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Pulmonary Alveolar Macrophages
Contribute to the Premetastatic Niche by
Suppressing Antitumor T Cell Responses in
the Lungs
Sharad K. Sharma, Navin K. Chintala, Surya Kumari
Vadrevu, Jalpa Patel, Magdalena Karbowniczek and Maciej
M. Markiewski
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Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 24 April 2015
http://www.jimmunol.org/content/early/2015/04/24/jimmun
ol.1403215
Published April 24, 2015, doi:10.4049/jimmunol.1403215
The Journal of Immunology
Pulmonary Alveolar Macrophages Contribute to the
Premetastatic Niche by Suppressing Antitumor T Cell
Responses in the Lungs
Sharad K. Sharma,1 Navin K. Chintala,1 Surya Kumari Vadrevu,1 Jalpa Patel,
Magdalena Karbowniczek, and Maciej M. Markiewski
T
umor-associated macrophages (TAM) recruited to primary
tumors facilitate cancer progression by suppressing antitumor T cell responses, enhancing angiogenesis, tumor
cell invasion, and metastasis (1). In addition, it has been shown
that the subpopulation of inflammatory monocytes is recruited to
lung metastatic foci by CCL2 and facilitates progression of
metastatic disease (2). A similar mechanism appears to be responsible for recruitment of blood inflammatory monocytes that
become TAM (3). In contrast, a role for tissue resident macrophages (TRM) in cancer remains unclear, with some conflicting
studies suggesting both tumor-promoting and antitumor activities by these cells (4).
Recent work has highlighted marked heterogeneity in the origin
of tissue macrophages that arise from hematopoietic versus selfrenewing embryo-derived populations (5); therefore, it is imagDepartment of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas
Tech University Health Science Center, Abilene, TX, 79601
1
S.K.S., N.K.C., and S.K.V. contributed equally to this work.
Received for publication December 24, 2014. Accepted for publication March 21,
2015.
This work was supported by Cancer Prevention and Research Institute of Texas Grant
RP 120168 (to M.K.) and Department of Defense Breast Cancer Research Program
Grant BC 111038 (to M.M.M.).
Views and opinions of and endorsements by the author(s) do not reflect those of the
U.S. Army or the Department of Defense.
Address correspondence and reprint requests to Dr. Maciej M. Markiewski and
Dr. Magdalena Karbowniczek, Texas Tech University Health Sciences Center, 1718
Pine Street, Abilene, TX 79601. E-mail addresses: [email protected]
(M.M.M.) and [email protected] (M.K.)
Abbreviations used in this article: AM, alveolar macrophage; BM, bone marrow;
C5aR, complement C5a receptor; DC, dendritic cell; GzB, granzyme B; IM, interstitial macrophage; m, murine; MDSC, myeloid-derived suppressor cell; TAM,
tumor-associated macrophage; TRM, tissue resident macrophage; WT, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403215
inable that functions of TRM, including alveolar macrophages
(AM) (CD11bnegativeF4/80+CD11c+) that originate from yolk-sac
or fetal liver macrophages, could be different from those of TAM
and inflammatory monocytes/macrophages (CD11b+F4/80+) of
hematopoietic origin. Although AM have also been studied in
the context of primary lung cancer, AM may play a key role in
cancer metastatic progression, as the lungs are the most common targets of hematogenous cancer metastases (6). Because
TRM colonize tissue-specific niches during embryonic development and have remarkable self-renewal capacity (5, 7), these
cells are present in metastases-targeted organs in large numbers
prior to metastases. Thus, we hypothesize that TRM could play
the pivotal role in the premetastatic niche, which is defined a set
of alterations in metastases-targeted organs facilitating metastases that occur prior to tumor cell arrival (8, 9). Although
several cells, mainly bone marrow (BM)–derived cells, have
been reported to contribute to the premetastatic niche, AM have
not yet been identified as potential players in this niche (9).
Because AM have important immunomodulatory functions,
contributions of these cells to the premetastatic niche might
involve inhibition of antitumor immunity. AM are the key
players in preventing unwanted immune reactions to environmental Ags (10); therefore, we theorize that immunosuppressive
mechanisms that prevent massive immune reactions to these
Ags in the lungs are harnessed by tumors to facilitate metastasis. Thus, the intrinsic properties of lung immune homeostasis
could be, at least partially, responsible for the susceptibility of
the lungs to metastasis. Complement plays an important part in
regulating macrophage function and migration during the inflammatory reaction (11), and we have demonstrated activation
of complement in the lung premetastatic niche (12); therefore,
we also determined the impact of complement C5a receptor
(C5aR) on AM accumulation in the premetastatic niche.
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In contrast to tumor-associated macrophages, myeloid-derived suppressor cells, or inflammatory monocytes, functions of tissue
resident macrophages, including alveolar macrophages (AM), in cancer were not well studied. Using a mouse model of breast cancer,
we show that AM promote cancer metastasis to the lungs by suppressing antitumor T cells in this organ. AM accumulated in the
premetastatic lungs through complement C5a receptor–mediated proliferation but not through recruitment from the circulation.
AM preconditioned by breast tumors inhibited Th1 and favored generation of Th2 cells that had lower tumoricidal activity than
Th1 cells. In addition, AM reduced the number and maturation of lung dendritic cells by regulating TGF-b in the lung environment. Depletion of AM reversed immunosuppression imposed by these cells and strengthened local Th1 responses, which
significantly reduced lung metastatic burden. C5a receptor deficiency, which also lessens myeloid-derived suppressor cells in the
premetastatic niche, synergized with the depletion of AM in preventing metastasis, leading to protection of mice from lung
metastases. This study identifies AM as a new component of the premetastatic niche, which is harnessed by tumors to impose
immunosuppression, and as a new target for cancer immunotherapies to eliminate or reduce metastasis. Because the lungs are the
most common target for hematogenous metastasis, this research offers a plausible explanation for susceptibility of the lungs to
cancer metastasis. The Journal of Immunology, 2015, 194: 000–000.
2
Materials and Methods
Mice and cell lines
Balb/c, C5aR-deficient (C5aR2/2), C57BL/6, Balb/cByJ (both C45.1 and
CD45.2), OTII TCR, and FVB/Nj Her2-transgenic mice were from The
Jackson Laboratory. C5aR2/2 mice were backcrossed to Balb/c for 10
generations. Mice were housed in the animal facility of Texas Tech University Health Sciences Center. All experiments were approved by the
Committee for Institutional Animal Care of Texas Tech University Health
Sciences Center. The 4T1 (CRL-2539, ATCC), B16-F10 (CRL-6475,
ATCC), and chicken ovalbumin–expressing B16-F10 cells (a gift from
Dr. Laurence Wood, Texas Tech University Health Sciences Center) were
maintained in the cell culture media as recommended by the supplier and
routinely tested for Mycoplasma.
Tumor model and AM depletion
Cell isolation, stimulation, and FACS
The lungs were digested and mechanically disintegrated to obtain singlecell suspensions, as described (15). Spleens were passed through cell
strainers (BD) for obtaining single-cell suspensions. For T cell stimulation
assays, cells were incubated in the presence of plate-bound CD3 and
soluble CD28 Abs (17A2 and 37.51; eBioscience) with Golgi inhibitors
(brefeldin A and monensin, BD Biosciences) for 6–8 h. For evaluation of
myeloid cell function, lung cell preparations were ex vivo stimulated with
1 mg/ml LPS for 8–12 h in the presence of Golgi inhibitors. As negative
controls, unstimulated cells were used. Cytokine production was assessed
by standard intracellular cytokine staining procedure, as described in our
earlier study (12). Staining for Ki-67 was performed after permeabilization
with 70% chilled ethanol. For surface staining, cells were incubated with
fluorescently labeled Abs. For examining direct tumor cell death by
clodronate liposomes, 4T1 cells were incubated with clodronate or PBS
liposomes for various time points (0–24 h), and 4T1 cells were harvested
and stained for annexin V.
All Abs were from BioLegend unless otherwise specified: mouse BV605
CD45 (30-F11), AF488-conjugated CD45.1 (A20), BV605 CD45.2 (104),
PerCPCy5.5 CD4 (RM4-5), PE-TR CD8a (53-6.7, RM4-5), PE IFN-g
(XMG1.2), PE/Cy7 IL-4 (11b11), AF 647 IL-17A (Tc11-18H10.1), PE
CD11b (M1/70), PE/Cy7 F4/80 (BM8), APC/Cy7 CD11c (N418),
PerCPCy5.5 IA/IE (MHCII) (M5/114.152), PE CD80 (16-10A1), AF 647
CD86 (GL-1), PE/Cy7 C5aR (20/70), PE Ki-67 (16A8), FITC granzyme
B (GB11), PerCP/Cy5.5 TGF-b1 (TW7-16B4), APC IL-12/IL-23 p40
(C15.6), polyclonal rabbit caspase-3 IgG (R&D Systems), and FITC goat
anti-rabbit (Santa Cruz Biotechnology). Live-dead stain was from eBiosciences, and staining for live-dead marker was performed in the absence
of other Abs according to the manufacturer’s instructions. Cells were incubated with CD16-CD32 Abs (Fc block; 2.4G2) to block Fc receptors
prior to staining. After staining, cells were fixed in 1% paraformaldehyde,
stored at 4˚C until data acquisition on an LSRFortessa equipped with 3
lasers (12 parameters), and analyzed using FlowJo (TreeStar). Cells were
first gated based on forward- and side-scatter properties, followed by
gating on CD45+ live cells before identifying the individual population of
interest (Fig. 1C). Dendritic cells (DC) were CD11c+MHCII+, interstitial
macrophages (IM) were CD11b+F4/80+, myeloid-derived suppressor cells
(MDSCs) were identified as CD11b+Gr-1+, and AM were CD11bnegative
CD11c+F4/80+ (16). Tumor cells were identified as CD45negative cells in
the lungs, as described earlier (17).
Immunofluorescence, immunohistochemistry, and quantitative
PCR
Lungs were fixed in 10% (v/v) formalin or frozen in OCT medium.
Formalin-fixed samples were processed for histological study, and frozen
samples were sectioned with a cryostat (Leica) for immunofluorescence.
AM were analyzed by immunofluorescence with rat anti-mouse F4/80
(BM8; BioLegend) and CD11b (M1/70; BD Biosciences) Abs at the recommended concentration. Stained sections were analyzed in a blinded
fashion by confocal microscopy (Nikon) and Nikon Elements Advanced
Research Image-Analysis software either by counting stained cells in
a higher power field (363) or by quantifying an area in a section occupied
by AM. H&E-stained lung sections were scanned and digitalized (Aperio
Technologies). Analysis was performed using ImageScope (Aperio). Quantitative real-time PCR for C5 was performed as described by us earlier (12).
C5 (forward: 59-CCTGTTACCAGTGATGAAGGCAG-39; reverse: 59-TCGTTAGTGAGTCAGGCAGCGT-39) primers were procured from SigmaAldrich (St. Louis, MO). Gene expression analysis was performed on
the StepOnePlus™ Real-Time PCR System (Applied Biosystems). Gene
expression was normalized to 18S using the DDCT method. Interpretation
of data was performed using QIAGEN RT2 Profiler PCR Array Data
Analysis version 3.5.
CD45.1 BM transfer
A single-cell suspension was prepared from femur BM of tumor-free
CD45.1 (ly5.1) Balb/cByJ mice, and 1 3 106 BM cells were injected into the tail vein of tumor-bearing and tumor-free wild-type (WT) Balb/cByJ
mice (CD45.2) 14 d after 4T1 tumor cell injection into the mammary fat
pad. After 7 d, blood, lungs, and spleens were harvested and analyzed for
the presence of CD45.1 (transplanted) cells. Further CD45.1 cells in the
lungs were analyzed for their differentiation into various populations
(MDSC, IM, and AM), using FACS.
In vitro CD4+ T cell polarization in the presence of AM
To study the impact of AM from tumor-bearing versus tumor-free mice on
the polarization of T cell responses, AM were isolated by two-step magnetic
sorting (F4/80+ cells followed by CD11bnegative; Miltenyi Biotec) from the
lungs of tumor-bearing (21 days after tumor cell injection into the mammary fat pad) and tumor-free control mice. Naı̈ve CD4+ T cells were
enriched (Miltenyi Biotec) from splenocytes of tumor-free Balb/c mice.
The purity of cells was verified by FACS and found to be .98%. CD4+
T cells were differentiated into effectors in the presence of AM from either
tumor-bearing or tumor-free mice in a 24-well plate, coated with CD3 Abs
in the presence of soluble CD28 Abs. CD4+ T cell cultures were harvested
at day 6, and intracellular staining for IFN-g, IL-4, and IL-17 was performed.
Generation of CD4+ T cell subsets and tumor cell killing assay
For generation of OVA-specific Th1 and Th2 cell lines, CD4+ T cells were
isolated from OTII splenocytes using magnetic columns (Miltenyi Biotec).
A total of 5 3 105 CD4+ T cells were stimulated with 1027M OVA323–339
peptide (AnaSpec)–loaded C57BL/6 splenocytes (5 3 106). These splenocytes were treated with 50 mg/ml mitomycin B (Sigma-Aldrich) before
coculturing with T cells. To generate Th1 cells, cultures were supplemented with murine (m) IL-2 (20 U/ml, eBiosciences), mIL-12 (10 ng/ml,
eBiosciences), and neutralizing IL-4 Ab (1 mg/ml, BioLegend). For Th2
generation, mIL-2 (20 U/ml), mIL-4 (10 ng/ml), neutralizing IL-12 (1 mg/ml,
BioLegend), and IFN-g (1 mg/ml, BioLegend) Abs were added (18). Cells
were grown in RPMI 1640 medium containing L-glutamine, HEPES, 10%
FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-ME, 1%
NEAA, and 1 mM sodium pyruvate. Cultures were maintained for 7–10
days in 24-well plates. Differentiated Th1 and Th2 cells were stimulated
with Ova323–339 peptide–loaded B16-F10 cells to test their ability to produce IFN-g and IL-4, respectively. Differentiated Th1 and Th2 cells were
separated from dead cells by Ficoll and cocultured with B16-F10 and
OVA-expressing B16-F10 cells. To evaluate induction of cytotoxicity, the
expression of caspase-3 was examined in tumor (CD45negative) cells by
intracellular staining and FACS.
Tumor measurement and metastatic burden evaluation
Tumor measurements were obtained in two dimensions (length and width).
The depth of the tumor was estimated based on the smaller (width) measurement, and the volume of the tumor was calculated using the following
formula: volume = (length 3 width 3 width)/2. Lung metastases were
counted using a dissection microscope and then verified by routine histological examination.
Statistical analysis
Data were analyzed with an unpaired t test or nonparametric Mann–
Whitney test, depending on the results of the normality test (Kolmogorov–
Smirnov). For data with the normal distribution, a two-tailed unpaired t test
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In a syngeneic model of breast cancer, mice were injected with 1 3 105 4T1
cells in the mammary fat pad. This model recapitulates stage IV human
breast cancer, because primary tumors metastasize to the vital organs, as
in human malignancy (13). FVB/Nj Her2-Tg mice develop spontaneous
breast adenocarcinomas starting at 4 months of age. Of tumor-bearing
mice that live to 8 months or longer, 72% develop lung metastatic disease. For AM depletion, mice were intranasally administered 60 ml
clodronate liposomes (FormuMax), starting on day 6 after injection of 4T1
cells into the mammary fat pad. The dose was repeated every fourth day
until mice were sacrificed. This treatment selectively depleted AM without
affecting other lung immune cell populations (Fig. 3) and has been used in
various studies for AM depletion (14). Control mice received similar doses
of PBS liposomes (FormuMax).
TISSUE RESIDENT MACROPHAGES IN CANCER METASTASIS
The Journal of Immunology
3
(t test) was used. A two-tailed unpaired t test with Welch’s correction
(t test with Welch’s correction) was used for data having significant differences in variances between groups. For data lacking the normal distribution, a two-tailed nonparametric Mann–Whitney test (Mann–Whitney
test) was used. Outliers were identified using Grubb’s test at a = 0.05. All
statistical analysis was done with GraphPad Prism 6 software. Statistical
significance was based on a p value # 0.05.
Results
Primary breast tumors affect the cellular composition of the
lung environment
AM accumulate in the premetastatic niche through complement
C5aR-mediated proliferation
In contrast to inflammatory macrophages and lung IM that originate from recruited blood monocytes, TRM, including AM, are
maintained through adult life by self-renewal (5). Therefore, we
FIGURE 1. Breast tumors affect the composition of the lung immune
microenvironment. (A) Cellular composition of the lung immune environment in tumor-free (TF) and tumor-bearing (TB) mice (pie charts
showing fractions) and percentages of CD45+ cells determined by FACS in
the lungs of TF and TB mice (bar graph). ***p , 0.0001 (t test). (B)
Absolute numbers of CD11b+Gr-1+ MDSC, ***p , 0.0001 (t test);
CD11b+F4/80+ lung IM, ***p , 0.0001 (t test); CD11bnegativeCD11c+F4/
80+ AM, **p = 0.0084 (t test); CD11c+MHCII+ DC, ***p = 0.0001 (t test);
CD8+ T cells, **p = 0.0082 (t test); and CD4+ T cells, *p = 0.0110 (t test)
determined by FACS in the lungs of TF and TB mice. (C) Gating strategy
used to identify various immune cell populations in the lungs of TF and TB
mice. Cells were first gated based on forward light scatter (FSC)/side light
scatter (SSC), followed by gating on viable cells (live/dead staining), and
then based on CD45 expression (middle plots). CD11b-negative cells were
gated to identify AM (middle plots). Numbers in each plot depict the
absolute counts of the cell population calculated from the total number of
lung cells retrieved after single-cell preparation. (D) MHC II and (E) CD86
expression on lung DC from TF and TB mice. ***p , 0.0001 (t test with
Welch’s correction). (F) Representative histograms with MFI. (G) The
ratio of percentages of IFN-g–expressing CD4+ T cells (Th1 cells) to IL-4–
expressing CD4+ T cells (Th2 cells) determined by FACS in the lungs of
TF and TB mice after ex vivo stimulation. **p = 0.0027 (t test). (H)
Representative dot plots of IFN-g–and IL-4–expressing CD4+ T cells in
the lungs of TF and TB mice. Data pertaining to TF mice are representative
of two independent experiments with n = 10 mice, and data for TB mice
are representative of three independent experiments with n1 $ 9, n2 = 5,
and n3 = 10. MFI, median fluorescence intensity.
hypothesized that an increase in the number of these cells in the
lungs of tumor-bearing mice results from proliferation and sustained self-renewal capabilities rather than recruitment from the
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Impact of primary tumors on the immune environment of the
premetastatic niche requires clarification; therefore, we examined
various immune cell populations with immunomodulatory roles in
the lungs of tumor-bearing mice. Overall, primary breast malignancy induced the significant accumulation of hematopoietic cells
(CD45+) in the lungs (Fig. 1A). Although recruited MDSC constituted the largest cell population (Fig. 1A, 1B), which is in
concordance with previous reports (19), several other immune
cells, such as IM, DC, CD4+ T cells, and CD8+ T cells (Fig. 1B),
were also increased in the lungs of tumor-bearing versus tumorfree mice. In addition to hematopoietic cells, we found a significant increase in the number of AM (CD11bnegativeCD11c+F4/80+),
defined by the set of previously described phenotypic markers
(www.immgen.org; Ref. 16) (Fig. 1C). These cells could be distinguished from other myeloid populations, such as lung IM
(CD11b+F4/80+), MDSC (CD11b+Gr-1+), or DC (CD11c+MHCII+)
in both tumor-free and tumor-bearing mice (Fig. 1C). In addition to
the alterations in cellular composition of the lung immune microenvironment, tumor-bearing mice demonstrated downregulation of
MHC II (Fig. 1D, 1F) and CD86 (Fig. 1E, 1F) on lung DC, which is
likely to contribute to the reduction of Ag-presenting functions
of DC, as in the primary tumor microenvironment (20). Tumorbearing mice also displayed reduction in Th1 function (% of IFNg–expressing/% of IL-4–expressing CD4+ T cells) (Fig. 1G, 1H),
corresponding to the reduced function of DC. Because Th1 cells
have been shown to contribute to antitumor immunity (21), these
changes suggest that primary tumors modify developing antitumor
response at distant sites, which might facilitate metastasis. We did
not observe differences in IL-17–producing cells between tumorfree and tumor-bearing mice (Fig. 1H). Altogether, these immune
alterations represent an escape mechanism for metastasizing tumor
cells.
To determine the time interval in which premetastatic changes
occur, we tracked 4T1 cells that expressed GFP (4T1-GFP+) and
determined that tumor cells arrived to the lungs at day 20, at the
earliest, as a result of spontaneous metastasis of the implanted
breast tumors (12). Therefore, we concluded that the accumulation
of AM and other immune cells observed in this model, using the
same 4T1 clone (Fig. 2A), occurs prior to metastasis and thus
contributes to the premetastatic niche. To validate these findings
in another model, we examined the lungs of FVB/N HER2/neu
transgenic mice that develop spontaneous focal mammary adenocarcinomas metastasizing to the lungs (22), before and after
the development of breast tumors, but before lung metastasis.
We observed an increase in the number of AM in breast tumor–
bearing mice prior to metastases, as determined by histological
and immunohistochemical studies with AM marker F4/80 of
the lung serial sections (Fig. 2B).
4
TISSUE RESIDENT MACROPHAGES IN CANCER METASTASIS
circulation. This hypothesis was supported by the high percentage
of AM expressing proliferation marker Ki-67 (Fig. 2C), when
compared with MDSC that are primarily recruited to the lungs
from the circulation and, hence, have no proliferative capacity
once recruited (Fig. 2C). These data were further confirmed by
confocal microscopy that demonstrated nuclear expression of
Ki-67 in AM (F4/80+CD11bnegative) (Fig. 2D, upper panel). We
also found some lung IM (CD11b+F4/80+) that expressed Ki-67
(Fig. 2D, lower panel).
Although the complement anaphylatoxin C5a is considered
a potent chemoattractant for leukocytes and MDSC (12, 23), C5a/
C5aR signaling has a significant role in regeneration of the liver
and, importantly, in proliferation of other types of TRM Kupffer
cells (24). Therefore, we examined whether the accumulation of
AM was dependent on C5aR. We found a significant reduction in
the number of Ki-67–expressing AM in C5aR-deficient (C5aR2/2)
mice compared with WT tumor-bearing mice, prior to metastasis, at day 14 after tumor cell injection into the mammary fat pad
(Fig. 2E). Together with the high expression of C5aR on AM
(Fig. 2F) and the reduced number of AM in C5aR2/2 lungs at the
end point of study, these data suggest that C5a/C5aR signaling
contributes to the accumulation of AM in the premetastatic lungs.
These findings are also concordant with our previous report indicating complement activation and generation of C5a from C5 in
the premetastatic lungs (12). Although liver is a major site for
complement protein synthesis and large quantities of C5 circulate
in plasma, macrophages are also known to produce C5 (25);
therefore, we examined the expression of C5 in the lungs of
tumor-bearing mice. We found that C5 was upregulated at several
time points after tumor cell inoculation (Fig. 2H), indicating that
the local production of C5 contributes to the C5a/C5aR signaling
in AM proliferation.
To evaluate the contribution of recruited myeloid cells to the
increase in AM, we adoptively transferred 1 3 106 BM cells from
tumor-free mice carrying the Ptprca allele (CD45.1) to tumor-free
and breast tumor–bearing WT mice that carry the Ptprcb allele
(CD45.2), at day 14 after tumor cell inoculation into the mammary
fat pad. With this approach, it is possible to distinguish CD45.1+
donor-derived cells that are recruited from the circulation from
CD45.2+ cells of the recipient mouse, using FACS staining. We
detected transplanted CD45.1 cells in the blood, lung, and spleen
of both tumor-free and tumor-bearing mice at day 7 after the
transplant. However, in all these locations, CD45.1 cells were
significantly higher in tumor-bearing mice (Fig. 3A), indicating
that the environment of the tumor-bearing host was more favorable for survival of transferred cells. Notably, transplanted CD45.1
cells in the lungs of tumor-bearing mice (CD45.2) lacked the AM
phenotype (CD11bnegativeF4/80+CD11c+) (Fig. 3B, the left dot plot
and two next upper plots) in contrast to CD45.2 originating from
recipient mice (Fig. 3B, the left dot plot and two next lower plots).
The majority of the transplanted cells in the lungs expressed CD11b
and Gr-1 typical markers for MDSC (Fig. 3B, the upper second
from the right plot, 3C) similar to CD45.2 cells (Fig. 3B, the lower
second from the right plot, 3C). Small fractions of CD45.1 express
markers of IM also defined as inflammatory macrophages (Fig. 3B,
the right plots, 3C). These observations remain in agreement with
studies demonstrating that MDSC and inflammatory monocytes
(converted later to IM) are recruited from the circulation to the
lungs of the tumor-bearing host (2, 19) and exclude the recruitment
of BM-derived cells as a source of AM. As with the lungs, trans-
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FIGURE 2. Increase in AM in the premetastatic
lungs is caused by C5a/C5aR-mediated proliferation.
(A) Numbers of CD11bnegativeF4/80+ AM in the lungs
of tumor-free (TF) and tumor-bearing (TB) mice at
various time points after tumor cell injection into the
mammary fat pad, determined by immunofluorescence
staining of lung sections. *p = 0.0089 (t test), **p =
0.0016 (t test), ***p = 0.0015 (t test). (B) H&E staining
(upper panel) and F4/80 immunohistochemistry (lower
panel) in lung sections from TF and TB FVB/n-j-Her2
Tg mice. Scale bar, 50 mm. (C) Ki-67 expression in AM
(CD11bnegativeCD11c+F4/80+) and MDSC (CD11b+Gr-1+)
from the lungs of TB mice (day 14) determined by FACS.
(D) Immunofluorescence detection of Ki-67 (green)
in nuclei (DAPI, blue) of AM (CD11bnegativeF4/80+)
(upper panel) and lung IM (CD11b+F4/80+) (lower
panel). Arrowheads point to AM, and arrows point to
IM. Scale bar, 25 mm. (E) Absolute numbers of Ki-67+
AM (CD11bnegativeCD11c+F4/80+) in the lungs of TB
WT and C5aR2/2 mice (calculated based on FACS data).
*p = 0.02. (F) Histogram shows C5aR expression on AM
from WT and C5aR2/2 (control) mice. (G) Absolute
numbers of AM (CD11bnegativeCD11c+F4/80+) in the
lungs of TB WT and C5aR2/2 mice at the end point of
study (day 30). *p = 0.001. (H) Mean relative expression
of C5 in the lungs based on quantitative PCR. Data
pertaining to TF mice were obtained from one experiment with n = 10 mice, and data for TB mice are representative of three independent experiments with n1 $
9, n2 = 5, and n3 = 10. Data for quantitative PCR were
obtained from n = 4 mice per group.
The Journal of Immunology
5
FIGURE 3. Cells recruited from BM do not
contribute to the increase in number of AM prior to
cancer metastasis. BM cells from tumor-free (TF)
CD45.1 mouse (donor) were transferred i.v. to
CD45.2 (recipient) tumor-bearing (TB) and TF
mice. After 7 d, FACS was used to identify
transferred CD45.1+ cells in various organs. (A)
Percentages of CD45.1 cells in blood, lungs, and
spleens of TF and TB mice. *p = 0.006, **p =
0.0001, ***p = 0.0004 (t test). (B) FACS plots
show absence of CD45.1+ AM (CD11bnegative
CD11c+F4/80+) cells and presence of CD45.1+
MDSC (CD11b+Gr-1+) and lung IM (CD11b+
F4/80+) in the lungs of TB CD45.2 mice (recipient).
(C) Percentages of CD45.1+ cells that differentiated
into IM and MDSC in TB mice.
AM suppress Th1 responses in the lungs
Because AM are the key players in quenching T cell responses
to environmental Ags (10), we hypothesize that primary tumors
harness this property of AM to suppress developing antitumor
immune response in the lungs. To address this hypothesis, we
selectively depleted AM by intranasal administration of clodronate liposomes (Fig. 4A, 4B). Intratracheally or intranasally administered clodronate liposomes do not affect other phagocytic
cells in the lungs and other locations (14). Accordingly, we did not
observe the impact of this treatment on lung IM (Fig. 4C). Of note,
because MDSC have weak phagocytic properties (26) and intranasally administered clodronate liposomes are avidly taken up by
highly phagocytic AM (14), clodronate liposomes did not reduce
the percentage of MDSC in the lungs of tumor-bearing mice
(Fig. 4D). The depletion of AM increased the percentage of lung
DC in breast tumor–bearing mice (Fig. 4E, 4F) and upregulated
the expression of CD86, CD80 (Fig. 4G, 4H), and MHC II
(Fig. 4I, 4J) on DC, indicating their increased maturation. Consequently, this increase in DC maturation augmented the IFN-g
production by CD4+ T cells (Th1) (Fig. 5A, 5B), with no changes
in IL-4 (Fig. 5A, 5B). In addition, we found that compared with
IL-4–producing CD4+ T cells (Th2), the considerably higher
percentage of IFN-g–expressing CD4+ T cells coexpressed granzyme B (GzB) in AM-depleted mice (Fig. 5C, 5D), confirming the
tumoricidal activity of these CD4+ T cells. In concordance with
these findings, Th1 cells, compared with Th2 cells, have been
shown to be superior in tumor cell killing as a result of their cytolytic activity (21, 27). Importantly, we observed that enhancing
Th1 responses and associated IFN-g production upregulated MHC
II on metastasizing (CD45negative) tumor cells (Fig. 5E–G), ren-
dering them more susceptible for attack by these cytolytic Th1
cells. This finding corresponds to a previous study reporting on the
importance of MHC II expression on tumor cells in enhancing the
efficacy of tumor cell vaccine for therapy against metastatic carcinoma (28). We have identified CD45negative MHC II–expressing
cells as tumor cells, because MHC II is normally expressed on
APCs (in the majority CD45+) and aberrantly on CD45negative
tumor cells (28). Nontransformed epithelial cells, which are also
CD45negative, do not express MHC II. In addition, CD45negative
lung endothelial cells that might express MHC II are usually not
present in a single-cell suspension prepared using standard protocols for flow cytometry.
To determine whether primary tumors condition AM for suppressing antitumor T cells in the lungs, we cocultured AM (F4/80+
and CD11bnegative) isolated from the lungs of tumor-free and
tumor-bearing mice with CD4+ T cells (from tumor-free “naive”
mice) differentiated into effectors by CD3/CD28 stimulation (Fig
5H). Notably, AM isolated from tumor-bearing mice promoted the
Th2 response, in contrast to AM from tumor-free mice, as indicated by the ratio of Th1/Th2 cells (Fig. 5I, 5J). We did not observe an impact of AM on IL-17 responses (Fig. 5J). Thus, these
in vitro findings correspond to our in vivo data and support the role
of AM in polarization of T cell responses in the lungs of tumorbearing mice. These results also indicate that AM, in addition to
modulating DC maturation, can directly regulate T cell function.
Because T cell and DC functions are regulated by the cytokine
milieu, we next investigated whether depletion of AM affects
expression of immunoregulatory cytokines in the lung hematopoietic cells. We observed that AM depletion reduced the production of immunosuppressive TGF-b (Fig. 5K, 5L) and increased
the production of Th1-promoting IL-12 (Fig. 5M, 5N) in LPS ex
vivo stimulated CD45+ cells. We used LPS stimulation because
TLR-4 signaling is important for immunosuppressive properties of
myeloid cells and has been reported to contribute to the premetastatic niche (1, 9). These findings further support the suppressive role of AM toward effector CD4+ T cells.
AM facilitate lung metastasis
The enhancement of immunity in the lungs resulting from AM
depletion was associated with reduction in lung metastasis
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planted CD45.1 cells expressed markers of MDSC in peripheral
blood and spleen (Fig. 3C). In addition, these cells expressed
markers of macrophages in spleen but not in the circulation
(Fig. 3C). To determine if the source of transferred cells has an
impact on results, we also adoptively transferred CD11b+ splenocytes that were CD45.1 from tumor-free mice to tumor-free and
breast tumor–bearing WT mice (CD45.2). As with experiments
involving BM cells, we did not detect expression of AM markers on
transplanted cells in the lungs (data not shown).
6
TISSUE RESIDENT MACROPHAGES IN CANCER METASTASIS
Discussion
Immune privileged status of the lungs favors metastasis
FIGURE 4. AM inhibit DC in the lungs of tumor-bearing mice. (A)
Percentages of AM (CD11bnegativeCD11c+F4/80+) in PBS (AM+, nondepleted) and clodronate liposome (AM2, depleted)–treated tumor-bearing
(TB) mice. *p , 0.0001 (t test with Welch’s correction). (B) Representative FACS plots depicting AM in the lungs of TB AM+ and AM2 mice
with percentages of AM depicted. (C) Percentages of lung IM (CD11b+
F4/80+) and (D) MDSC (CD11b+Gr-1+). (E) Percentages of DC (CD11c+
MHCII+) in the lungs of TB AM+ and AM2 mice. *p = 0.0023 (t test). (F)
Representative FACS plots showing lung DC from TB AM+ and AM2 TB
mice, where numbers depict their percentages in the lungs. (G) CD86 and
CD80 expression on lung DC from TB AM+ and AM2 mice. *p = 0.024
(t test), ***p , 0.0001 (t test). (H) Representative histograms. (I) MHC II
expression on lung DC from TB AM+ and AM2 mice. *p , 0.0001 (t test).
(J) Representative histograms. Data are representative of at least three
independent experiments with n $ 10. MFI, median fluorescence intensity.
(Fig. 6A) without an impact on primary tumor growth (Fig. 6B).
Therefore, we propose that AM-mediated suppression of lung
immunity promotes metastasis independently of immunoregulatory mechanisms operating in primary tumors. C5aR deficiency or
C5aR blockade lessens lung metastasis by reducing recruitment of
MDSC to the lungs and inhibiting their suppressive function (12).
Therefore, we hypothesize that inhibition of C5aR will synergize
with AM depletion in reducing lung metastasis. Indeed, in the
independent experiment the depletion of AM in C5aR2/2 mice
entirely prevented lung metastasis (Fig. 6C), indicating increased
therapeutic efficacy of combined treatment versus depletion of
AM or blocking C5aR alone. Bisphosphonates, including clodronate, have been reported to cause apoptosis of human breast
cancer cells in vitro (29); therefore, we tested whether clodronate
liposomes, used to deplete AM, induce apoptosis of 4T1 cells.
No impact of clodronate on 4T1 cell apoptosis was observed
(Fig. 6D), ruling out this possibility.
We conclude that AM facilitate metastases by suppressing Th1
T cell responses, respectively. The tumoricidal activity of CD8+
T cells is well established; however, a direct killing of tumor cells
by Th1 cells has been less studied. We found that breast malignancy reduced the Th1/Th2 ratio in the lungs and AM depletion
Cancer metastasis is a hallmark of tumor malignancy and a cause of
∼90% of cancer-associated deaths. However, paradoxically, this
process is the most poorly understood aspect of cancer pathophysiology (6). One of the key unanswered questions pertaining to
metastasis is why certain organs are more often affected by metastases whereas other sites are spared or seldom affected. The
hemodynamic features of the circulation and specific properties of
the vasculature in metastases-targeted organs, together with the
distinct molecular signatures of metastasizing tumor cells, do not
seem to fully explain the receptiveness of the vital organs, such as
lungs for tumor cells (6). Given the role of the immune system in
cancer surveillance (30), the immune-privileged status of the lungs
(10), and growing evidence for the role of immunosuppression in
distant sites in facilitating metastasis (12, 19), we hypothesize that
alterations in antitumor immunity in the lungs play a significant role
in facilitating metastasis. The special immune status of the lungs is
dictated by their positions as a barrier between the external environment and the body. The lung immune system must be controlled
to avoid unwanted immune reactions to harmless environmental Ags
(10). We hypothesize that these immunosuppressive homeostatic
mechanisms that operate in physiology to protect the lungs from
autoimmunity are harnessed by tumors to suppress antitumor immune responses.
AM in metastasis
The important feature of lung immune homeostasis is the abundance of AM that appear to have the critical role in physiological
immunosuppression (31, 32). Therefore, we propose that, as instructed by tumors, AM suppress developing antitumor responses in the
lungs and through this mechanism facilitate metastasis. Early studies
reported different and often conflicting results on the role of AM in
metastasis. AM from mice injected s.c. with Lewis lung carcinoma
cells were shown to suppress Con A blastogenesis and NK cytotoxicity. Thus, it was concluded from this study that through these immunosuppressive properties AM might facilitate metastasis (33). In
contrast, AM isolated from the bronchial lavage fluid of patients with
lung cancer and chronic lung disease were cytotoxic to various human
cancer cell lines in vitro (34). Furthermore, pulmonary macrophages
activated by intranasal inoculation of Propionibacterium acnes failed
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reversed this trend. Therefore, we investigated tumor cell killing
by Th1 versus Th2 cells. To use identical Ag-specific T cells with
different polarization conditions, we obtained naı̈ve CD4+ T cells
from OTII TCR transgenic mice. This system enables us to study
cytotoxicity imposed by both Th subsets on tumor cells expressing
OVA (OVA+). In vitro differentiated Th1 and Th2 cells were
cocultured with OVA-expressing B16-F10 cells, and apoptosis represented by activation of caspase-3 was evaluated in tumor cells. We
found that the percentages of OVA+ tumor cells expressing caspase-3
were moderately, although significantly, increased in the cultures
with Th1 cells compared with Th2 (Fig. 6E, 6F). Although less
efficient, Th2 cells were also cytotoxic to OVA+ tumor cells (Fig. 6E,
6F). In contrast, killing of WT B16-F10 cells (no OVA expression)
by both Th1 and Th2 was minimal, showing the importance of Ag
specificity in CD4+ T cell–mediated tumor cell apoptosis. In addition, we found a higher percentage of Th1 cells than Th2 cells
expressing GzB upon stimulation with B16-F10–OVA, indicating the
better potential of Th1 cells for Ag-specific cytotoxicity toward tumor cells (Fig. 6G, 6H, upper panels). In contrast, control B16-F10
cells without OVA expression did not induce granzyme expression
(lower panels).
The Journal of Immunology
7
to reduce metastases of fibrosarcoma cells injected i.v. (35). These
studies were intriguing but were devoid of modern-day understanding and technical advancement to finely characterize or identify
the precise role of AM. Recent studies have demonstrated a role
in facilitating metastasis for a distinct subpopulation of macrophages
(CD11b+F4/80+), known as metastasis-associated macrophages,
recruited to the lungs from the circulation (2). However, on the basis
of phenotypic characteristics, these cells were different from AM that
do not express CD11b and that do express CD11c and F4/80 (5)
(www.immgen.org). In addition, depletion of AM by clodronate liposomes that were administered intratracheally did not affect lung
metastasis when tumor cells were injected i.v. (2). Our data differ
from those of previous studies demonstrating that AM depletion had
no impact on lung metastasis. Of note, these studies used experimental metastasis (i.v. injected tumor cells). This approach differs
from models of spontaneous metastasis used in the current work, as
the presence of primary tumors preconditions the lungs and, consequently AM, to receive metastasizing tumor cells. This part of the
metastatic process is omitted when tumor cells are injected into the
circulation to otherwise tumor-free mice. Therefore, AM from tumorfree mice might be functionally different from the cells of tumorbearing mice that develop spontaneous metastasis. However, it
remains to be established how primary tumors that are distant from
the lungs alter AM. A plausible scenario involves several recently
described tumor-derived factors that facilitate the formation of the
premetastatic niche, such as vascular endothelial growth factor (36),
placental growth factor (36), TNF, and TGF-b (9). Alternatively,
tumor-derived exosomes that have recently emerged as inducers of
the premetastatic niche in mouse models and melanoma patients may
take part in this process (37). Another possibility is that AM are altered through interactions with the BM-derived cells, including
MDSC that are recruited to the premetastatic niche, similar to the
interactions described for MDSC with TAM in the primary tumor
microenvironment (1). MDSC are known to skew the polarization
of TAM macrophages toward the M2 phenotype through various
mechanisms (38); therefore, our data indicating that AM isolated from
tumor-bearing mice were more efficient in favoring Th2 responses
compared with AM from tumor-free mice support this concept.
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FIGURE 5. AM suppress Th1 responses in the lungs of tumor-bearing (TB) mice. The CD4+ T cell response was examined after ex vivo stimulation with
CD3/CD28 Abs of lung cells from TB mice treated with PBS (AM+, no depletion) or clodronate liposomes (AM2, depleted). (A) Percentages of IFN-g–
and IL-4–expressing CD4+ T cells in the lungs of AM+ and AM2 TB mice. *p = 0.0344 (t test). (B) Representative FACS plots. (C) Percentages of CD4+
T cells coexpressing GzB and IFN-g or IL-4 in the lungs of AM2 mice. ***p , 0.0001 (t test). (D) Representative histograms showing percentage of GzB
expression. (E) Representative FACS plots of MHC II expression on CD45negative tumor cells in the lungs. (F) MHC II expression on metastasizing tumor
cells. *p = 0.0049 (t test with Welch’s correction). (G) Representative histograms. (H) AM from TB and tumor-free (TF) mice were cocultured with ex vivo
stimulated “naive” CD4+ T cells from TF mice. Histograms show enriched CD11bnegativeF4/80+ AM isolated from TF and TB mice. (I) The ratio of the
percentage of CD4+ T cells expressing IFN-g (Th1 cells) to the percentage of cells expressing IL-4 (Th2 cells) after coculture with AM from TF versus TB
mice. **p = 0.002 (t test). (J) Representative FACS plots. (K) Percentages of CD45+ TGF-b–producing lung cells (***p = 0.0003, t test) or (M) IL-12–
producing lung cells (*p = 0.2515, 95% confidence interval, 20.09–0.02, t test) from TB AM+ and AM2 mice. (L and N) Representative FACS plots,
respectively, showing percentages of positive cells after ex vivo stimulation with LPS. Control cells in (L) are unstimulated CD45+ lung cells from TB mice.
Data are representative of one experiment with n = 10 mice. MFI, median fluorescence intensity.
8
TISSUE RESIDENT MACROPHAGES IN CANCER METASTASIS
recruited from the BM (9, 19, 36), with the exception of resident
lung fibroblasts (36). In addition, the accumulation of AM was
C5aR dependent, which indicates the possible impact of C5aR on
AM proliferation. Given the early activation of complement in the
premetastatic lungs (12) with the documented contribution of
C5aR to proliferation of liver TRM Kupffer cells (24, 39), this
scenario is plausible, although further mechanistic studies are
required to elucidate this mechanism. Furthermore, adoptive
transfer studies excluded contributions of circulating monocytes to
the increase in number of AM, which further underscores the selfrenewal capabilities of AM (5).
AM modulate antitumor immunity in the lungs of
tumor-bearing mice
FIGURE 6. AM promote lung metastasis. (A) Number of lung metastases in tumor-bearing (TB) mice in the presence of AM (AM+) or after
their depletion (AM2). *p = 0.0013, t test. (B) Primary tumor volumes in
AM+ and AM2 mice. (C) Number of lung metastases in TB WT or C5aR2/2
mice in the presence of AM (WT-AM+, C5aR2/2-AM+) or in AM-depleted lungs (WT-AM2, C5aR2/2-AM2). *p = 0.0325, Mann–Whitney
test, **p = 0.0294, Mann–Whitney test. (D) Annexin V staining in 4T1
cells treated with PBS or clodronate liposomes. (E) Percentages of B16 and
B16-OVA tumor cells expressing caspase-3 after incubation with in vitro
generated OVA-specific Th1 and Th2 cells. *p = 0.0275, t test, ***p ,
0.0001, t test, ****p , 0.0001, t test. (F) Representative histograms. (G)
Percentages of OVA-specific Th1 and Th2 cells expressing GzB. **p =
0.0010, t test. (H) Representative FACS plots (upper panels); granzyme
expression in T cells incubated with B-16 cells that did not express OVA
(lower panel, control). Data are representative of three independent
experiments with n1 = 5, n2 $ 8, n3 $ 9 mice for (A); one experiment with
n = 10 mice per cohort for (B), (C), and (D); and one experiment with n $ 3
biological replicates for (E)–(H).
AM arrive to the lungs during the embryonic stage and selfrenew through proliferation thereafter (5, 7). We found that the
number of AM gradually increased in tumor-bearing mice via
proliferation, which is consistent with their self-renewal capacity
(5). This finding implies their contribution to the premetastatic
niche, as these cells home to the metastases-targeted organ
(i.e., lungs) prior to tumor cell arrival. In contrast to AM, cells
previously reported to contribute to the premetastatic niche were
FIGURE 7. The role of AM in the premetastatic niche. C5a/C5aRregulated proliferation of AM leads to an increase in the number of AM
in the premetastatic lungs. Preconditioned by primary breast tumor, AM
suppress antitumor T cell immunity in the lungs. AM suppress IFN-g
production in CD4+ T cells (Th1) that are directly cytotoxic to tumor cells
in the context of MHC II by regulating TGF-b in lung cells or by inhibiting
lung DC. AM also downregulate MHC II expression on tumor cells (TC),
thereby preventing their interactions with tumor-specific cytotoxic CD4+
T cells. The alterations in antitumor immunity mediated by AM predispose
lungs to metastasis.
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We found that AM depletion resulted in an increase in IFN-g–
producing CD4+ T cells (Th1). This finding is in agreement with
the key immunoregulatory functions of AM in the absence of
malignancy (10). AM have been shown to inhibit adaptive immune responses, as confirmed by the induction of T cell responses
to inhaled Ags in AM-depleted mice (32). However, in contrast to
our previous work on the role of C5aR in the lung premetastatic
niche (12), we did not observe a significant increase in IFN-g–
producing CD8+ T cells (data not shown). Therefore, given the
striking reduction in lung metastases in AM-depleted mice, we
concluded that Th1 cells protect the lungs from metastasis. Although the cytotoxic function of Th1 cells is documented, it has
been less frequently studied than cytotoxicity exerted by CD8+
T cells (27, 40). Therefore, we further verified the killing capacity
of tumor-specific Th1 cells, using the OTII transgenic system. Of
note, the increased expression of IFN-g in the lungs of AMdepleted tumor-bearing mice led to the upregulation of MHC II
on metastasizing tumor cells, rendering them more susceptible to
Th1-mediated killing (41). The results of coculture experiments
involving AM and T cells demonstrated that AM directly affected
The Journal of Immunology
Acknowledgments
We thank Dr. Laurence Wood, Texas Tech University Health Science
Center, for providing quantitative PCR primers and the B16 cell line.
Disclosures
The authors have no financial conflicts of interest.
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the generation of effector Th1 cells, which supports the concept
that AM can directly suppress T cells (10). However, AM depletion also increased the number and maturation of lung DC,
which corresponds to previous studies in nontumor conditions
demonstrating enhanced DC function and migration of these cells
to alveolar spaces in AM-depleted animals (42). The impact of
AM depletion on the function of DC also corresponds to our
findings that AM regulate Th1/Th2 polarization. Furthermore, we
observed that AM increased the production of TGF-b in hematopoietic lung cells. Because TGF-b suppresses production of
inflammatory cytokines by AM (43), this mechanism could be
responsible for the paracrine regulation of immunosuppressive
functions of AM in the tumor-bearing host. Although AM represent the largest population of macrophages in the lungs, blood
monocytes are recruited to the lungs by CCL2 and differentiate
into macrophages during inflammation or in the tumor-bearing
host (2, 44). However, the functional and phenotypic characteristics of these macrophages are different from those of AM (2, 10).
Importantly, we found that clodronate liposomes administered
intranasally efficiently depleted AM but did not affect monocytederived macrophages. Therefore, the consequences of clodronate
liposome treatment on lung immunity and metastasis should be
attributed to the absence of AM.
In view of recent studies demonstrating the nonhematopoietic
origin of TRM (5), functions of these cells in health and disease
require further investigation. Our current work suggests the contribution of these cells represented by AM to the premetastatic
niche in the lungs (Fig. 7). In summary, in the presence of primary
breast tumors, AM acquire T cell suppressive functions, facilitating metastasis to their resident organ. Selective depletion of
AM leads to a reduction of metastases in the lungs and synergizes
with C5aR inhibition in preventing lung metastasis. Therefore,
this study points to AM as a novel target for anticancer therapy.
9
10
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C. W. Strey, S. Franchini, R. A. Wetsel, A. Erdei, and J. D. Lambris. 2004.
C3a and C3b activation products of the third component of complement
(C3) are critical for normal liver recovery after toxic injury. J. Immunol.
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cells present endogenous antigen and are potent inducers of tumor-specific immunity. Proc. Natl. Acad. Sci. USA 94: 6886–6891.
TISSUE RESIDENT MACROPHAGES IN CANCER METASTASIS
42. Jakubzick, C., F. Tacke, J. Llodra, N. van Rooijen, and G. J. Randolph. 2006. Modulation of dendritic cell trafficking to and from the airways. J. Immunol. 176: 3578–3584.
43. Munger, J. S., X. Huang, H. Kawakatsu, M. J. Griffiths, S. L. Dalton, J. Wu,
J. F. Pittet, N. Kaminski, C. Garat, M. A. Matthay, et al. 1999. The integrin alpha
v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating
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44. Winter, C., K. Taut, M. Srivastava, F. Länger, M. Mack, D. E. Briles, J. C. Paton,
R. Maus, T. Welte, M. D. Gunn, and U. A. Maus. 2007. Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to
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