) of CD11b.sup.high extract/DU of
CD11b.sup.low extracts from three experiments. Significant
differences by Student's t test between CD11b.sup.high and
CD11b.sup.low extracts (* P value<0.05, ** P value<0.005, ***
P value<0.001) are indicated.
[0056] FIGS. 2A-2C show that CD11b.sup.low macrophages engulf
higher numbers of apoptotic neutrophils than CD11b.sup.high
macrophages. Sorted CD11b.sup.high (black bars and black boxes) and
CD11b.sup.low (white bars and white diamonds) macrophages were
stained with Hoechst and LysoTracker, analyzed by confocal
microscopy, enumerated for apoptotic PMN uptake and analyzed
according to average (A) neutrophils engulfed per macrophage (N/M),
and percentage of cells reaching engulfment threshold of N/M=7 (B)
or engulfing the indicated number of apoptotic PMN (C). Results are
mean.+-.SE (A, B) or representative (C) from three experiments.
Significant differences by Student's t test between CD11b.sup.high
and CD11b.sup.low macrophages (* P value<0.05, ** P
value<0.005, *** P value<0.001) are indicated.
[0057] FIGS. 3A-3G show that CD11b.sup.low macrophages secrete
lower levels of pro-inflammatory cytokines and chemokines, but
higher levels of TGF.beta. in comparison to CD11b.sup.high
macrophages. Sorted CD11b.sup.high (black bars in A, B) and
CD11b.sup.low (white bars in A, B) macrophages were activated with
lipopolysaccharide (LPS) at the indicated concentrations (A-B) or
at 1 .mu.g/ml (C-G), and evaluated for secreted TNF.alpha. (A),
IL-1.beta. (B) IL-10 (C), TGF.beta. (D), CCL2 (E), CCL3 (F), and
CCL5 (G) using standard ELISA. Results are mean.+-.SE of four
replicates from a representative of three experiments. N.A.: not
available. In FIGS. 3C-G, black bars: LPS, white bars: vehicle
(RPMI 10% FBS). Significant differences by Student's t test between
CD11b.sup.high and CD11b.sup.low macrophages or between vehicle and
LPS-stimulated macrophages (* P value<0.05, ** P value<0.005,
*** P value<0.001) are indicated.
[0058] FIGS. 4A-4D shows that CD11b.sup.low macrophages secret
lower levels of pro-inflammatory cytokines in response to various
Toll-like receptor (TLR) ligands. Sorted CD11b.sup.high and
CD11b.sup.low macrophages were not activated (added vehicle, white
bars), activated with poly (I:C) (4 mg/ml; A-B), or with CpG-ODN (1
mM; C-D) (black bars) and evaluated for secreted TNF.alpha. (A,C)
or IL-1.beta. (B,D) using standard ELISA. Results are mean.+-.SE of
four replicates from a representative of three experiments.
Significant differences by Student's t test between CD11b.sup.high
and CD11b.sup.low macrophages or between macrophages stimulated
with vehicle, poly (I:C), or CpG-ODN (* P value<0.05, ** P
value<0.005, *** P value<0.001) are indicated.
[0059] FIGS. 5A-5C show that CD11b.sup.low macrophages are
satiated. PKH2-PCL-green was injected intraperitoneally (i.p.) to
mice 48 hrs post peritonitis initiation. After 4 hrs, the
peritoneal cells were recovered, immune-stained for F4/80 and CD11b
and analyzed using FACSCalibur. Results are representative dot plot
for CD11b.sup.low (A) and CD11b.sup.high macrophages (B), or mean
fluorescence intensity (MFI) values.+-.SE of 5 experiments (C).
Significant differences by Student's t test between CD11b.sup.high
and CD11b.sup.low macrophages (*** P value<0.001) are
indicated.
[0060] FIGS. 6A-6B show that CD11b.sup.low macrophages are prone to
migrate to lymphoid organs. (A) Cells were recovered from the
peritoneum (white), inguinal lymph node (LN, striped) and spleen
(black) of mice undergoing peritonitis for 66 hrs. The cells were
stained as above and CD11b expression on macrophages was determined
by flow cytometry. (B) Macrophages were recovered from peritoneal
exudates 48 hrs post peritonitis initiation, labeled with CFSE, and
transferred to recipient mice undergoing peritonitis for the same
period. After 18 hrs, the cells from the peritoneum (white),
inguinal LN (striped, and spleen (black) were recovered and the
expression of CD11b on transferred macrophages was determined as
above. Results presented are mean.+-.SE from three experiments, 5
mice per experiment. Significant differences by Student's t test
between CD11b.sup.high and CD11b.sup.low macrophages (* P
value<0.05, ** P value<0.005, *** P value<0.001) are
indicated.
[0061] FIGS. 7A-7D show that interaction with apoptotic leukocytes
converts CD11b.sup.high macrophages to CD11b.sup.low macrophages.
Sorted CD11b.sup.high (A) or CD11b.sup.low (B) macrophages were
immunostained for CD11b immediately (A-B, dotted line), or after
incubation for 20 hrs without cells (A-B, dashed line) or with
apoptotic Jurkat cells (1:5 ratio; A-B, solid line). Then the
unbound apoptotic cells were washed and the macrophages were
recovered and immunostained for CD11b expression on their surface.
Results are representative (A, B; depicted macrophage populations
indicated) or average MFI values.+-.SE (C) of 3 experiments; white
bars: incubation w/o cells, black bars: incubation with apoptotic
cells). Significant differences by Student's t test between
CD11b.sup.high and CD11b.sup.low macrophages (* P value<0.05, **
P value<0.005, *** P value<0.001) are indicated. (D).
Peritoneal macrophages were recovered 66 hrs post peritonitis
initiation and incubated with apoptotic Jurkat cells (+) or without
cells (-), as indicated. After 20 hrs the macrophages were
recovered and lysed. The protein extracts were run by SDS-PAGE and
analyzed by Western blot for CD11b, arginase-1, 12/15-LO, actin and
tubulin as loading control. Results are a representative set of
blots from 3 experiments.
[0062] FIGS. 8A-8B show promotion of macrophage switch to the
CD11b.sup.low phenotype by pro-resolving lipid mediators.
Macrophages were recovered from peritoneal exudates 72 hrs post
peritonitis initiation and incubated overnight with vehicle, RvD1,
RvD2, PD1, LXB.sub.4, or 15-epi-LXA.sub.4, (all at 50 nM),
apoptotic cells (M/N ratio of 1:5), or Dex (25 .mu.g/ml
dexamethasone). Then, the cells were stained for CD11b and analyzed
by flow cytometry, and the percentage of CD11b.sup.low (A) and
CD11b.sup.high (B) macrophages was determined. Results are
mean.+-.SE of two experiments (n=2). # P<0.005, $ P<0.01, *
P<0.05 compared to vehicle.
[0063] FIGS. 9A-9E show the generation of CD11b.sup.high enriched
macrophages for ex vivo experiments. A. Selection of macrophages
from exudates based on their size and granularity, arrow points to
the relevant area of size and granularity. B. Histogram of
identified macrophages, stained positively for F4/80. C. Percentage
of macrophages (M.sup.-) (identified by positive staining for
F4/80) and other cells (negative staining for F4/80 in peritoneal
exudates), (n=5, *: p.ltoreq.0.05). D. Characterization of
macrophages stained positively for F4/80 by CD11b expression
levels. CD11b.sup.high and CD11b.sup.low are shown. E. Percentage
of CD11b.sup.high and CD11b.sup.low macrophages before incubation
with apoptotic cells (AC) (n=3, **: p.ltoreq.0.01).
[0064] FIGS. 10A-10D show conversion of CD11b.sup.high macrophages
to CD11b.sup.low macrophages upon their incubation with apoptotic
cells at a ratio of M.phi. to apoptotic cells (AC) of 1:5. A-D show
FACS analysis of CD11b.sup.high macrophages either untreated (A) or
incubated with apoptotic cells (AC) overnight (O.N.) (B-D). A.
Characterization of untreated CD11b.sup.high macrophages (M.phi.)
stained positively for F4/80, for CD11b expression levels after O.N
incubation. B. Histogram of macrophages identified by staining
positively for F4/80, after incubation with AC at a ratio of 1:5.
C. Characterization of M.hoarfrost. stained positively for F4/80,
for CD11b levels after O.N incubation with AC cells at a ratio of
1:5. D. The percentage of CD11b.sup.high (black bars) and
CD11b.sup.low (white bars) macrophages of untreated M.phi. or
M.phi. incubated with AC at a ratio of 1:5 (M.phi.+AC(1:5)).
Representative result (n=3).
[0065] FIGS. 11A-11D show conversion of CD11b.sup.high macrophages
to CD11b.sup.low macrophages upon their incubation with apoptotic
cells at a ratio of M.phi. to apoptotic cells (AC) of 1:1 or 1:2.
A-D show FACS analysis of CD11b.sup.high macrophages either
untreated or incubated O.N with apoptotic cells (AC). A. Histogram
of macrophages identified by staining positively for F4/80, either
untreated (top left panel) or incubated with AC at a ratio of 1:1
or 1:2 (top right panel and bottom panel, respectively). B.
Characterization of F4/80 positive macrophages either untreated
(top panel) or incubated with AC at a ratio of 1:1 or 1:2 (middle
panel and bottom panel, respectively), for CD11b expression levels.
C. The percentage of CD11b.sup.high (black bars) and CD11b.sup.low
(white bars) macrophages of untreated M.phi. (left pair) or M.phi.
incubated with AC at ratios of 1:1 or 1:2 (middle bar and right
bar, respectively). Representative result (n=3). D. Percentage of
the residual AC which did not undergo engulfment by CD11b.sup.high
macrophages when incubated with AC at ratio of 1:1, 1:2 and 1:5
(from left to right).
[0066] FIG. 12 shows reduction in MEF number upon incubation with
conditioned media of enriched CD11b.sup.low macrophages (CM-Mres)
with TGF.beta.1 (panel at the lower left). Representative bright
field microscopy images of MEF grown on coaster 6 plate.
Magnification.times.20. Bars equal 50 .mu.m: upper panels, left:
MEF alone; right: MEF with TGF.beta.1; lower panels, left: MEF with
TGF.beta.1 and CM-Mres; right: MEF with TGF.beta.1 and conditioned
media of enriched CD11b.sup.high macrophages (CM-M.phi.).
[0067] FIGS. 13A-13B show that MEFs treated with CM-Mres are cell
cycle arrested and not apoptotic. A. Proliferation of MEF cells
either untreated (diamonds on dashed line) or treated with
TGF.beta.1 alone (dotted line), or with TGF.beta.1 in the presence
of conditioned medium from either CD11b.sup.high-enriched
(CM-M.phi., triangles), CD11b.sup.low-enriched (CM-Mres, diamonds
on solid line) or apoptotic cells (CM-AC, circles). (n=5). B.
Quantification of the percentage of MEFs undergoing apoptosis.
Percentage of apoptotic cells from total cell number was determined
by TUNEL assay (n=3, no significance). From left to right: MEF
untreated, or treated with 1 ng/ml TGF.beta.1, TGF.beta.1+CM-Mres,
or 1.5 .mu.g/ml staurosporine (STS) (positive control).
[0068] FIGS. 14A-14B show low expression of .alpha.-SMA in MEF
cells treated with TGF.beta.1+ CM-Mres. A. Western blot analysis
showing expression levels of .alpha.-SMA, representative blots from
two independent experiments. B. Quantification of .alpha.-SMA
expression levels by densitometry from two independent experiments.
Values normalized to GAPDH (n=2; **: p.ltoreq.0.01). From left to
right: MEFs untreated, or treated with TGF.beta.1 alone or together
with CM-M.phi., CM-Mres, or CM-AC.
[0069] FIG. 15 shows that CM-Mres reduced the expression of type I
collagen (Col-I) in TGF.beta.1-treated MEF. Percentage of Col-I
expression is shown in MEFs treated from left to right: untreated,
or treated with TGF.beta.1 alone or together with CM-M.phi.,
CM-Mres, or CM-AC. Values were normalized to TGF.beta.1-treated MEF
with CM-M.phi. (n=3; *: p.ltoreq.0.05; **: p.ltoreq.0.01).
[0070] FIG. 16 shows abolishment of Col-I expression in MEF cells
cultured in 3D BME culture upon CM-Mres treatment. Quantification
of Col-I expression by MEF cultured in the 3D BME system is shown
in the following treatments, from left to right: untreated, treated
with CM-M.phi., CM-Mres or CM-AC. Values were normalized to
CM-M.phi. treatment (n=3; *: p.ltoreq.0.05; **: p.ltoreq.0.01; ***:
p.ltoreq.0.001).
[0071] FIGS. 17A-17C show that conditioned media of pro-resolving
CD11b.sup.low macrophages induces apoptosis of outbreaking (day 4)
dormant D2A1 cells in the 3D BME system. A. Generation and
characterization of macrophage populations enriched for
CD11b.sup.high (white bars) or CD11b.sup.low (black bars)
macrophages. From left to right: macrophages only; with addition of
live Jurkat cells (LC); with addition of apoptotic Jurkat cells
(AC). B. Proliferation of D2A1 cells in the 3D system overlaid on
day 4 (prior to their transition from dormancy to proliferation)
with conditioned media from the different macrophage populations
(from left to right): untreated, treated with apoptotic Jurkat
cells (AC); treated with CD11b.sup.high macrophages; treated with
CD11b.sup.high macrophages and live Jurkat cells (LC); treated with
CD11b.sup.low macrophages. ***: P.ltoreq.0.001. C. Proliferation
(normalized to control) of D2A1 cells in the 3D system overlaid on
day 2 (when dormant) with conditioned media from the different
macrophage populations (from left to right): untreated macrophages;
enriched with CD11b.sup.high macrophages; enriched with
CD11b.sup.low macrophages; (** P value<0.005, *** P
value<0.001).
DETAILED DESCRIPTION OF THE INVENTION
[0072] Recently, some of the inventors of the present application
identified and characterized a novel subset of pro-resolving
macrophages designated CD11b.sup.low macrophages that appear during
the resolution of murine peritonitis (Schif-Zuck et al., 2011).
Furthermore, they found that these macrophages display an enzyme
expression signature distinct from either M1 or M2 macrophages.
CD11b.sup.low macrophages can be generated in vivo or ex vivo from
M2-like macrophages that are characterized by high levels of CD11b
expression and termed CD11b.sup.high macrophages, following the
engulfment of apoptotic leukocytes, which is a hallmark of
resolution. Furthermore, these newly discovered pro-resolving
CD11b.sup.low macrophages, also termed resolution-promoting
macrophages or Mres, lost their phagocytic potential and were prone
to migrate to lymphoid tissues.
[0073] In the present invention, the inventors show for the first
time that conditioned media of these recently described
CD11b.sup.low macrophages can inhibit fibrosis, as manifested by
inhibition of the transition of fibroblasts into myofibroblasts,
including inhibiting Type I collagen (Col-I) secretion and
.alpha.-SMA expression. The inventors have further surprisingly
found that a conditioned medium of CD11b.sup.low macrophages has
highly effective cytotoxic and cytostatic activities. Specifically,
using a three-dimensional culture system that models tumor dormancy
and outbreak of disseminated tumor cells it was unexpectedly found
that the conditioned medium not only inhibited the proliferation of
outbreaking dormant tumor cells, but also eradicated effectively
even non-proliferating dormant tumor cells, which are largely
resistant to currently used chemotherapeutic agents. These
characteristics indicate that the conditioned medium may be useful
for preventing or inhibiting metastasis and preventing the
recurrence of cancer after remission.
[0074] CD11b.sup.high and CD11b.sup.low macrophages are two
populations of macrophages typically isolated in murine models from
peritoneal exudates during the resolution phase of peritonitis. As
can be seen in FIGS. 9D and 9E, fluorescence activated cell sorter
(FACS) analysis shows the two populations: the majority of cells
expressing high levels of CD11b (CD11b.sup.high) and the minority
of cells expressing low levels of CD11b (CD11b.sup.low). The whole
population of macrophages isolated from the resolution phase of
inflammation is termed hereinbelow as "resolution-phase
macrophages".
[0075] The level of CD11b or other markers may be determined by
various methods known in the art, e.g. immunostaining and FACS
analysis, or gel electrophoresis and Western blotting. The term
"CD11b.sup.high macrophages", as used herein, relates to
macrophages expressing high levels of CD11b. The term
"CD11b.sup.low macrophages", as used herein, relates to macrophages
that express on their surface a level of CD11b that is
substantially lower than that of Cd11b.sup.high macrophages.
Sometimes the term "CD11b deficient macrophages" is used to define
"CD11b.sup.low macrophages" and both terms should be considered as
synonyms.
[0076] The term "culture macrophages" refers herein to human
macrophages differentiated in culture from peripheral blood
mononuclear cells (PBMC). Culture macrophages are typically
obtained by incubating PBMC for 5-7 days. In some embodiments, the
cells are cultured in the presence of autologous serum (e.g. 1-10%,
preferably 10%), this being serum obtained from the blood of the
patient to whom the culture of CD11b.sup.low macrophages or the
conditioned medium thereof will be administered. In some other
embodiments, the cells are cultured in a suitable medium such as
RPMI 1640, supplemented with 10% human serum, 2 mM L-glutamine, 100
.mu.g/ml streptomycin, and 100 units/ml penicillin, to which
granulocyte-macrophage colony stimulating factor (GM-CSF; e.g. 10
.mu.g/ml) or, preferably, macrophage colony stimulating factor
(M-CSF; e.g. 25 .mu.g/ml), has been added.
[0077] It is shown herein that expression of CD11b on
Cd11b.sup.high macrophages was about tenfold higher than its
expression on CD11b.sup.low macrophages. The amount of CD11b in
protein extracts of CD11b.sup.low macrophages was also
significantly lower than its amount in the extracts from
CD11b.sup.high macrophages (see Example 1).
[0078] Thus, according to some embodiments of the invention, the
mean level of expression of CD11b.sup.low macrophages is defined as
being 2-100 times lower than the mean CD11b expression on
CD11b.sup.high macrophages or on culture macrophages. According to
some embodiments, the mean level of expression of CD11b.sup.low
macrophages is 2-10 times lower than the mean CD11b expression on
CD11b.sup.high macrophages or on culture macrophages. According to
some embodiments, the mean level of expression of CD11b.sup.low
macrophages is 11-100 times lower than the mean CD11b expression on
CD11b.sup.high macrophages or on culture macrophages. According to
some embodiments, the level of CD11b expression on CD11b.sup.low
macrophages is undetectable. Thus, according to some embodiments,
the level of CD11b expression on CD11b.sup.low macrophages may be
defined as 0 (zero). All the CD11b level measurements define
surface expression detected by flow cytometry.
[0079] As mentioned above and explained in more detail below,
CD11b.sup.low murine macrophages can be isolated from a macrophage
population retrieved during the resolution phase of murine
peritonitis. Additionally, CD11b expression on the surface of both
CD11b.sup.high and CD11b.sup.low macrophages can be significantly
reduced following their incubation with apoptotic cells (see
Examples 6, 9). However, following incubation with apoptotic cells,
some of the macrophages have even lower expression levels of CD11b
compared to the level of expression found for CD11b.sup.low
macrophages isolated from peritoneal exudates, and are termed
CD11b.sup.- macrophages. Therefore the ex vivo-generated population
of macrophages expressing low levels of CD11b may be comprised from
both CD11b.sup.low and CD11b.sup.- macrophages.
[0080] Accordingly, the definition of CD11b.sup.low macrophages
also includes CD11b.sup.- macrophages that are substantially devoid
of surface CD11b or have very low, barely detectable, levels of
CD11b or no CD11b at all.
[0081] According to some embodiments of the present invention, the
population of CD11b.sup.low macrophages is comprised of a
heterogeneous population of cells. Typically, at least 50% of the
heterogeneous populations of cells are CD11b.sup.low macrophages.
In some embodiments, more than 55%, 60%, 65%, 70%, 75% or more than
80% of the cells are CD11b.sup.low macrophages in heterogeneous
populations [see FIGS. 10, 11].
[0082] In some embodiments, the population of CD11b.sup.low
macrophages is comprised of a homogeneous population, meaning that
it is substantially devoid of macrophages that are not
CD11b.sup.low macrophages.
[0083] The terms "CD11b deficient macrophages",
"resolution-promoting macrophages", "pro-resolving macrophages", or
"Mres" are used hereinbelow interchangeably with, and have the same
meaning as, "CD11b.sup.low macrophages".
[0084] The effect of treating mouse embryonic fibroblasts (MEF)
with a conditioned medium from CD11b.sup.low macrophages was shown
in Examples 10-14. To mimic the process of fibrosis, mouse
embryonic fibroblasts (MEFs) were induced to differentiate to
myofibroblasts by treating the cells with TGF.beta.1. As shown in
Example 10, treatment with conditioned medium from Mres
(CD11b.sup.low macrophages) of MEFs which were induced with
TGF.beta., resulted in less proliferation than treatment with
conditioned medium from CD11b.sup.high macrophages. Further,
according to Example 11, this lack of proliferation was most likely
due to growth arrest, since most of the MEFs were alive and did not
stain by a TUNEL assay, which detects apoptosis. Example 12 shows
that conditioned medium from CD11b.sup.low significantly inhibited
the expression of .alpha.-SMA in MEF (.alpha.-SMA marks their
differentiation to myofibroblasts) treated with TGF.beta.1 compared
to the expression levels in MEF treated with TGF.beta.1+conditioned
medium from CD11b.sup.high macrophages. According to Examples 13
and 14, conditioned medium from CD11b.sup.low macrophages
significantly inhibited Col-I expression of TGF.beta.1-treated MEFs
compared to untreated MEFs and to TGF.beta.1-treated MEFs that were
treated with conditioned medium from CD11b.sup.high
macrophages.
[0085] Additionally, the effect of a conditioned medium from
CD11b.sup.low macrophages on the proliferation of dormant tumor
cells was shown in Examples 15 and 16 for tumor cells cultured in a
modified 3D culture system constituted from growth factor reduced
basement membrane (BME; a specialized ECM). This 3D BME system,
described recently in Barkan et al., 2011, can model tumor dormancy
and outgrowth. Supplementing the 3D BME system with Col-I was shown
to induce the transition of dormant tumor cells from quiescence to
proliferative growth. Example 15 shows that conditioned media from
CD11b.sup.low cells induced cell death in outbreaking tumor cells
during their transition from dormancy to proliferation in the 3D
system, and also of dormant tumor cells. Additionally, Example 16
shows that treating dormant tumor cells co-cultured in the 3D BME
system with a conditioned medium from CD11b.sup.high macrophages
causes them to emerge from their dormant state, but treatment with
a conditioned medium from CD11b.sup.low macrophages prevented Col-I
expression by the differentiated MEFs and resulted in lower numbers
of tumor cells.
[0086] In one aspect of the invention, there is provided a
conditioned cell culture medium of CD11b.sup.low macrophages or a
biologically active fraction thereof.
[0087] The term "conditioned medium" in general refers to a growth
medium in which cells have been cultured/incubated for a period of
time followed by harvest of such medium from the cultured cells. A
conditioned medium contains metabolites, growth factors, and
extracellular matrix proteins secreted into the medium by the
cultured cells.
[0088] In accordance with the present invention, the term
"conditioned medium of CD11b.sup.low macrophages" refers to the
medium harvested from the cultured macrophages after the incubation
with either apoptotic cells or with a pro-resolving lipid mediator,
when the CD11b.sup.low macrophages are obtained. The conditioned
medium may be derived from autologous serum or from a medium
suitable for culture of monocytes such as, but not limited to,
Iscove's Modified Dulbecco's Medium (IMDM) and RPMI 1640, and may
contain additional nutrients.
[0089] In some embodiments of the invention, the conditioned medium
of CD11b.sup.low macrophages is based on RPMI 1640 with 10% fetal
bovine serum (FBS) or 10% human serum and supplemented with 2 mM
L-glutamine, 100 .mu.g/ml streptomycin, and 100 units/ml
penicillin. It also contains soluble factors secreted by the
cultured cells into the medium. The conditioned medium may be
purified, e.g. by filtration or centrifugation, to provide
substantially cell free preparations.
[0090] In the method for preparing the conditioned medium of the
invention, in a first step a population of mononuclear cells of the
monocyte/macrophage lineage, preferably human monocytes, is
cultured for 5-7 days, so as to induce differentiation of said
mononuclear cells/monocytes to macrophages. The culture is made in
autologous serum or in a medium suitable for culture of monocytes
as defined above in paragraphs [0071] and [0083].
[0091] In a second step of the method, the macrophages obtained in
the first step, which are mainly CD11b.sup.high macrophages, are
incubated with an agent that will reduce the CD11b expression of
the macrophages so as to obtain a macrophage population mainly
constituted of CD11b.sup.low macrophages.
[0092] In some embodiments, the agent used for reduction of the
CD11b expression of macrophages is apoptotic cells.
[0093] As shown in Example 1 hereinafter, CD11b.sup.high and the
CD11b.sup.low macrophage populations are distinct populations, as
shown by the difference in protein expression profiles between
these two populations, For example, CD11b.sup.low macrophages may
express lower levels (typically 2-10 times lower) of COX-2 and/or
MMP-9 compared to CD11b.sup.high macrophages. CD11b.sup.low
macrophages may also express 12/15-LO at a level significantly
higher than CD11b.sup.high macrophages. CD11b.sup.low typically
express iNOS, arginase-1 or actin a significantly reduced level
compared to CD11b.sup.high macrophages, or in the case of iNOS or
arginase-1, possibly not at all.
[0094] CD11b.sup.low macrophages may therefore be further
characterized by their expression level of certain additional
markers, such as an increased expression level of 12/15-LO and/or a
reduced expression level of iNOS, arginase-1 or actin.
[0095] Example 2 shows that CD11b.sup.low macrophages engulfed
significantly more PMN on average than CD11b.sup.high macrophages
and the two populations were distinguished by an engulfment
threshold of seven PMN. Example 4 shows that CD11b.sup.low
macrophages may be considered "satiated", meaning that they lost
their phagocytic potential upon meeting the apoptotic PMN
engulfment threshold and reducing their CD11b expression.
[0096] To avoid potential residual effect of the apoptotic cells
(AC) in the conditioned media, it is important that most AC is
engulfed by the macrophages. Example 9 shows the effect of
different ratios of CD11b.sup.high macrophages to AC, on the
fraction of AC that remains after engulfment. A ratio of 1:1 left
only 8% AC not engulfed, while higher macrophage/AC ratios of
1:1.5, 1:2 or 1:5 left more AC not engulfed.
[0097] Accordingly, in some embodiments the incubation of
mononuclear cells in the presence of apoptotic cells is performed
at a ratio of mononuclear cells to apoptotic cells of between 1:1
and 1:5. In some embodiments, the ratio between mononuclear cells
and apoptotic cells is between 1:1 and 1:1.5. In some embodiments,
the ratio between mononuclear cells and apoptotic cells is 1:1. In
some embodiments incubation of mononuclear cells in the presence of
apoptotic cells is performed for 16-24 hours. For example, the
incubation with the apoptotic cells may take from 5 to 10,
preferably 8, hours, followed by washing of the cells (e.g., with
PBS), addition of fresh medium and further incubation for 10-15,
preferably, 12, hours. In some embodiments the incubation of
mononuclear cells in the presence of apoptotic cells is performed
such that the average number of engulfed apoptotic cell per
mononuclear cell is 7. In some embodiments incubation of
mononuclear cells in the presence of apoptotic cells is performed
in the presence of one or more anti-inflammatory agents such as,
but not limited to, IL-10, TGF.beta., PPAR.lamda. ligand, vitamin D
and its derivatives, glucocorticoid, or other pro-resolving lipid
mediators.
[0098] In certain embodiments the apoptotic cells are
polymorphonuclear cells. In some embodiment the apoptotic cells are
neutrophils. In some embodiment the apoptotic cells are
lymphocytes, e.g. T cells. Such cell populations may be produced by
standard procedures known in the art. Apoptosis may be induced by a
variety of well-known methods including but not limited to,
incubation with staurosporine (e.g. 1 .mu.M for 4 hr) or etoposide
(0.5 .mu.M), or exposure to UV irradiation (e.g. 5 minutes).
Apoptosis may also be induced in neutrophils by prolonged
incubation (e.g. for 12-24 hours) in culture media in the absence
of cytokines. Any type of apoptotic cells may be used such as
apoptotic Jurkat T cells after treatment with staurosporine.
[0099] In some other embodiments, the agent used for reduction of
the CD11b expression of macrophages is a pro-resolving lipid
mediator. The pro-resolving lipid mediator may be selected from,
without being limited to, a lipoxin (LX), a resolvin (Rv), a
protectin (PD), a maresin (MaR), or a glucorticoid. Examples of
such agents, without being limited to, are lipoxin LXB.sub.4 and
15-epi-LXA.sub.4, resolvins RvD1 and RvD2, protectin PD1, and
dexamethasone.
[0100] Example 7 shows that resolvins (RvD1, RvE1) and
glucocorticoids (Dex) enhanced the appearance of CD11b.sup.low
macrophages and RvD1 and RvE1, but not Dex, also reduced CD11b
expression on CD11b.sup.high macrophages. The secretion of IL-10, a
pro-resolving cytokine generated following the ingestion of
apoptotic cells, was up-regulated by each of the pro-resolving
mediators, in unstimulated and LPS-stimulated macrophages. Thus,
treatment with RvD1, RvE1, and Dex, promoted macrophage
immune-silencing, as well as the secretion of pro-resolving
cytokines from these cells. Example 8 shows that ex vivo treatment
with the pro-resolving lipid mediators RvD1 or RvD2, the protectin
PD1, the lipoxins LXB.sub.4 or 15-epi-LXA.sub.4, or dexamethasone
(Dex), promotes macrophage switch to the CD11b.sup.low phenotype
similarly to the effect of incubation with apoptotic cells.
Therefore, such treatment can possibly either enhance the effect of
incubating with apoptotic cells in order to enrich for
CD11b.sup.low macrophages, or such treatment with pro-resolving
lipid mediators may replace incubation with apoptotic cells.
[0101] In the last step of the method, the conditioned medium is
collected from the culture of the CD11b.sup.low macrophages and
further purified by standard procedures.
[0102] In a further aspect, the present invention relates to a
pharmaceutical composition comprising the conditioned cell culture
medium of CD11b.sup.low macrophages of the invention, or a
biologically active fraction thereof, and a pharmaceutically
acceptable carrier, excipient or diluent.
[0103] The conditioned medium of the present invention or prepared
according to the methods of the present invention or fractions
thereof can be administered to individuals in need per se or in a
pharmaceutical composition with suitable carriers, excipients or
diluents.
[0104] As used herein, the term "a biologically active fraction
thereof" refers to a fraction of the conditioned cell culture
medium of CD11b.sup.low macrophages obtained by fractionation of
the conditioned medium and shown to retain the same biological
activities as shown herein for the whole conditioned medium such as
cytotoxic, anti-metastatic or anti-fibrotic activity. Conveniently,
the biological activity may be determined using the three
dimensional culture system that models tumor dormancy and breakout,
e.g. as exemplified in the Examples section below. The
fractionation of the conditioned medium may be carried out by
standard procedures, e.g. organic extractions, size exclusion
fractions and/or HPLC fractionation, as known in the art.
[0105] The term "pharmaceutically acceptable carrier" refers to a
carrier or diluent that does not cause irritation or other adverse
effect to an organism and does not have an adverse effect on the
biological activity and properties of the administered compound.
The "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of
an active ingredient. Examples, without limitation, of excipients
include calcium carbonate, calcium phosphate, various sugars and
types of starch, cellulose derivatives, gelatin, vegetable oils and
polyethylene glycols.
[0106] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, grinding,
pulverizing, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.
[0107] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active agents into preparations that can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0108] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal, or
parentersl delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal or
intraocular injections.
[0109] Alternatively, the pharmaceutical composition may be
administered in a local rather than systemic manner, for example,
via injection directly into a tissue region of a patient.
[0110] For injection, the active ingredients of the compositions of
the invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants for example
DMSO, or polyethylene glycol are generally known in the art.
[0111] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a "therapeutically effective
amount" means an amount of an active ingredient effective to
prevent, alleviate or ameliorate symptoms of a disease or disorder
or prolong the survival of the subject being treated.
[0112] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0113] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations or a single administration of a slow release
composition, with course of treatment lasting from several days to
several weeks or until cure is effected or diminution of the
disease state is achieved.
[0114] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0115] The CD11b.sup.low macrophages or the conditioned media
thereof may be re-infused (in the case of autologous treatment, or
infused, in the case of an allogeneic treatment) into the patient
(e.g. in a physiologic PBS solution) systemically, e.g.
intravenously, by conventional clinical procedures. Patients may
generally receive from about 10.sup.6 to about 10.sup.12
macrophages, depending on the condition of the patient. The
macrophages administered are typically viable and may optionally be
activated prior to administering to the subject (e.g. by treatment
with corticosteroids, PPAR.gamma. agonists, TGF.beta. or vitamin
D).
[0116] The conditioned cell culture medium of CD11b.sup.low
macrophages. a biologically active fraction thereof or a
pharmaceutical composition comprising them can be used for
treatment of cancer or fibrosis.
[0117] The term "treating" or "treatment" as used herein includes
abrogating, substantially inhibiting, slowing or reversing the
progression of a condition, substantially ameliorating clinical
symptoms of a condition or substantially preventing the appearance
of clinical symptoms of a condition, With regard to cancer, the
term refers to preventing or delaying cancer recurrence or
occurrence of metastasis, inhibiting tumor growth or causing death
of cancer cells, especially of metastatic cancer cells. Such
treatment can also lead to regression of tumor growth, i.e., to
decrease in size or complete regression of the tumor, and to
elimination of metastases. The terms "tumor" and "cancer" are used
interchangeably herein.
[0118] According to some embodiments, the treatment according to
the present invention prevents the proliferation or outbreak of
dormant tumor cells.
[0119] The terms "dormant cells" or "dormant tumor cells" as used
herein relate to either of the two states that have been described
regarding tumor dormancy. Dormant tumor cells may exist in a
quiescent state for many years as solitary quiescent tumor cells as
demonstrated by their negative staining for the proliferation
marker Ki67 and their negative staining for apoptosis.
Alternatively, tumor dormancy may exist as micrometastases where
cellular proliferation is balanced by apoptosis. These
micrometastases remain dormant because of lack of recruitment of
the vasculature needed to nourish the tumor, known as the
angiogenic switch and/or involvement of the adaptive immune system
such as cytotoxic CD8.sup.+ T cells.
[0120] In some embodiments of the invention, the treatment of
cancer comprises inhibiting cancer metastasis.
[0121] The term "treating" or "treatment" as used herein with
regard to fibrosis, refers to preventing the occurrence of fibrosis
or inhibiting fibrosis by inhibiting the progress and/or the extent
of the fibrotic process.
[0122] The term "fibrosis" as used herein, relates to a process in
which excess fibrous connective tissue is formed in an organ or
tissue in a reparative or reactive process. In the process of
fibrosis, the connective tissue deposited can obliterate the
architecture and function of the underlying organ or tissue.
Fibrosis can happen in response to injury (scarring) or as part of
a pathological process such as, for example, in liver disease,
kidney disease, pulmonary fibrosis or heart failure.
[0123] In a further aspect, the present invention is directed to a
culture of isolated CD11b.sup.low human macrophages or a cellular
therapy preparation comprising said culture and a pharmaceutically
active carrier, excipient or diluent, for use in the treatment of
cancer or fibrosis.
[0124] In yet a further aspect, the present invention provides a
method for treating cancer comprising administering to a subject in
need an effective amount of an active agent selected from: (i) a
conditioned cell culture medium of CD11b.sup.low macrophages; (ii)
a biologically active fraction thereof; (iii) a pharmaceutical
composition comprising (i) or (ii) and a pharmaceutically active
carrier, excipient or diluent; (iv) a culture of human
CD11b.sup.low macrophages; or (v) a pharmaceutical composition
comprising (iv) and a pharmaceutically active carrier, excipient or
diluent, wherein said treatment comprises preventing or delaying
cancer recurrence or occurrence of metastasis or inhibiting cancer
metastasis.
[0125] The types of cancer that can be treated by the method of the
invention are typically cancers that relapse, meaning that the
patient can be in remission, that is, free of active disease, but
the cancer may become active again. As explained above, recurrence
of the disease is generally due to the presence of dormant cells
which may become activated. Additionally, treatment-eligible tumors
are also those which metastasize.
[0126] Accordingly, in some embodiments, the active agents of the
invention may be used to prevent and treat recurring metastatic
cancers. It is also appreciated that they can be used also for
treatment of patients with an active cancer, for example, to
prevent further metastases or inhibit the level of tumor
spreading.
[0127] In some embodiments, the cancer is selected from breast,
prostate, esophageal, skin, lung, head and neck, colon or liver
cancer. In some embodiments, the cancer is breast cancer.
[0128] In some embodiments, the cancer is resistant to
chemotherapy, immunotherapy, anti-angiogenic therapy and/or
radiotherapy. In some embodiments, the cancer is resistant to
chemotherapy and/or radiotherapy. In some embodiment the cancer is
resistant to hormone responsive therapy. For example, the
compositions and methods of the invention may be suitable for the
treatment of heterogeneous hormone responsive tumors (e.g. breast
or prostate tumors), which are not amenable for treatment with
conventional hormone responsive therapies since the respective
receptors are not expressed on substantially all the tumor cells.
Advantageously, the treatments of the invention may be used for
treatment of triple-negative breast cancer, i.e. breast tumors not
expressing the genes for estrogen receptor (ER), progesterone
receptor (PR) and Her2/neu, which are thus not amenable for
treatment with hormone-responsive therapies. Often, such triple
negative breast tumors are also resistant to immuno-therapies (e.g.
Herceptin) or treatment with small molecules (e.g. Lapatinib.
[0129] Optionally, the active agents or compositions of the
invention may be administered to the subject in combination
(concurrently or sequentially) with other anti-cancer agents or
treatments. For example, they may be administered in combination
with one or more chemotherapeutic agents such as, but not limited
to, alkylating agents, e.g. Cyclophosphamide, Ifosphamide,
Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine,
Busulfan, and Thiotepa; antimetabolites, e.g. Methotraxate,
5-Fluorouracil, Cytarabine, Gemcitabine, 6-mercaptopurine,
6-thioguanine, Fludarabine, and Cladribine; anthracyclins, e.g.
daunorubicin. Doxorubicin, Idarubicin, Epirubicin and Mitoxantrone;
camptothecins, e.g. irinotecan and topotecan; taxanes, e.g.
paclitaxel and docetaxel; and platinums, e.g. Cisplatin,
carboplatin, and Oxaliplatin, as well as to immunotherapies, e.g.
Herceptin and Cetuximab, hormone responsive therapies, e.g.
Tamoxifen, Raloxifene, Fulvestrant, Anastrozole, Letrozole or
Exemestane for breast cancer, or anti-androgens e.g. flutamide for
prostate cancer, small molecules inhibiting epidermal growth factor
receptor (EGFR, e.g. Lapatinib or gefitinib), anti-angiogenic
therapy, e.g. Bevacizumab, sunitinib, sorafenib and pazopanib,
antibodies and small molecules targeted against beta 1 integrins
(e.g. ATN-161, Volociximab and JSM6427), or inhibitors, antagonists
and small molecules against urokinase receptor (UPAR).
[0130] Advantageously, the compositions of the invention may be
used in conjunction (concurrently or sequentially) with surgery or
radiotherapy. For example, the cells or cell-free preparations of
the invention may be used concomitantly with, or within 1-4 days
of, a surgical treatment for cancer. Dissemination of tumor cells,
which are the source for the recurrence and progression to
metastatic disease, may occur as a result of the surgical
operation. By performing such procedures in conjunction with the
compositions of the invention, cancer recurrence or metastasis may
be prevented or inhibited. Thus the compositions and methods of the
invention may be used to inhibit post-surgery metastatic
process.
[0131] Given that dissemination of tumor cells may have already
occurred even at an early stage of tumor progression, the
anti-cancer agents or treatments that may be administered in
combination with the compositions of the invention, include, in
some embodiments, neoadjuvant treatment, namely radiotherapy,
chemotherapy, hormone therapy and/or immunotherapy used for
shrinking the size of the tumor prior to surgical operation.
[0132] In yet a further aspect, the present invention provides a
method for treating fibrosis comprising administering to a subject
in need an effective amount of an active agent selected from: (i) a
conditioned cell culture medium of CD11b.sup.low macrophages; (ii)
a biologically active fraction thereof; (iii) a pharmaceutical
composition comprising (i) or (ii) and a pharmaceutically active
carrier, excipient or diluent; (iv) a culture of human
CD11b.sup.low macrophages; or (v) a pharmaceutical composition
comprising (iv) and a pharmaceutically active carrier, excipient or
diluent, wherein said treatment of fibrosis comprises preventing or
inhibiting fibrosis.
[0133] Examples 15 and 16 hereinafter show that development of
fibrosis can be the cause of activation and proliferation of
dormant tumor cells. Since the active agents of the invention can
prevent the process of fibrosis, they also prevent proliferation of
the dormant tumor cells. Thus, treatment of the fibrosis associated
with cancer is encompassed by the present invention.
[0134] Examples 10-14 show that conditioned medium of CD11b.sup.low
macrophages can inhibit proliferation of fibroblasts and their
differentiation into myofibroblasts induced by TGF.beta., including
inhibiting expression of .alpha.-SMA and deposition of Col-I, which
marks the process of fibrosis. These results demonstrate for the
first time that conditioned medium of CD11b.sup.low macrophages can
prevent fibroblasts from differentiation into myofibroblasts and
secretion of Col-I. Without being bound to any theory, the
inventors believe that these results indicate that these
macrophages may secrete anti-fibrotic factors that can prevent the
establishment of a fibrotic microenvironment.
[0135] Accordingly, in some embodiments, the fibrosis is associated
with a disease or a condition selected from acute lung injury,
Alzheimer's disease, chronic diabetic wounds, chronic granulomatous
disease (CGD), chronic obstructive pulmonary disease (COPD),
constrictive pericarditis, Dupuytren's disease, emphysema,
hypertrophic burn scars, keloid, liver cirrhosis, plantar
fibromatosis, retinal detachment inflammation, scleroderma, cystic
fibrosis, endometrial fibrosis, idiopathic pulmonary fibrosis,
myocardial fibrosis, nephrogenic fibrosis, pancreatic fibrosis,
perineural fibrosis, renal interstitial fibrosis, secondary
fibrosis in the gastrointestinal tract or fibrosis resulting after
surgery.
[0136] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the scope of the
invention.
EXAMPLES
Materials and Methods
Reagents
[0137] ELISA kits for mouse TNF-.alpha., IL-1.beta., IL-10,
TGF.beta., CCL2, CCL3, and CCL5 were obtained from R&D Systems.
CFSE, staurosporine, LPS (from Escherichia coli, clone 055:B5),
PKH2-PCL green fluorescence linker kit, and dexamethasone (Dex)
from Sigma. Poly (I:C) and CpG-ODN from InvivoGen. RvE1
(5S,12R,18R-trihydroxy-4Z,8E,10E,14Z,16E-eicosapentaenoic acid) and
RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic
acid) were obtained from Cayman Chemicals and the synthetic Rv were
matched according to the previously published biological and
physical material, PD1, LXB.sub.4, or 15-epi-LXA.sub.4 were a gift
from Prof. Serhan, Harvard.
Isolation and Culture of Human Monocytes
[0138] PBMC is obtained following informed consent from
individual/patient by Ficoll density centrifugation (400 g, 30 min,
without brake), and is washed three times with RPMI. Monocytes are
further purified by adherence: cell pellet is resuspended in
culture media (RPMI 1640, supplemented with 10% % human serum, 2 mM
L-glutamine, 100 .mu.g/ml streptomycin, and 100 units/ml
penicillin) and 10.times.10.sup.6 cells per well are transferred to
6-well plates, and incubated horizontally for 1.5 hrs at 37.degree.
C. in 5% CO.sub.2, humidified environment. Then, nonadherent
lymphocytes are discarded and the plates are washed gently with
fresh RPMI.
[0139] The isolated monocytes are then collected and differentiated
to macrophages either with 10% autologous serum or with MCSF.
Murine Peritonitis
[0140] Male C57BL/6 mice (6-8 wk) were injected intraperitionealy
(I.P) with zymosan A (1 mg). 66 hours post zymosan A injection, the
mice were euthanized by CO.sub.2, their peritoneal cavity was
lavaged with 5 ml of phosphate buffered saline (PBS) and the
peritoneal exudates were collected by centrifugation for further
analysis and experimentation.
[0141] Peritoneal exudates were collected and exudate cells were
stained with FITC-conjugated rat anti-mouse Ly-6G, PE-conjugated
rat anti-mouse F4/80, and PerCP-conjugated rat anti-mouse CD11b
(Biolegend) and analyzed by FACSCalibur (Beckton-Dickinson).
Macrophages were isolated using EasySep PE selection magnetic beads
following the manufacturer's instructions (StemCell
Technologies).
[0142] In some experiments, the macrophages were sorted to
CD11b.sup.high and CD11b.sup.low populations of >95% purity,
using FACSaria (Beckton-Dickinson), and the separate populations
were used for microscopic analysis and for ex vivo stimulation. In
some experiments, PKH2-PCL green (0.25 mM, 0.5 mL) was injected I.P
at 48 h and peritoneal cells were recovered 4 h later,
immunostained for F4/80 and CD11b as above and analyzed for
fluorescence intensity of different macrophage populations. In
relevant experiments, vehicle, RvD1, RvE1 (100 ng/mouse each), or
Dex (25 mg/mouse) were introduced to the peritoneum 48 h after
peritonitis initiation, and the exudates were recovered after 66 h
and analyzed as before.
Western Blot Analysis for CD11b.sup.high and CD11b.sup.low
Macrophages
[0143] Protein extracts of sorted populations (>95% purity) of
CD11b.sup.high and CD11b.sup.low macrophages were run using 10%
SDS-PAGE (50 .hoarfrost.g/lane), transferred to nitrocellulose
membranes, and immunoblotted with either goat anti-mouse CD11b
(Santa-Cruz Biotechnology), rabbit anti-mouse iNOS (Abcam), goat
anti-mouse arginase-1 (Abcam), rabbit anti-mouse COX-2 (Cayman
Chemicals), sheep anti-mouse 12/15-LO (Cayman Chemicals), goat
anti-mouse MMP-9 (R&D Systems), goat anti-mouse .sup.--actin
(SantaCruz Biotechnology) or goat anti-mouse tubulin (Santa-Cruz
Biotechnology). Then, the membranes were washed and incubated with
the appropriate HRP-conjugated secondary antibody. The blots were
washed and developed using EZ-ECL (Biological Industries).
Macrophage Transfer
[0144] Macrophages were isolated from peritoneal exudates 48 h
post-peritonitis initiation, stained with CFSE (1 mM), and injected
into the peritoneum of mice that underwent peritonitis for 4 h.
After an additional 18 h, cells from peritoneal exudates, inguinal
lymph nodes (LN), and spleen were recovered from the recipient
mice, immunostained as above, and the percentage of CD11b.sup.high
and CD11b.sup.low macrophages in each site was determined.
Confocal Microscopy
[0145] Sorted CD11b.sup.high and CD11b.sup.low macrophages were
isolated and loaded with 50 nm LysoTracker Red DND 99 dye
(Molecular Probes) for 2 h at 37.degree. C. in RPMI. Cells were
then fixed with 2% paraformaldehyde at room temperature and stained
with Hoechst (Molecular Probes) and FITC-conjugated anti-mouse
Ly-6G. Mounted slides were kept in the dark at 4.degree. C. until
analyzed by confocal microscopy. Confocal images were acquired
using Z-sections of 1 m thickness. The images were processed with
Zeiss LSM Image Browser software.
Apoptotic PMN (Polymorphonuclear) Engulfment Enumeration
[0146] Exudate cells or sorted CD11b.sup.high and CD11b.sup.low
macrophages were stained with Hoechst, and enumerated under a
fluorescent microscope (Zeiss). Two areas of two cover slips, each
containing at least 50 (overall 200) macrophages were analyzed, and
the average number of PMN engulfed per macrophage, as well as the
number of macrophages with cutoff numbers of engulfed PMN, were
calculated. In sorted cells, F4/80.sup.+, Ly-6G.sup.+ entities
(identified as macrophages that are attached to but did not fully
engulf a PMN by forward versus side scatter analysis) were excluded
from the samples to avoid counting of attached PMN.
TLR-Mediated Responsiveness Ex Vivo
[0147] Exudate macrophages were sorted or separated using PE
selection magnetic beads (StemCell Technologies) and incubated
(1.times.10.sup.6 cells in 0.5 mL of culture media) with LPS
(0-1000 ng/mL), poly (I:C) (4 .mu.g/mL), or CpG-ODN (1 .mu.M).
After 16 h, the supernatants were collected and their TNF-.alpha.,
IL-1.beta., IL-10, TGF.beta., CCL2, CCL3, and CCL5 contents were
determined by standard ELISA.
Regulation of Macrophage Phenotype by Apoptotic Cells Ex Vivo
[0148] Jurkat cells (T lymphocyte cell line) were treated with 1 mM
staurosporine (4 h, Sigma) to induce apoptosis and washed. Then,
peritoneal macrophages or sorted subpopulations thereof were
incubated in the presence or absence of apoptotic Jurkat cells (1:5
macrophage to apoptotic cell ratio). At the beginning of the
incubation and after 20 h, macrophages were immunostained for CD11b
and analyzed by FACS analysis. Alternatively, protein extracts were
prepared from the macrophages after the incubation period and run
by SDS-PAGE followed by western blot analysis for CD11b,
arginase-1, 12/15-LO, actin, and tubulin, as above.
Cell Line Cultures
[0149] Mouse mammary cancer cells D2.0R (obtained from Prof. Ann F.
Chambers, described in Morris et al., Clinical & Experimental
Metastasis 1993, 11(1):103-112), Mouse Embryonic Fibroblast (MEF)
cell line (obtained from Dr. Sarit Larisch, Haifa University,
Haifa, Israel) were maintained in DMEM high glucose (GIBCO), 10%
fetal bovine serum (FBS), and 1% penicillin-streptomycin at
37.degree. C., 5% CO.sub.2 incubator. Jurkat T cells (obtained from
Prof. Debbie Yablonski, Technion, Israel) were maintained in
RPMI-1640 (GIBCO) high glucose, 10% fetal bovine serum (FBS), and
1% penicillin-streptomycin at 37.degree. C., 5% CO2 incubator.
[0150] Fibroblasts differentiation to myofibroblasts was promoted
as follows: Mouse embryonic fibroblasts (MEFs) were culture in DMEM
10% FBS medium in 6 well plates. The next day the cells were
overlaid with RPMI containing 1% penicillin-streptomycin media for
overnight incubation. Thereafter the cells were treated with RPMI
10% FBS medium containing Ing/ml TGF.beta.1 for 48-72 hours
(PeproTech, Israel).
Animals
[0151] 7-8 weeks old male C57BL mice were purchased from Harlan
Biotech Israel and maintained under special pathogen-free
conditions in the animal facility at the Gutwirth Science Park in
the Technion (Israel Institute of Technology, Haifa).
Apoptotic Cells Preparation
[0152] Apoptosis was initiated in Jurkat T cells by treating the
cells with staurosporine (1 .mu.g/ml; Sigma-Aldrich) for 4 hours.
Then the cells were washed twice with PBS, re-suspended in RPMI 10%
FBS medium and added to the plated macrophages.
Ex-Vivo Generation of Pro-Resolving CD11b.sup.low Macrophages
[0153] The isolated macrophages (M.phi.) were incubated with
apoptotic Jurkat T cells (AC). 510.sup.6 cells were used at the
following M.phi. to AC ratios of: 1:1, 1:2 and 1:5, respectively.
After 8 hours of incubation the cells were washed with PBS and
overlaid with fresh RPMI 10% FBS medium for additional 12 hours of
incubation. Next, conditioned medium was collected and the
macrophages were further characterized for their conversion to
CD11b.sup.low phenotype by determining their surface expression of
CD11b by FACS analysis.
FACS Analysis
[0154] Determining surface expression of CD11b was carried out as
follows: Exudates were blocked with FcR blocker (anti-CD16/CD32;
Biolegend) mAb (0.5 .mu.g/0.5.times.10.sup.6 cells), and co-stained
with PE-conjugated anti mouse F4/80 (Cat#12216) (0.5
.mu.g/0.5.times.10.sup.6 cells) and PerCP-conjugated anti-mouse
CD11b (Cat#101230) (0.5 .mu.g/0.5.times.10.sup.6 cells) for 20 min
at 4.degree. C. Then, cells were washed with FACS Buffer (1% BSA in
PBS) and analyzed by FACSCanto ii (BD Biosciences) and FACSDiva
software.
Western Blot Analysis for MEFs
[0155] MEFs were lysed in WCE (whole-cell extract) buffer [25 mM
Hepes, pH 7.7, 0.3M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton
X-100, 100 .mu.g/ml PMSF and protease inhibitor cocktail (Rosche,
1:100 dilution)]. The proteins were separated by SDS-PAGE (8-10%)
followed by transfer on to a nitrocellulose membrane. The membrane
was blocked with 5% (w/v) non-fat dried skimmed milk powder in PBS
supplemented with 0.05% Tween20 (PBS-T) for 1 hour at room
temperature (R.T). Membrane was then probed either with mouse anti
.alpha.-SMA (Cat#ab7817; Abcam), or with rabbit anti-mouse GAPDH
(Cat#sc25778; Santa Cruz Biotechnology) at 4.degree. C.
overnight.
[0156] Next, the membrane was incubated with the appropriate
HRP-conjugated secondary antibody, for 1 hour at R.T. and washed 15
min.times.3 with PBS-T, WesternBright ECL (Advansta) was added to
the membrane for 30 seconds and analyzed using ImageQuant LAS-4000
analyzer (GE Healthcare Life Sciences) & "ImageQuant LAS-4000"
software (GE Healthcare Life Sciences). Densitometry analysis was
performed using ImageQuant total lab 7 (GE Healthcare Life
Sciences), an image analysis software.
Three-Dimensional Cell Cultures
[0157] D2.0R stably expressing Green Fluorescence protein
(D2.0R-GFP cells) and MEF cells were harvested from their growth
plates using 0.25% trypsin EDTA. Collected cells were cultured in
Cultrex.RTM. growth factor reduced Basement Membrane Extract (BME:
Trevigen, Inc) as follows: An 8-well chamber glass slide system
(Lab-TEK.RTM. II, Naperville, Ill.) was coated with 50 .mu.l
Cultrex.RTM. (Basement membrane Extract; BME) (Barkan et al.,
2010a) (protein concentration between 15 mg/ml; thickness.about.1-2
mm). 5.times.10.sup.3 cells/well of D2.0R-GFP cells, and
6.times.10.sup.4 MEF cells were re-suspended in DMEM low glucose
supplemented with 2% FBS and 2% Cultrex.RTM. and either cultured
separately or co-cultured on the coated slides. Slides were
incubated at 37.degree. C., 5% CO.sub.2 incubator. Cell morphology
was monitored by light microscopy.
Immunofluorescence Staining in Cell Culture
[0158] Cells cultured in 8-well chamber glass slides in 2
dimensional culture or in 3 dimensional culture, were fixed and
treated for 5 minutes with 4% Paraformaldehyde (PFA) containing 5%
sucrose and 0.1% Triton X-100, and re-fixed for an additional 25
minutes with cold acetone for 10 minutes. The cells were washed for
10 minutes with PBS and an additional 15 minutes with PBS
containing 0.05% Tween 20 (Sigma). Next, fixed cells were blocked
with IF buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 7.7 mM
NaN3, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20) contain 10%
donkey serum for 1 hour and incubated overnight at 4.degree. C.
with goat anti type I collagen (1:100) (Cat#1310-01;
SouthernBiotech) together with Alexa Fluor.RTM. 488 phalloidin
(1:40; Molecular Probes). The cells were washed three times with
PBS for 15 minutes each, and incubated for 1 hour with donkey anti
goat conjugated to Alexa Fluor.RTM. 649, (Invitrogen) at R.T. Next,
the cells were washed as mentioned above and mounted with
VECTASHIELD mounting medium with 4', 6-diamidino-2-phenylindole
(DAPI). The slides were imaged using a Nikon A1-R confocal laser
scanning microscope (Haifa University, Haifa, Israel).
Apoptosis Detection
[0159] MEF cell line were cultured as described above in 8 well
chamber glass slides, fixed with 4% PFA containing 5% sucrose and
incubated for 1 hour with a mixture of TUNEL reaction mix according
to the manufacturer's protocol (In Situ Cell death Detection Kit;
TMR Red) covered with aluminum foil and placed in the 37.degree.
C., 5% CO.sub.2 incubator for 1 hour. Next, the slides were washed
three times with PBS for 15 minutes each, and mounted with
VECTASHIELD mounting medium with DAPI. The slides were imaged using
a Nikon A1-R confocal laser scanning microscope (University of
Haifa, Haifa, Israel).
Proliferation Assay
[0160] MEF cells were cultured in 96 wells plate
(1.5.times.10.sup.3 Cells/well). At indicated time points the
CellTiter 96 AqueousOne Solution cell proliferation assay kit
(Promega) was added to the wells for 2 hours to measure cell
proliferation according the manufacturer's instructions. The
absorbance was recorded at 490 nm.
2D Images Analysis
[0161] 3 images were captured from each well. Each captured frame
was analyzed using NIS-Elements AR software to measure in the
defined area the number of cells with positive DAPI staining,
co-staining for DAPI and TUNEL (selected cells were larger than 8
.mu.m), or Col-I expression by measuring sum intensity of area
stained for Col-I per captured field.
3D Images Analysis
[0162] Confocal images were acquired using Z-sections of 1 .sup.-m
thickness. Three images comprised of 15 Z-stacks images were
captured from each well. Each Z-stack was analyzed using
NIS-Elements AR software to measure sum intensity of area stained
for Col-I.
Statistical Analyses
[0163] Ex vivo and in vivo experiments were performed at least
three times with at least four replicates. Results were analyzed by
one-way analysis of variance (for multiple groups) or Student's
t-test (for comparison between two groups, used for proliferation
assays, TUNEL assay, densitometry for W.B membranes and
quantification of Col-I) with statistical significance defined as
P.ltoreq.0.05 (*), P.ltoreq.0.001 (**), or P.ltoreq.0.0001
(***).
Example 1: Characterization of Protein Expression Profile and
Differentiation Markers of CD11b.sup.low and CD11b.sup.high
Macrophages
[0164] Macrophages positive for the macrophage differentiation
marker F4/80 obtained from mouse peritoneal exudates following
induction of peritonitis were characterized and sorted according to
their CD11b expression as detailed above. It was found that at 66 h
after peritonitis initiation, 70% of the exudate cells were
macrophages, of which 17% expressed low levels of CD11b
("CD11b.sup.low"). As can be seen in FIG. 1A, top panel and FIG.
1B, the expression of CD11b on CD11b.sup.high macrophages was
tenfold higher than its expression on CD11b.sup.low macrophages,
and the amount of CD11b in protein extracts of CD11b.sup.low
macrophages was significantly lower than its amount in the extracts
from CD11b.sup.high macrophages. The calculation of the fold
difference in expression was calculated according to the following
formula: densitometric units (DU) of CD11b.sup.high extract/DU of
CD11b.sup.low extracts from three experiments.
[0165] Analysis of some proteins that are functionally relevant to
inflammation and well-characterized by their expression in
classically (M1)- and alternatively (M2)-activated macrophages was
performed in CD11b.sup.high and CD11b.sup.low macrophages. The
analysis has shown (FIGS. 1A, 1B) that CD11b.sup.high macrophages
express low levels of iNOS, moderate levels of COX-2 and MMP-9 and
high levels of arginase-1, but no 12/15-LO, whereas CD11b.sup.low
macrophages express low levels of COX-2 and MMP-9, moderate levels
of 12/15-LO and no iNOS or arginase-1. CD11b.sup.low macrophages
lysates contained lower levels of actin than CD11b.sup.high cells,
suggesting a modulation of cytoskeletal dynamics in these cells.
Another housekeeping gene--tubulin--was equally expressed in both
protein extracts indicating equal protein loading.
[0166] Further analysis have shown that the macrophage
differentiation marker F4/80 was expressed to a higher extent on
CD11b.sup.high macrophages, further indicating that these cells
possess different properties in comparison to CD11b.sup.low
macrophages (data not shown).
[0167] Further analysis of surface receptor expression revealed
reduced expression of CD206 and CD163 (both M2 markers) on
CD11b.sup.low macrophages, in comparison to their CD11b.sup.high
counterparts (19- and 17-fold difference, respectively; N=3, data
not shown).
[0168] Thus, CD11b.sup.high and CD11b.sup.low macrophages display
different expression profiles of functional proteins and
differentiation markers.
Example 2: Engulfment of Apoptotic Polymorphonuclear Cells (PMN) by
CD11b.sup.low Macrophages
[0169] To examine whether CD11b.sup.low macrophages differ in their
efferocytosis capacity from CD11b.sup.high, sorted CD11b.sup.high
and CD11b.sup.low macrophages were stained with Hoechst and
LysoTracker, analyzed by confocal microscopy, and photographed (not
shown). Similar preparations were enumerated for apoptotic PMN
uptake, and analyzed according to average neutrophils engulfed per
macrophage (N/M), and percentage of cells reaching engulfment
threshold or engulfing an indicated number of apoptotic PMN. The
results are shown in FIGS. 2A-C.
[0170] The analysis has shown that CD11b.sup.low macrophages
engulfed significantly more PMN on average than CD11b.sup.high
macrophages (12.2.+-.0.2 N/M and 2.2.+-.0.2 N/M, respectively, see
FIG. 2A). LysoTracker staining indicated that the apoptotic PMN
visualized in macrophages were indeed phagocytosed and not merely
attached to the macrophage surface. In fact, in this experimental
setting, CD11b.sup.high and CD11b.sup.low macrophages were
distinguished by an engulfment threshold of seven PMN (FIG. 2B):
90.6.+-.4.2% of the CD11b.sup.low macrophages engulfed seven or
more PMN, whereas 97.5.+-.1.2% of the CD11b high macrophages
engulfed less than seven PMN. The results shown in FIG. 2C indicate
that the differences between CD11b.sup.high and CD11b.sup.low
macrophages in terms of engulfment of apoptotic PMN are distinct
not only when engulfment thresholds are depicted but rather that
there is very little overlap between the populations. Consequently,
the threshold of engulfment is enforced in a very narrow range and
hence, in this experimental setting only macrophages that engulfed
six PMN have the same tendency to be either CD11b.sup.high or
CD11b.sup.low macrophages.
Example 3: Responsiveness of CD11b.sup.low Macrophages to TLR
(Toll-Like Receptors) Ligands
[0171] Sorted macrophages were activated with LPS, and the
secretion levels of TNF-.alpha., IL-1.beta., IL-10, and TGF.beta.
by CD11b.sup.high and CD11b.sup.low macrophages were determined.
The results are presented in FIGS. 3A-3D. CD11b.sup.high
macrophages were found to secrete twice as much TNF-.alpha. as
CD11b.sup.low macrophages in the absence of LPS. Stimulation with
LPS at 500 ng/mL resulted in a significantly elevated and maximal
secretion of TNF-.alpha. by CD11b.sup.high macrophages, whereas
CD11b.sup.low macrophages did not increase their TNF-.alpha.
secretion. Similar results were obtained when the secretion of the
pro-inflammatory cytokines and chemokines IL-1.beta., CCL2, CCL3
and CCL5 was determined (FIGS. 3B, 3E-3G). The secretion of IL-10,
an anti-inflammatory cytokine, was also increased in CD11b.sup.high
macrophages stimulated with LPS (FIG. 3C). The secretion of the
resolution-promoting cytokine TGF.alpha. was higher in
CD11b.sup.low macrophages, but did not increase following
activation with LPS (FIG. 3D).
[0172] In addition, CD11b.sup.low and CD11b.sup.high macrophages
were stimulated with the TLR3 and TLR9 ligands poly (I:C) and
CpG-oligodeoxynucleotides (CpG-ODN), respectively, and the
secretion of TNF-.alpha. and IL-1.beta. was determined (FIG. 4).
The results indicate that both TNF-.alpha. and IL-1.beta. secretion
were increased following exposure to poly (I:C) and CpG-ODN of
CD11b.sup.high macrophages, whereas the secretion of these
cytokines by CD11b.sup.low macrophages treated in the same manner
was significantly lower. No significant reduction in the expression
of TLR3, 4 and 9 was found in CD11b.sup.low macrophages, in
comparison to their CD11b.sup.high counterparts. Thus,
CD11b.sup.low macrophages are poorer responders to different TLR
ligands in terms of cytokine and chemokine secretion, and therefore
may be considered immune-silent.
Example 4: The Phagocytic Capacity of CD11b.sup.low and
CD11b.sup.high Macrophages
[0173] To determine whether CD11b.sup.low and CD11b.sup.high
macrophages differ in their ability to phagocytose external
particles, the phagocyte-specific dye PKH2-PCL green was injected
intraperitoneally (i.p.) to mice undergoing peritonitis for 48 and
4 h later the peritoneal cells were recovered, immunostained, and
analyzed for PKH2-PCL acquisition. The results are shown in FIG. 5.
Most CD11b.sup.low macrophages did not acquire PKH2-PCL (FIG. 5A),
whereas the majority of CD11b.sup.high macrophages acquired higher
amounts of PKH2-PCL (FIG. 5B). As a result, the mean fluorescence
intensity (MFI) of PKH2-PC was 410-fold higher in CD11b.sup.high
macrophages (FIG. 5C).
[0174] Thus, CD11b.sup.low macrophages may be considered
"satiated", meaning that they lost their phagocytic potential upon
meeting the apoptotic PMN engulfment threshold and reducing their
CD11b expression.
Example 5: Migration of CD11b.sup.low Macrophages to Lymphoid
Organs
[0175] To determine whether CD11b.sup.low macrophages are prone to
depart resolving inflammation sites and emigrate to lymphoid
organs, the relative distribution of CD11b.sup.high and
CD11b.sup.low macrophages 66 h post-peritonitis initiation was
determined at the peritoneum, inguinal LN, and spleen. The results
(FIG. 6A) indicate that CD11b.sup.high macrophages are the
predominant macrophage subtype in the peritoneum during late
resolution, whereas, at the same time CD11b.sup.low macrophages are
the predominant macrophage subtype at the LN and spleen. To examine
whether CD11b.sup.low macrophages at the LN and spleen originated
in the peritoneum, adoptive transfer experiments were performed in
which peritoneal macrophages were isolated and labeled
fluorescently, and then transferred to the peritoneum of mice
undergoing peritonitis at the same period (48 h). The results (FIG.
6B) indicate that, 18 h after transfer, the distribution of labeled
CD11b.sup.high and CD11b.sup.low macrophages at the peritoneum,
inguinal LN and spleen was similar to the distribution of unlabeled
macrophages.
Example 6: Ex-Vivo Generation of Macrophages Expressing Low Levels
of CD11b
[0176] Sorted CD11b.sup.high and CD11b.sup.low macrophages were
incubated with or without apoptotic Jurkat cells and the changes in
surface expression of CD11b were determined. The results (FIGS. 7A,
B) indicate that CD11b expression on the surface of both
CD11b.sup.high and CD11b.sup.low macrophages was significantly
reduced, following their incubation with apoptotic cells, and some
of the macrophages had even lower expression levels compared to the
level of expression found for CD11b.sup.low macrophages recovered
from peritoneal exudates. Therefore the ex vivo-generated
population of macrophages expressing low levels of CD11b may be
comprised from both CD11b.sup.low and CD11b.sup.- macrophages (FIG.
7C). No significant reduction in surface expression of F4/80 or of
CD11b was observed following macrophage incubation with latex beads
(LB) or IgG-opsonized LB (data not shown). The impact of apoptotic
cells was partially mimicked by CD11b ligation with monoclonal
antibodies, which resulted in a decrease in CD11b, but not of
F4/80, surface expression, thus suggesting CD11b is involved in the
signaling cascade that leads to its own down-regulation. Notably,
CD206 and CD163 surface expression was not modulated by AC ex vivo,
but was reduced by anti-CD11b antibodies. Exposure to zymosan A,
TGF.beta., or live cells did not result in a significant reduction
in the surface expression of CD11b (data now shown).
[0177] The reduction in macrophage CD11b expression ex vivo appears
to be specific for interaction with apoptotic cells, and could not
be achieved by treatment with other phagocytic targets,
prototypic-activating bacterial moieties, or pro-resolving
cytokines, and is not due to the cell type of the apoptotic
cells.
[0178] To determine whether macrophage interaction with apoptotic
cells also triggers the major differences in protein expression
distinguishing CD11b.sup.high and CD11b.sup.low macrophages that
are shown in FIG. 1, peritoneal macrophages were incubated with
apoptotic cells and changes in the cytoplasmic content of CD11b,
arginase-1, 12/15-LO and actin were determined. The results (FIG.
7D) show that macrophages incubated with apoptotic cells expressed
reduced levels of CD11b and arginase-1, and increased levels of
12/15-LO. The levels of detergent-soluble actin in macrophages were
also reduced, following incubation with apoptotic cells. These
findings are consistent with the findings in FIG. 1 that show
reduced actin levels in lysates of CD11b.sup.low macrophages, in
comparison to their CD11b.sup.high counterparts.
[0179] Ex vivo exposure of macrophages to senescent neutrophils,
but not to latex beads (LB) or IgG-opsonized LB, resulted in a
significant reduction in CD11b and arginase-1 expression, whereas
12/15-LO expression was increased in this setting by all phagocytic
targets. These results indicate that interaction with apoptotic
cells is sufficient to drive the conversion of CD11b.sup.high to
CD11b.sup.low macrophages.
Example 7: RvD1, RvE1, and Dex Reduce the PMN Engulfment Threshold
Required for Macrophage Immune-Silencing
[0180] Resolvins are resolution-phase-generated mediators derived
from .sup.--3 polyunsaturated fatty acids; they were found to
promote resolution by acting on PMN and macrophages. Similar
properties were attributed to glucocorticoids, such as
dexamethasone (Dex), that inhibit leukocyte infiltration to
inflammation sites and promote the clearance of apoptotic PMN by
macrophages. To determine whether pro-resolving mediators regulate
emergence of CD11b.sup.low macrophages and thereby promote
resolution, RvD1, RvE1, or Dex were introduced into
peritonitis-affected mice, and the recovered leukocytes were
collected and analyzed for macrophage and neutrophil numbers,
macrophage CD11b expression, engulfment of PMN, and responsiveness
to LPS. The results are presented in FIG. 8 of Schif-Zuck et al.,
2011, which is incorporated herein by reference. These results
indicate that RvD1, RvE1 and Dex induced a reduction in neutrophil
numbers, whereas RvD1 and Dex, but not RvE1 also reduced the number
of macrophages in peritoneal cavities. In addition, RvD1, RvE1 and
Dex enhanced the appearance of CD11b.sup.low macrophages
(32.8.+-.8.8%, 46.2.+-.1.8%, and 39.9.+-.4.6% increases over
vehicle treatment, for RvD1, RvE1, and Dex, respectively) in
peritoneal exudates. RvD1 and RvE1, but not Dex, also reduced CD11b
expression on CD11b.sup.high macrophages. To determine whether
RvD1, RvE1, and Dex regulate apoptotic PMN engulfment by
macrophages during resolution, exudate cells were enumerated for
PMN engulfment and analyzed as in FIG. 2. The results indicate that
RvD1 and, to a greater extent, RvE1 and Dex, reduced the numbers of
apoptotic PMN engulfed by macrophages present in the peritoneum. A
detailed analysis of engulfment according to thresholds indicated
that RvE1 and Dex, but not RvD1, induced the appearance in the
peritoneum of low-engulfing macrophages, designate "inexperienced"
since they phagocytosed less than 2 Neutrophil engulfed per
macrophage (N/M).
[0181] To validate the improvement in immune-silencing of
macrophages that followed treatment with RvD1, RvE1 and Dex,
macrophages were activated with LPS, and cytokine secretion was
determined. The results indicate that RvD1 and RvE1, and, to a
lesser extent, Dex, inhibited the secretion of TNF.alpha. from
unstimulated and LPS-stimulated macrophages. A similar response was
observed with RvD1, RvE1 and Dex when IL-1.beta. secretion from
LPS-stimulated macrophages was determined. The secretion of IL-10,
a pro-resolving cytokine generated following the ingestion of
apoptotic cells, was up-regulated by each of the pro-resolving
mediators, in unstimulated and LPS-stimulated macrophages. Thus,
treatment with RvD1, RvE1, and Dex, promoted macrophage
immune-silencing, as well as the secretion of pro-resolving
cytokines from these cells.
[0182] FIG. 8 show promotion of macrophage switch to the
CD11b.sup.low phenotype by pro-resolving lipid mediators.
Macrophages were recovered from peritoneal exudates 72 hrs post
peritonitis initiation and incubated overnight with vehicle (RPMI
10% FBS+0.05% ethanol), RvD1, RvD2, PD1, LXB.sub.4, or
15-epi-LXA.sub.4, (all at 50 nM), apoptotic cells (M/N ratio of
1:5), or Dex (25 .mu.g/ml). Then, the cells were stained for CD11b
and analyzed by flow cytometry, and the percentage of CD11b.sup.low
(A) and CD11b.sup.high (B) macrophages was determined. Results are
mean.+-.SE of two experiments (n=2). # P<0.005, $ P<0.01, *
P<0.05 compared to vehicle.
Example 8: Ex Vivo Treatment with Pro-Resolving Lipid Mediators
Promotes Macrophage Switch to the CD11b.sup.low Phenotype
[0183] Macrophages were recovered from peritoneal exudates 72 hrs
post peritonitis initiation and incubated overnight with either
RPMI 10% FBS+0.05% ethanol, the resolvins RvD1 or RvD2, cell death
protein PD1 or lipoxins LXB.sub.4, or 15-epi-LXA.sub.4, (all at 50
nM), apoptotic cells (M/N ratio of 1:5), or Dex (25 .mu.g/ml).
After 20 hours, the cells were stained for CD11b and analyzed by
flow cytometry. As can be seen from FIG. 8, all of the treatments
significantly elevated the percentage of CD11b.sup.low macrophages
(8A) and reduced the percentage of CD11b.sup.high (8B)
macrophages.
Example 9: Ex-Vivo Generation of Secreted Factors of Pro-Resolving
CD11b.sup.low Macrophages
[0184] Conditioned media of enriched CD11b.sup.high macrophages
(CM-M.phi.) and of ex-vivo generated pro-resolving CD11b.sup.low
macrophages (CM-Mres) was collected in order to test their impact
on the formation of a fibrotic ECM by myofibroblasts and the
transition from tumor dormancy to metastatic growth as detailed
below. To this end peritonitis was induced and 66 hours later
peritoneal exudates were collected and the percentage of
macrophages was determined in peritoneal exudates, based on their
size and granularity (FIG. 9A, see gated area and red arrow) and
positive staining for F4/80 (FIG. 9B). The majority of the cells
(55% of the exudates) were macrophages (FIG. 9C) as determined by
FACS analysis (FIGS. 9A and B). Furthermore, 66% of the macrophages
expressed high levels of CD11b and 34% expressed low levels of
CD11b (FIG. 9D-E) indicating that resolution was already initiated
in vivo. Next, CD11b.sup.high macrophages were collected and were
either untreated (FIG. 10A) or treated with apoptotic Jurkat cells
(a common apoptotic leukocyte target for macrophages in
experimental procedures; as described in Schif-Zuck et al., 2011,
with some modifications). Originally, CD11b.sup.high macrophages
were incubated with apoptotic Jurkat cells at a ratio of 1:5
respectively (FIG. 10B) resulting in 80% conversion of
CD11b.sup.high macrophages to CD11b.sup.low macrophages compared to
untreated macrophages where only 25% of CD11b.sup.high macrophages
were converted to CD11b.sup.low macrophages, as determined by
surface expression of CD11b by FACS analysis and as illustrated
also in FIG. 10C-D. However, 30% of the apoptotic cells (AC) were
not engulfed by CD11b.sup.high macrophages (FIG. 11D). Given that
we planned on using the conditioned media of the untreated and
treated macrophages it was essential to reduce the presence of
non-engulfed AC cells to avoid potential residual effect of the AC
in the conditioned media. Hence, we tested whether we can
successfully generate ex-vivo the CD11b.sup.low macrophages by
incubating CD11b.sup.high macrophages with AC at a ratio of 1:2 and
1:1 respectively. Importantly, after incubating the CD11b.sup.high
macrophages with AC we collected the conditioned media (that will
be utilized for our experiments as will be detailed below), and
characterized the macrophages according to their staining for F4/80
(as illustrated in FIG. 11A) and CD11b expression levels (as
illustrated in FIG. 11B). Indeed, we found that the majority of the
CD11b.sup.high macrophages converted to CD11b.sup.low macrophages
upon incubation with lower amounts of AC. CD11b.sup.high
macrophages to AC ratios of 1:2 and 1:1 yielded conversion of 60%
and 55% of CD11b.sup.high macrophages to CD11b.sup.low macrophages
respectively compared to untreated macrophages where only 20% of
CD11b.sup.high macrophages were converted to CD11b.sup.low
macrophages (FIG. 11C). Importantly, only 8% of the AC remained
after incubation of CD11b.sup.high macrophages with AC at 1:1
ratio, compared to 25% at 1:2 ratios (FIG. 11D).
Example 10: CM-Mres Prevents Fibroblasts Differentiation to
Myofibroblasts in 2 Dimensional Cultures
[0185] We next determined whether conditioned media recovered
during the conversion of CD11b.sup.high macrophages to
CD11b.sup.low macrophages (CM-Mres) (as described in Example 9) was
able to inhibit the differentiation of fibroblasts to
myofibroblasts. In order to address this question we tested
proliferation of fibroblasts, their expression of .alpha.-SMA and
expression and deposition of Type I collagen (Col-I) upon treatment
with CM-Mres.
[0186] Initially, we carried out a qualitative experiment to
determine the growth of fibroblasts. To this end mouse embryonic
fibroblasts (MEFs) were induced to differentiate to myofibroblasts
by treating the cells with Ing/ml TGF.beta.1 for 72 hours. To test
the impact of the conditioned media on the activation of MEFs we
carried out the following: 1 hour prior to adding TGF.sup.-1, the
MEFs were either untreated or treated with either condition media
of CD11b.sup.high-enriched macrophages (CM-M.phi.) or with CM-Mres
for 72 hours. Our results demonstrate that fewer MEFs were present
in plates treated with CM-Mres and TGF.beta.1, compared to
untreated MEF or to MEF treated with either CM-M.phi. and
TGF.beta.1 or with TGF.beta.1 alone (FIG. 12).
Example 11: MEFs Treated with CM-Mres are Cell Cycle Arrested
[0187] We further studied whether reduction in the number of MEFs
that were treated with TGF.beta.1+CM-Mres was due to inhibition of
proliferation. To this end condition media from either cultured
residual AC (CM-AC), CM-M.phi. or CM-Mres was overlaid on starved
MEFs cultured on 96 well plates 1 hour prior to TGF.beta.1
treatment. Proliferation of the cells was monitored 24, 48 and 72
hours after addition of TGF.beta.1.
[0188] Our results demonstrate that TGF_1 promotes the
proliferation of MEF as expected in a time dependent manner.
Furthermore CM-M.phi. did not significantly affect the
proliferation of MEF that were treated with TGF.beta.1, however the
CM-Mres inhibited significantly the TGF.beta.1 induced
proliferation of MEFs cells (FIG. 13A). CM-AC during the first 48
hours had no significant effect on TGF.beta.1 induced MEFs
proliferation, however at 72 hours it inhibited the proliferation
significantly (p.ltoreq.0.001) but still their proliferation was
significantly (p.ltoreq.0.001) higher compared to MEF treated with
CM-Mres.
[0189] Next we explored whether the inhibition of MEF proliferation
by the CM-Mres was due to cell cycle arrest or increase in
apoptosis of MEF. Therefore, we tested apoptosis of the MEFs at 48
hours. At this time point significant changes in the proliferation
of the cells was already observed as shown in FIG. 13A. Therefore,
MEFs were either untreated or were induced to differentiate on 8
chamber glass slides and were pre-incubated with CM-Mres 1 hour
prior to adding TGF.beta.1. As for positive control for apoptosis,
MEFs were treated with staurosporine (STS; 1.5 .mu.g/ml) for 4
hours. The cells were fixed and evaluated for apoptotic death by
TUNEL staining. Our results demonstrate that low percentages of
apoptotic cells were present in all treatments with no significant
changes (FIG. 13B) including our positive control. Most of the MEFs
(more than 96%) were alive and did not stain for TUNEL upon
treatment with TGF.beta.1 and CM-Mres, indicating CM-Mres does not
cause MEF cell death. However, it is noted that prolonged cell
cycle arrest is expected to eventually induce apoptosis.
Example 12: CM-Mres Inhibit .alpha.-SMA Expression by MEF Treated
with TGF.beta.1
[0190] We further explored the effect of CM-Mres on differentiation
of MEF to myofibroblasts by testing the expression of .alpha.-SMA.
To this end, we either undifferentiated or differentiated MEF in
the presence of CM-Mres, CM-M.phi., or CM-AC 1 hour before adding
TGF.beta.1, for 72 hours. Protein lysates were collected and
.alpha.-SMA levels were determined by western blot analysis.
CM-Mres inhibited significantly the expression of .alpha.-SMA in
MEF treated with TGF.beta.1 compared to the expression levels in
MEF treated with TGF.beta.1+CM-M.phi. (FIG. 14).
Example 13: CM-Mres Inhibit Type I Collagen Expression by MEF
Treated with TGF.beta.1
[0191] Type I Collagen (Col-I) is expressed in differentiated
fibroblasts. Therefore, we tested whether CM-Mres will inhibit
Col-I expression in TGF.beta.1-treated MEF. To this end MEF were
cultured on 8 chamber glass slides and were either untreated or
treated with TGF.beta.1 in the presence of either CM-Mres,
CM-M.phi. or CM-AC. We determined Col-I expression by
immunofluorescence staining and F-actin organization by phalloidin
staining (data not shown). Our results demonstrated that CM-Mres
significantly inhibited Col-I expression of TGF.beta.1 treated MEF
compared to untreated MEF and to TGF.beta.1 treated MEF that were
treated with either CM-M.phi. or CM-AC (FIG. 15). Furthermore, a
decrease in F-actin stress fibers was evident in MEF treated with
TGF1+CM-Mres compared to MEF treated with TGF.beta.1 alone or in
combination with either CM-M.phi. or CM-AC (data not shown).
Example 14: CM-Mres Prevent Production of Col-I by Myofibroblast
Cultured in 3D System
[0192] A novel 3D BME system to model tumor dormancy and outgrowth
was described recently in Barkan et al., 2011. Furthermore,
supplementing the 3D BME system with Col-I was shown previously to
induce the transition of dormant D2.0R cells from quiescence to
proliferative growth. Therefore, we wanted to explore whether MEF
cultured in the 3D BME system will successfully differentiate to
myofibroblasts, express Col-I and thus will induce dormant D2.0R
cells outgrowth. If successful, we will test whether we can prevent
the D2.0R cells outbreak by using CM-Mres. To this end MEF cells
were cultured in the 3D BME system and were either untreated or
treated with CM-M(p, CM-AC or CM-Mres for 7 days. Col-I expression
was determined by immunofluorescence staining. Our results
demonstrate that MEFs cultured in the 3D BME system differentiated
to myofibroblasts given they expressed Col-I (FIG. 16).
Furthermore, treatment with CM-Mres reduced significantly the
expression of Col-I (FIG. 16). Hence, CM-Mres prevented the
differentiation of MEF to myofibroblasts in the 3D BME system.
Interestingly, as can also be seen from FIG. 16, cells treated
either with CM-M.phi. or with CM-AC showed increased Col-I
expression, compared to untreated cells.
Example 15: Conditioned Media of Ex-Vivo Generated CD11b.sup.low
Macrophages (CM-Mres) Inhibit Proliferation and Induce Cell Death
of D2A1 Dormant Tumor Cells
[0193] Peritoneal mouse macrophages were recovered 66 hours post
peritonitis initiation and incubated either with no cells, or in
the presence of apoptotic (AC) or live (LC) Jurkat cells. The
unbound apoptotic cells were then washed and the macrophages were
recovered and immunoassayed for CD11b expression on their surface.
Expression of CD11b was determined by FACS analysis. As can be seen
in FIG. 17A, incubation with apoptotic (AC) but not with live (LC)
Jurkat cells, generated a macrophage population enriched with
CD11b.sup.low cells (n=5). Conditioned media from cells incubated
with AC or LC were collected and assessed for their potential
ability to directly affect the growth of dormant or out-breaking
D2A1 cells by the 3D basement membrane (BME) system modeling tumor
dormancy (Barkan et al., 2008).
[0194] The conditioned media were overlaid on D2A1 cells cultured
in the 3D BME system on day 2 when cells are dormant, and on day 4,
prior to their transition from quiescence to proliferation.
Proliferation of the D2A1 cells was determined by using the Cell
Titer 96 Aqueous One Solution cell proliferation assay kit
(Protege; Madison, Wis.) as described by Barkan et al. 2008.
[0195] FIG. 17B shows proliferation of D2A1 cells in the 3D system
overlaid on day 4 for 96 h with either assay media for dormancy
(DMEM low glucose (GIBCO), 2% fetal bovine serum (FBS), 1%
penicillin-streptomycin and 2% BME (Trevigen Inc.)) (untreated),
with RPMI incubated with apoptotic cells (AC), or with conditioned
media obtained from macrophages incubated ex-vivo with RPMI+10%
FBS+1% penicillin-streptomycin (CD11b.sup.highmac), live
(CD11b.sup.highmac+LC), or apoptotic (CD11b.sup.lowmac) Jurkat
cells. n=4;***, P.ltoreq.0.001.
[0196] The results demonstrate that conditioned media obtained from
ex-vivo generated macrophages expressing low levels of CD11b
(CD11b.sup.low) induced cell death of outbreaking D2A1 cells during
their transition from dormancy to proliferation in the 3D
system.
[0197] FIG. 17C shows proliferation of D2A1 cells in the 3D system
overlaid on day 2 for 96 h with either DMEM low glucose (GIBCO), 2%
fetal bovine serum (FBS), and 1% penicillin-streptomycin+2% BME
(untreated), or with conditioned media obtained from cell
preparations enriched for CD11b.sup.high or CD11b.sup.low
macrophages as described above, supplemented also with 2% BME.
Since the D2A1 cells are dormant at day 2, and remain dormant for
4-6 days, a measured reduction in proliferation indicates cell
death of the cultured D2A1 cells.
[0198] The results indicate that soluble factors secreted by the
CD11b.sup.low macrophages, which are present in the conditioned
media, induce cell death of dormant D2A1 cells. Thus, dormant
disseminated tumor cells may be eradicated by natural soluble
factors produced by immune cells participating in physiological
healing processes.
Example 16: CM-Mres Prevent Metastatic Outbreak of Dormant Tumor
Cells Co-Cultured with Myofibroblasts
[0199] Our findings demonstrated that culturing MEFs in the 3D BME
system promoted their differentiation and the expression of Col-I
and this differentiation was inhibited by treatment with CM-Mres.
D2.0R cells cultured in the 3D BME system are dormant and
supplementing the BME with Col-I was shown previously to induce
their outbreak (Barkan et al., 2010a). Hence, based on our recent
and previous results we next tested whether 1) co-culture of
dormant D2.0R cells with MEF in the 3D BME system will induce their
emergence from tumor dormancy to proliferative growth and 2)
whether treatment with CM-Mres will inhibit Col-I expression and
will prevent D2.0R cells outbreak. To this end we co-cultured D2.0R
cells stably expressing green fluorescent protein (D2.0R-GFP) with
MEF for 7 days. Our results showed that D2.0R-GFP cells cultured in
the BME system remain dormant as expected, whereas co-culture of
D2.0R-GFP cells with MEF in the 3D BME system, promoted their
outbreak (dramatic increase in cell number) and Col-I expression
was evident (data now shown). Similarly D2.0R-GFP cells emerged
from their dormant state in the presence of MEF treated with
CM-M.phi.. However, treatment of the co-culture with CM-Mres
prevented Col-I expression by the differentiated MEF and lower
number of D2.0R-GFP were evident compared to untreated co-culture
or co-culture treated with CM-M.phi..
Example 17: In Vivo Treatment of Dormant and Outbreaking Tumor
Cells with CM-Mres
[0200] Conditioned media of ex-vivo generated CD11b.sup.low
(CM-Mres) and control CD11b.sup.high macrophages (CM-M.phi.) (as
described in Example 9) is assayed for their impact on the
metastatic outbreak in vivo, using tumor-inoculated mice.
Experimental metastasis assay in athymic nude mice are carried out
in female athymic nude mice (n=30) injected via tail vein with D2A1
cells stably expressing GFP (D2A1-GFP cells) to allow tracking by
live video microscopy as described previously as described in
Barkan et al. 2008 and 2010a. Prior to the outbreak of dormant
D2A1-GFP cells (5 days post D2A1-GFP injection) 15 of the recipient
mice are treated (50-100 .mu.l, intranasal or via tail vain
injection) with CM-M.phi.. (control group), whereas the rest of the
recipient mice (n=15) are treated with CM-Mres (treated group). The
control and treated groups are divided to 3 even groups (n=5;
control groups A/B/C and treated groups A/B/C). Control and treated
group B and C receive additional treatments at days 8 and 11 of
either CM-M.phi. or CM-Mres, respectively. Lungs from all groups
(and untreated mice; n=5) are harvested 2 weeks post injection of
D2A1-GFP cells, and tumor cells are analyzed for their dormant or
metastatic growth using live video microscopy as described
previously in Barkan et al. 2008 and 2010a. Imaged lungs are frozen
in optimal cutting temperature (OCT) compound for determination of
extent of fibrosis at the metastatic site by immunofluorescence
staining for Col-I and macrophages (F4/80 staining) of frozen
sections prepared from the harvested lungs.
[0201] Metastatic outbreak in the control groups treated with
CM-M.phi., with possible enhancement of the extent of metastasis
compared to untreated mice may be expected, whereas in the
CM-Mres-treated group the number of lesions will decrease and the
majority of the lesions are expected to be dormant with inhibition
of a fibrotic stroma at the metastatic site (depicted by low
expression of Col-I and clearance of macrophages).
[0202] The following experiments are repeated with highly
metastatic human breast cancer cell line such as MDA-MB-231 cells.
The mice are treated as above but at two weeks when metastatic
lesions are already established.
[0203] In the CM-Mres-treated group, the number of lesions is
expected to decrease and the majority of the lesions are expected
to be dormant with inhibition of a fibrotic stroma at the
metastatic site (depicted by low expression of Col-I and clearance
of macrophages).
Example 18: Treatment of Tumor Metastasis by Administering Mres
Macrophages
[0204] Ex-vivo generated peritoneal/lung pro-resolving
CD11b.sup.low macrophages (Mres macrophages) are prepared as
described in Example 9. Initially, a pilot experiment is conducted
to determine the transfer regimen of the ex-vivo generated Mres
macrophages required to promote inhibition of the metastatic
outbreak in the lungs. The transfer is carried out by intranasal
administration. To this end, experimental metastasis assays are
carried out on recipient female athymic mice (n=30) that are
injected via tail vein with D2A1-GFP cells in order to analyze
later on their dormant and metastatic outbreak by live video
microscopy as described previously in Barkan et al. 2008 and 2010a.
Prior to the outbreak of dormant D2A1-GFP cells (5 days post
D2A1-GFP injection), 15 of the recipient mice are transferred with
isolated and sorted CD11b.sup.high macrophages obtained from 45
donor mice with peritonitis (control group), whereas the rest of
the recipient mice (n=15 mice) are transferred with an equal number
of sorted ex-vivo generated Mres macrophages (treated group). The
control and treated groups are divided to 3 even groups: Control
group A/B/C (n=5 mice per each group) and treated group A/B/C (n=5
mice per each group). Control and treated group B receive an
additional transfer 3 days later of either freshly prepared
pro-resolving CD11b.sup.high or ex-vivo generated Mres macrophages
(as described above), respectively, whereas control and treated
group C receive two additional transfers, at 3 day intervals, with
either freshly prepared CD11b.sup.high or ex-vivo generated Mres
macrophages, respectively. Lungs from all groups of mice are
harvested 2 weeks post injection of D2A1-GFP cells, and tumor cells
are analyzed for their dormant or metastatic growth using live
video microscopy. Imaged lungs are frozen in OCT for determination
of extent of fibrosis at the metastatic site by immunofluorescence
staining for Col-I and macrophages (F4/80 staining) of frozen
sections prepared from the harvested lungs.
[0205] Metastatic outbreak in the control group transferred with
CD11b.sup.high macrophages is expected, with possible enhancement
of the extent of metastasis, whereas in the treated group the
majority of the lesions are expected to be dormant. Once the
transfer regimen needed to inhibit the metastatic outbreak of
D2A1-GFP cells is established, the experiment is repeated with a
larger cohort of mice transferred with ex-vivo-generated
peritoneal/lung Mres macrophages, and proceed to further confirm
the promotion of resolution of the metastatic microenvironment as
detailed below.
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