CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.
ABSTRACT Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2-CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.
- SourceAvailable from: Ganji Purnachandra Nagaraju[Show abstract] [Hide abstract]
ABSTRACT: Deregulation of angiogenesis–the growth of new blood vessels from an existing vasculature–is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding “the most important target” may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the “Halifax Project” within the “Getting to know cancer” framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy; 1) endothelial cell migration/tip cell formation, 2) structural abnormalities of tumor vessels, 3) hypoxia, 4) lymphangiogenesis, 5) elevated interstitial fluid pressure, 6) poor perfusion, 7) disrupted circadian rhythms, 8) tumor promoting inflammation, 9) tumor promoting fibroblasts and 10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds; 1) oleic acid, 2) tripterine, 3) silibinin, 4) curcumin, 5) epigallocatechin-gallate, 6) kaempferol, 7) melatonin, 8) enterolactone, 9) withaferin A and 10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the “hallmarks” of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.Seminars in Cancer Biology 01/2015; · 9.14 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Deregulation of angiogenesis – the growth of new blood vessels from an existing vasculature – is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding “the most important target” may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the “Halifax Project” within the “Getting to know cancer” framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy: (1) endothelial cell migration/tip cell formation, (2) structural abnormalities of tumor vessels, (3) hypoxia, (4) lymphangiogenesis, (5) elevated interstitial fluid pressure, (6) poor perfusion, (7) disrupted circadian rhythms, (8) tumor promoting inflammation, (9) tumor promoting fibroblasts and (10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds: (1) oleic acid, (2) tripterine, (3) silibinin, (4) curcumin, (5) epigallocatechin-gallate, (6) kaempferol, (7) melatonin, (8) enterolactone, (9) withaferin A and (10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the “hallmarks” of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.Seminars in Cancer Biology 01/2015; (ePub ahead of print). · 9.14 Impact Factor
Article: Innate Immune Recognition of Cancer.[Show abstract] [Hide abstract]
ABSTRACT: The observation that a subset of cancer patients show evidence for spontaneous CD8(+) Tcell priming against tumor-associated antigens has generated renewed interest in the innate immune pathways that might serve as a bridge to an adaptive immune response to tumors. Manipulation of this endogenous T cell response with therapeutic intent-for example, using blocking antibodies inhibiting PD-1/PD-L1 (programmed death-1/programmed death ligand 1) interactions-is showing impressive clinical results. As such, understanding the innate immune mechanisms that enable this T cell response has important clinical relevance. Defined innate immune interactions in the cancer context include recognition by innate cell populations (NK cells, NKT cells, and γδ T cells) and also by dendritic cells and macrophages in response to damage-associated molecular patterns (DAMPs). Recent evidence has indicated that the major DAMP driving host antitumor immune responses is tumor-derived DNA, sensed by the stimulator of interferon gene (STING) pathway and driving type I IFN production. A deeper knowledge of the clinically relevant innate immune pathways involved in the recognition of tumors is leading toward new therapeutic strategies for cancer treatment. Expected final online publication date for the Annual Review of Immunology Volume 33 is March 21, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual Review of Immunology 01/2015; · 41.39 Impact Factor
CCL2 recruits inflammatory monocytes to facilitate
Bin-Zhi Qian1, Jiufeng Li1, Hui Zhang1, Takanori Kitamura1, Jinghang Zhang2, Liam R. Campion3, Elizabeth A. Kaiser3,
Linda A. Snyder3& Jeffrey W. Pollard1
enhance malignancy1. At metastatic sites, a distinct population of
cytes are preferentially recruited to pulmonary metastases but not to
primary mammary tumours in mice. This process also occurs for
subsequent recruitment of metastasis-associated macrophages and
their interaction with metastasizing tumour cells, is dependent on
CCL2 synthesized by both the tumour and the stroma. Inhibition of
CCL2–CCR2 signalling blocks the recruitment of inflammatory
monocytes, inhibits metastasis in vivo and prolongs the survival of
tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also
inhibits metastatic seeding. Inflammatory monocytes promote the
extravasation of tumour cells in a process that requires monocyte-
derived vascular endothelial growth factor. CCL2 expression and
static disease in human breast cancer3–6. Our data provide the mech-
anistic link between these two clinical associations and indicate new
therapeutic targets for treating metastatic breast cancer.
To understand the origin of macrophages in primary tumours and
their metastatic sites, we measured monocyte trafficking. Mouse
monocytes were identified by their expression of CD11b and CD115
sorting (FACS) into sub-populations of inflammatory monocytes
expressing Gr1 and Ly6c and resident monocytes lacking Gr1 and
Ly6c (refs 7, 8) (Supplementary Fig. 3b–d). Both populations had
similarexpressionof GFPin Csf1r–GFP transgenicmice (Supplemen-
into syngeneic FVB mice bearing autochthonous late-stage Polyoma
Middle T (PyMT) mammary tumours with spontaneous pulmonary
metastases (Fig. 1a). Eighteen hours after adoptive transfer, we deter-
mined the ratio of recovered GFP-positive inflammatory monocytes
(Supplementary Fig. 3e) to resident monocytes from the same donor,
to measure their relative recruitment. This indicated that there were
similar numbers of donor cells in the blood (showing equivalent
availability),butthat intheprimary tumour, residentmonocyteswere
preferentially recruited, whereas in pulmonary metastases, inflam-
endogenous inflammatory monocytes was identified in metastasis-
bearing lungs but not in normal lungs (Supplementary Fig. 4a). This
preferential recruitment of inflammatory monocytes in the lung was
not observed in 7-week-old PyMT mice bearing pre-metastatic mam-
mary tumours (Supplementary Fig. 4b). In experimentally induced
mouse mammary tumour cell line)12, inflammatory monocytes were
also preferentially recruited (Supplementary Fig. 4c). GFP-labelled
cells were readily detectable in pulmonary metastases at least 5d after
transfer(datanot shown)andwithin2d,asignificant portionofthem
had differentiated into F4/801CD11b1Gr12metastasis-associated
tary Fig. 4d). To test whether inflammatorymonocytes were recruited
early in the metastasis process, we transferred monocyte populations
1Department of Developmental and Molecular Biology, Center for the Study of Reproductive Biology and Women’s Health, Albert Einstein College of Medicine, New York, New York 10461, USA.2Flow
Cytometry Core Facility, Albert Einstein College of Medicine, New York, New York 10461, USA.3Ortho Biotech Oncology R&D, 145 King of Prussia Road, Radnor, Pennsylvania 19087, USA.
Donor IM per
106 CD45+ cells
Human CD45+ cells per
106 total CD45+ cells
Ratio of IM to RM
per 106 CD45+ cells
0 h18 h
Ratio of IM to RM
per 106 CD45+ cells
Ratio of CD14+CD16–
to CD14loCD16+ cells
Figure 1 | Pulmonary metastases preferentially recruit inflammatory
monocytes through CCL2. a, Schematic for the adoptive transfer of
monocytes into PyMT-tumour-bearing mice with pulmonary metastases. i.v.,
intravenous. b, Ratiosof inflammatory monocytes (IM) to resident monocytes
(RM) in different tissues of recipient mice bearing PyMT tumours and
metastases. n56; ***, P,0.0001. c, Ratios of inflammatory monocytes to
injected7h before measurement. n54;**, P50.0039.d, Relativenumbers of
donor inflammatory monocytes recruited in lungs challenged with Met-1 cells
for 7h, with control or anti-mouse CCL2 antibody treatment. n53; *,
P50.045. e, Ratios of adoptively transferred CD141CD162and
CD14lowCD161human monocytes recruited into the lungs of normal mice
(open bars) and of mice challenged with 4173 cells that contain metastases
(Mets: solid bars) for 7h. n55; **, P50.0163. All barsshowmean 1 s.e.m.
f, Numbers of adoptively transferred human CD141CD162monocytes that
migrated into different tissues of mice challenged with 4173 cells via
intravenous injection, with control or anti-mouse CCL2 antibody treatment.
Each line connects data from the same donor. n55; *, P50.016.
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7h after intravenous injection of Met-1 cells, a time point before
significant interaction between the tumour and macrophages, and
before extravasation of tumour cells2. Compared to control lungs, the
recruitment of inflammatory monocytes to tumour-cell-challenged
lungs increased markedly, with the ratio of inflammatory monocytes
this preferential recruitment of inflammatory monocytes was not
for injection and particle lodgement, respectively (Supplementary Fig.
4e and data not shown). Consistent with this early recruitment of
inflammatory monocytes, MAMs expressing high levels of CCR2 were
preferentially recruited to lungs 36h after tumour-cell inoculation2.
were not differentially recruited at this time (Supplementary Fig. 5a–c
and datanot shown).Thesedataindicate that MAMsare derived from
inflammatory monocytes that are specifically recruited early in the
process of pulmonary metastasis, before other immune cells.
Lung metastases of PyMT tumours express CCL2 homogeneously, in
contrast to its heterogeneous expression in primary tumours (Sup-
plementary Fig. 6a–d), and inflammatory monocytes have high levels
of CCR2 expression, whereas resident monocytes do not (Supplemen-
markedly inhibited both the recruitment of inflammatory monocytes
to lungs challenged with metastatic tumour cells (Fig. 1d) and the
increase in the number of MAMs at the metastatic site (Supplemen-
cells) and also Tregcells were unaffected by anti-CCL2 antibody treat-
ment in this model (Supplementary Fig. 5d). Furthermore, the pref-
erential recruitment of inflammatory monocytes to the tumour-cell-
challenged lung was completely abrogated during adoptive transfer of
monocytes sorted from Ccr2-null mice (Supplementary Fig. 6f).
The pattern of human monocyte recruitment to tumours in vivo is
unknown. To investigate this, human CD141CD162inflammatory
monocytes and CD14lowCD161resident monocytes9were sorted from
enriched CD141cells from the peripheral blood of healthy donors
(Supplementary Fig. 7a). 105cells of each population were adoptively
transferred into pairs of nude mice supplemented with recombinant
human colony-stimulating factor 1 (CSF1), which is essential for the
survival of monocytes and macrophages (Supplementary Fig. 7e).
Human monocytes were quantified 18h after adoptive transfer, using
FACS analysis with an antibody against human CD45 (Supplementary
Fig.7b). In normal mice, after adoptive transfer of monocytes fromthe
same donor, there were comparable numbers of human inflammatory
to the lung, but about twice the numbers of inflammatory monocytes
compared to resident monocytes in the spleen (Fig. 1e, open bars). In
mice given an intravenous injection of human MDA-MB-231-derived
metastatic 4173 breast cancer cells147h before monocyte transfer, the
monocytes in the lungs increased more than sixfold (Fig. 1e). In estab-
lished pulmonarymetastases derived fromorthotopically injected 4173
Mouse inflammatory monocytes were also preferentially recruited to
CCL2 with an antibody against mouse CCL2 markedly reduced the
recruitment of human inflammatory monocytes into lungs challenged
Treatment with an antibody specific to human CCL2 (ref. 15) also
inhibited inflammatory monocyte recruitment (Supplementary Fig.
7f), indicating the importance of CCL2 from both the tumour and the
to the same CCL2–CCR2 signalling as mouse cells for their specific
recruitment during pulmonary metastasis.
of inflammatory monocytes, we performed experimental metastasis
control antibody shortly before the tumour-cell injection. Anti-CCL2
treatment reduced the total metastasis burden, owing to a markedly
reduced number of metastasis nodules (Fig. 2a, b). An antibody spe-
the metastasis of Met-1 cells (Supplementary Fig. 8). This indicates
that the specific CCL2-mediated recruitment of inflammatory mono-
cytes is critical for the pulmonary seeding of tumour cells.
Extravasation is a critical step for the metastatic seeding of tumour
cells in the lung2. We used an intact-lung imaging system16to test
the role of CCL2-recruited inflammatory monocytes in tumour-cell
extravasation. Csf1r–GFP transgenic mice were injected intravenously
with cyan fluorescent protein (CFP)-expressing Met-1 cells and ana-
lysed after 24h. Quantification of three-dimensional reconstructed
was significantly reduced by anti-mouse CCL2 neutralizing antibody,
compared with control antibody (Fig. 2e). Notably, tumour-cell
extravasation was delayed and less efficient after the blocking of
and macrophages. In an in vitro trans-endothelial migration assay
(Supplementary Fig. 9a)17, the trans-endothelial migration of tumour
cells was enhanced about fivefold by mouse bone-marrow-derived
lial monolayer. This effect was blocked by anti-mouse-CCL2 neutral-
izing antibody, but not by control antibody (Supplementary Fig. 9b).
only the macrophages express CCR2 (Supplementary Fig. 9c), indi-
cating thatonlymacrophagesrespond tothe CCL2 chemokine signal-
ling. In confirmation of this, macrophages from Ccr2-null mice were
not capable of promoting trans-endothelial migration of tumour cells
(Supplementary Fig. 9d). Notably, FACS-sorted inflammatory mono-
cytes, but not resident monocytes, markedly promoted tumour-
cell trans-endothelial migration and this was also inhibited by
anti-mouse-CCL2 neutralizing antibody (Fig. 2g, h).
Total blockade of CCL2 (both mouse and human) inhibited
spontaneous lung metastasis of orthotopically injected MDA-MB-231
cells (Fig. 3a). Ligands secreted by both the tumour cells and the host
mouse antibodies markedly inhibited the experimental metastasis of
4173 cells (Fig. 3b) without affecting tumour-cell proliferation in vitro
(data not shown). This conclusion was also confirmed by knocking
down CCL2 using small interfering RNAs in 4173 cells: this markedly
reduced lung colonization in experimental metastasis assays (Sup-
plementary Fig. 10e, f). Consistentwith this, a similarCcl2-knockdown
10a–c). Trans-endothelial migration of 4173 cells in vitro was also pro-
moted by human inflammatory monocytes and inhibited by neutral-
via the recruitment of inflammatory monocytes. Consistent with the
role of CCL2 synthesized by the microenvironment in the lung, bone
andtheinhibition ofCCL2 inhibits metastaticprogression.Incontrast,
liver metastases of Met-1 cells did not recruit inflammatory monocytes
and CCL2 inhibition did not reduce metastasis (data not shown).
Furthermore, CCL2 blockade 2d after intravenous injection of MDA-
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inflammatory monocytes and their differentiation into MAMs for per-
sistent metastatic growth (Fig. 3f, g).
and inflammatory monocytes18. Among the differentially regulated
by inflammatory monocytes, a fact that we verified experimentally
Csf1r promoter, crossed with Vegfaflox/floxmice19. Inducible ablation of
Vegfa was achieved in cultured BMDMs treated with 4-hydroxyta-
the trans-endothelial migration of tumour cells and did not enhance
tamoxifen specifically ablated Vegfa in monocytes, without ablation in
other circulating immune cells (Fig.4d). This monocyte-specific deple-
tion of VEGFA markedly inhibited the potential for experimental
cells per ×20 field
Transmigrated tumour cells
(normalized to control)
0 10 20 30 40 50 60 70 80 90
Relative mets burden
Figure 3 | CCL2 from both the tumour cell and the host promotes
metastatic seeding. a, Numbers of spontaneous pulmonary metastases from
orthotopic MDA-MB-231 tumours with total CCL2 blockade or control
treatment. Bar shows the mean; n58 per group; ***, P,0.001. b, Metastasis
burden of intravenously injected 4173 cells with different antibody treatments.
Bars show mean 1 s.e.m.; n56; ***, P52.1431025. c, Representative
fluorescent micrographs of transmigrated human 4173 cells pre-stained with
cell-tracker dye in the presence of inflammatory or resident monocytes. Scale
bar, 20mm. d, Numbers of transmigrated 4173 cells in the presence of
inflammatory monocytes or resident monocytes. Bars show themean 1 s.e.m.
of three experiments with duplicates; **, P 5 0.0051. e, Relative number of
transmigrated 4173 cells in the presence of inflammatory monocytes with
control, anti-human CCL2 or anti-mouse CCL2 antibodies, normalized to the
average number with control antibody treatment, which is set to 100. Bars
represent the mean 1 s.e.m. of five experiments with duplicates. One-way
analysis of variance with Bonferroni’s multiple comparison test; **, P,0.01;
by real-time PCR of human Alu repeats, normalized to mouse b-actin, on
day22 (f, n510; ***, P,0.001). CCL2 blockade also prolongs survival
(g, n510, P,0.001) compared to control treatment with PBS.
cells per ×20 field
cells per ×20 field
Number of macrophage/
Percentage of total
IM + ctrl
IM + anti-CCL2
Figure 2 | CCL2-recruited monocytes promote metastatic seeding.
a, Representative haematoxylin-&-eosin-stained sections showing Met-1
1 metastasis (Mets) burden with or without antibody treatment. n56; **,
P50.006. c, d, Representative snapshots of three-dimensional reconstructed
confocal images of tumour cells (blue) and macrophages (green) in lung
tumour cells (e) and tumour-cell extravasation (f) in mice with control or anti-
mouse CCL2 antibody treatment. (e, P50.0066, and f, P50.00163, are based
uponthree-dimensional images of 15–20 tumour clusters per mouse,n53mice
per group.) g, Numbers of transmigrated Met-1 cells in the presence of resident
monocytes or inflammatory monocytes. n55; ***, P,0.0001. h, Numbers of
transmigrated Met-1 cells in the presence of inflammatory monocytes, with
antibody treatments. n53; *, P50.0204. All bars show mean 1 s.e.m.
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metastasis of Met-1 cells and reduced their seeding efficiency (Fig. 4e
and Supplementary Fig. 11b). Adoptive transfer experiments indicated
that Vegfa-null inflammatory monocytes infiltrate Met-1 lung meta-
ing that VEGFA is not required for the recruitment of these cells
(Supplementary Fig. 11c). Notably, co-injection of Met-1 cells and
wild-type inflammatory monocytes into inducible macrophage-Vegfa-
These experiments indicate that CCL2 synthesized by metastatic
the subsequent extravasation of the tumour cells. Mechanistically, this
occurs at least in part through targeted delivery of molecules such as
tinually recruited by a CCL2-dependent mechanism and differentiate
into macrophages that promote the subsequent growth of metastatic
ciation of CCL2 overexpression in human cancers with poor prognosis
(Supplementary Fig. 2), strongly argue for therapeutic approaches
targeted against monocyte recruitment and function.
by adoptive transfer of mouse (Ly6c/Gr11or Ly6c/Gr12) monocytes or human
(CD141CD161and CD162) monocytes, using MMTV-PyMT autochthonous,
human and mouse experimental metastasis models and human orthotopic tumour
the tumours, followed by FACS analysis. To investigate mechanisms for monocyte
Vegfa expression in monocytes, a myeloid-specific (Csf1r promoter), tamoxifen-
was induced by tamoxifen. The effect of monocyte depletion on tumour-cell extra-
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 24 May 2010; accepted 19 April 2011.
Published online 8 June 2011.
1.Qian,B.Z.& Pollard,J.W.Macrophage diversity enhancestumorprogressionand
metastasis. Cell 141, 39–51 (2010).
cell extravasation, establishment and growth. PLoS ONE 4, e6562 (2009).
Ueno, T. et al. Significance of macrophage chemoattractant protein-1 in
Cancer Res. 6, 3282–3289 (2000).
Valkovic, T., Lucin, K., Krstulja, M., Dobi-Babic, R. & Jonjic, N. Expression of
monocyte chemotactic protein-1 in human invasive ductal breast cancer. Pathol.
Res. Pract. 194, 335–340 (1998).
Saji, H. et al. Significant correlation of monocyte chemoattractant protein-1
expression with neovascularization and progression of breast carcinoma. Cancer
92, 1085–1091 (2001).
mining platform. Neoplasia 6, 1–6 (2004).
cells, and their possible roles in the regulation of T-cell responses. Immunol. Cell
Biol. 86, 398–408 (2008).
Science 327, 656–661 (2010).
Geissmann, F., Jung, S. & Littman, D. R. Blood monocytes consist of two principal
subsets with distinct migratory properties. Immunity 19, 71–82 (2003).
10. Getts, D. R. et al. Ly6c1‘‘inflammatory monocytes’’ are microglial precursors
recruited ina pathogenicmannerinWestNilevirus encephalitis.J.Exp.Med. 205,
a remote control mechanism for monocyte recruitment to lymph nodes in
inflamed tissues. J. Exp. Med. 194, 1361–1374 (2001).
13. Tsui, P. et al.Generation, characterization and biological activity of CCL2 (MCP-1/
14. Minn, A. J. et al. Genesthat mediate breast cancermetastasis tolung. Nature436,
15. Carton, J. M. et al. Codon engineering for improved antibody expression in
mammalian cells. Protein Expr. Purif. 55, 279–286 (2007).
16. Al-Mehdi, A. B. et al. Intravascular origin of metastasis from the proliferation of
endothelium-attached tumor cells: a new model for metastasis. Nature Med. 6,
17. Ma, C. & Wang, X.-F. In vitro assays for the extracellular matrix protein-regulated
extravasation process. CSH Protoc. doi:10.1101/pdb.prot5034 (2008).
18. Swirski, F. K. et al. Identification of splenic reservoir monocytes and their
deployment to inflammatory sites. Science 325, 612–616 (2009).
19. Gerber, H. P. et al. VEGF is required for growth and survival in neonatal mice.
Development 126, 1149–1159 (1999).
20. Huang, Y. et al. Pulmonary vascular destabilization in the premetastatic phase
facilitates lung metastasis. Cancer Res. 69, 7529–7537 (2009).
Supplementary Information is linked to the online version of the paper at
also thank P. Marsters for statistical analyses and M. Thompson, F. Shi, C. Ferrante,
F. McCabe, H. Millar-Quinn and D. Wiley for discussions and technical assistance.
Author Contributions B-Z.Q., L.A.S. and J.W.P. conceived the ideas and designed the
experiments. B-Z.Q., J.L., H.Z., T.K., J.Z., L.R.C. and E.A.K. performed the experiments.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to J.W.P. (firstname.lastname@example.org).
MonoT cellB cell Granu
Relative Vegfa exon 3
DNA copy number
cells per ×20 field
Met-1 + IM
Figure 4 | Monocyte-specific ablation of Vegfa blocks pulmonary seeding.
a, PCR of Vegfa exon3 in BMDMs from Vegfaflox/floxmice, with or without the
Csf1r-Mer-iCre-Mer transgene, treated with 4-hydroxytamoxifen. Wild-type
(WT) and knockout (KO) bands are indicated. b, c, Numbers of trans-
endothelial migrated Met-1 cells (b) and permeability of the endothelial
monolayer to albumin (c), with no BMDMs, VegfafloxBMDMs or Vegfa-
of tamoxifen-treated Vegfaflox/floxCsf1r-Mer-iCre-Mer mice compared with
Vegfaflox/floxmice. Mono, monocyte; granu, granulocyte. e, Met-1 Mets burden
in Vegfaflox/floxmice with or without Cre, with the same tamoxifen treatment.
n56; ***, P50.0004. f, Met-1 Mets burden in Vegfaflox/floxCsf1r-Mer-iCre-
Mer mice with tamoxifen treatment, with or without co-injection of
inflammatory monocytes. n56; **, P,0.0001. All data are mean1s.e.m.
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Animals. All procedures involving mice were conducted in accordance with
National Institutes of Health regulations concerning the care and use of experi-
Medicine and Ortho Biotech R&D Institute animal care and use committees.
Transgenic mice expressing the Polyoma Middle T (PyMT) oncogene under the
promoter were provided by W. J. Muller and were bred in-house. FVB (Tg(Csf1r-
EGFP)1Jwp) mice have been previously reported to have the whole mononuclear
Laboratory. The FVB macrophage-specific (Csf1r promoter), tamoxifen-inducible
and crossed with Vegfaflox/floxmice (gift from N. Ferrara). Knockout of Vegfa in
myeloid cells was induced by daily subcutaneous injection of 3mg tamoxifen per
mouse for 2d, before sorting for blood leukocytes or tumour-cell injection.
were used for lung experimental-metastasis assays with intravenous injection of
53105Met-1 cells or106MDA-MB-231-derivedLM2 human breastcancercells,
after intravenous injection of Met-1 cells or 4 weeks after injection of human
tumour cells, for optimal metastatic burden. In experimental metastasis assays,
antibodies were given at 10mgkg21body weight via intraperitoneal injection 3h
before tumour-cell injection, for single treatments, or twice a week thereafter for
prolonged treatments, if not otherwise specified. For paraffin sections, lungs were
the inner airspaces and inflate the lung lobes. Lungs were excised and fixed in
formalin overnight. A precise stereological method21with modification was used
for quantification of lung metastases. Briefly, paraffin-embedded lungs were sys-
tematically sectioned through the entirelungwith one 5mmsectiontakeninevery
0.5mm of lung thickness. All sections were stained with haematoxylin and eosin
and images were taken using a Zeiss SV11 microscope with a Retiga 1300 digital
camera and analysed using ImageJ22. The Mets index is the total volume of meta-
stases normalized to total lung volume and Mets number is the number of meta-
human tumour cells was performed as reported previously, using human-specific
mammary gland of SCID beige or nude mice, respectively. Anti-mouse CCL2and
CCL12 (ref. 23) and anti-human CCL2 (ref. 15) antibodies neutralize only their
with the control antibody. In spontaneous metastasis assays, antibody treatment
began on day3 after tumour-cell intra-mammary-gland injection and continued
each group reached a mean primary-tumour volume of ,1,000 mm3, the mice
were killed. Lungs were perfused with India ink and placed in Fekete’s solution.
least two independent experiments with 3–10 mice for each group.
ferred into FVB mice. 105of either cell type were transferred into mice bearing
mammary tumours and/or pulmonary metastases. Monocytes were sorted from
(Invitrogen) following the manufacturer’s instructions, before adoptive transfer
intravenously transferred into nude mice supplemented with 23106units of
recombinant human CSF1 via subcutaneous injection. In the indicated experi-
ments, specified antibodies were given at 10mgkg21body weight 3h before
adoptive transfer of monocytes.
FACS analysis and antibodies. For FACS analysis, lungs or whole mice were
perfused thoroughly with cold PBS before cell collection, then lungs were minced
on ice and digested with an enzyme mix of Liberase and Dispase (Invitrogen).
Blood was drawn by cardiac puncture. Red blood cells were removed using RBC
lysis buffer (eBioscience). Cells were blocked using anti-mouse CD16/CD32
antibody staining. Antibodies against mouse antigens were: CD45 (30-F11),
CD11b (M1/70), Gr1 (RB6-8C5), CD115 (AFS98) and Foxp3 (FJK-16 s; all from
eBioscience); CD3 (145-2C11) and Ly6c1 (HK1.4; both from Biolegend); CD25
Ly6G (1A8; all from BD Pharmingen) and F4/80 (Cl:A3-1; AbD Serotec).
Antibodies against human antigens were: CD14 (Tu ¨k4) and CD16 (3G8; both
from Invitrogen), CD45 (HI30; BioLegend) and CCR2 (48607; R&D Systems).
FACS analysis was performed on a LSRII cytometer (BD Biosciences) and data
were analysed using Flowjo software (TreeStar). Gating of single cells using FSC/
W and SSC/W and exclusion of dead cells with DAPI staining were performed
routinely during analysis. Mouse CCL2 was stained using the specific antibody
R-17 (Santa Cruz) after a standard immunohistochemistry protocol.
modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum
(FBS). The extravasation assay was performed as previously described17,24with
modifications. Briefly, 23104endothelial cells (3B-11, ATCC) were plated into
the upper chamber of a GFR matrigel invasion chamber (BD Biosciences) in
DMEM with 10% (v/v) FBS. A monolayer was formed in 2d and was verified by
CSF1 to allow attachment. Vegfa-knockout BMDMs derived from Csf1r-
Mer-iCre-Mer:Vegfaflox/floxmice were induced by treating the cells with 1mM
4-hydroxyltamoxifen for 7d after isolation of bone marrow. 23104Met-1 cells
stained with CellTracker CMRA (Invitrogen) were loaded into the insert with
DMEM in 0.5% (v/v) FBS and 104unitsml21CSF1. CCL2-neutralizing antibody
and control antibody were used at 5ugml21, applied to both sides of the insert.
Plates were incubated under normal tissue-culture conditions for 36–48h before
being fixed with 1% (w/v) paraformaldehyde. Tumour-cell trans-endothelial
migration was quantified by counting the number of cells that migrated through
the insert under a fluorescent microscope (6–10 randomly-selected fields in each
insert)andwas expressedas cellnumber per320 field,ifnot otherwisespecified.
The permeability assay was performed by loading 4% (w/v) bovine serum ambu-
medium in the lower chamber at 650nm after a 30-min incubation in normal
culturing conditions. All in vitro experiments were at least three independent
experiments with duplicate or triplicate measures.
a small hairpin RNA (shRNA) that targets the Ccl2 mRNA sequence from
nucleotide 166 was cloned into the miR30 context in the retroviral vector
P2GM25. To knockdown CCL2 in 4173 cells, a 97-mer oligo containing a
shRNA targeting the human CCL2 mRNA sequence from nucleotide 255 was
cloned into the miR30 context in the same vector. For real-time PCR of mouse
CACACCTTGC were used, and for Ccr2, primers CCTGCAAAGACCAGAAG
AGG and GTGAGCAGGAAGAGCAGGTC. All real-time PCR was performed
on an MJ Research DNA Engine 2 Opticon real-time PCR machine using SYBR
master mix (Invitrogen). Primers used were: mouse Ccl2 primers GTTGGC
TCAGCCAGATGCA and AGCCTACTCATTGGGATCATCTTG; mouse Ccr2
GCAGA; mouse Vegfa exon 3 ACATCTTCAAGCCGTCCTGT and CTGCAT
GGTGATGTTGCTCT; human CCL2 AGGTGACTGGGGCATTGAT and
GCCTCCAGCATGAAAGTCTC. To verify mouse Vegfa exon 3 knockout, pri-
mers that flank this exon, GCTGCACCCACGACAGAAGG and TGAGGTT
TGATCCGCATGAT, were used.
Ex vivo whole-lung imaging. A well-established intact-lung microscopy tech-
nique16,26was applied to observe tumour cells, macrophages and blood vessels in
mouse lungs. CFP-expressing Met-1 cells, prepared by retrovirus infection of a
CMV-promoter CFP vector, were injected intravenously into the tail vein of each
mouse. At the times indicated, mice were anaesthetized and injected with 10mg
later, the mouse was put under artificial ventilation through tracheal cannulation.
The lung was cleared of blood by gravity perfusion through the pulmonary artery
with artificial medium (Kreb-Ringer bicarbonate buffer with 5% dextran and
10mmoll21glucose (pH7.4)). The heart–lung preparation was dissected en bloc
and placed in a specially designed plexiglass chamber with a port to the artificial
the posterior surface of the lung touching the plexiglass. The lung was ventilated
throughout the experiment with 5% CO2in medical air and perfused by gravity
perfusion except during imaging. Three to five animals were imaged for each time
point and 10–20 unrelated fields were imaged for each animal.
Images were collected with a Leica TCS SP2 AOBS confocal microscope
(Mannheim) with 360 oil-immersion optics. Laser lines at 458nm, 488nm and
633nm for excitation of CFP, GFP and AF647, respectively, were provided by an
fluorophores. Three-dimensional reconstruction was performed using Volocity
Statistical analysis. Statistical analysis methods were the standard two-tailed
Student’s t-test for two data sets and ANOVA followed by Bonferroni/Dunn post
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hoc tests for multiple data sets using Prism (GraphPad Inc.), except for human-
of variations among different donors. For the spontaneous-metastasis assay of
MDA-MB-231 cells, percentage differences in numbers of lung metastases were
compared between groups using parametric survival regression methods, with
metastasis counts of more than 100 considered censored at 100. P values of less
than 0.05 were deemed significant.
21. Nielsen, B. S. et al. A precise and efficient stereological method for determining
murine lung metastasis volumes. Am. J. Pathol. 158, 1997–2003 (2001).
22. Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image processing with ImageJ.
Biophotonics Int. 11, 36–42 (2004).
23. Havens,A.M.etal. An invivomousemodelforhuman prostatecancermetastasis.
Neoplasia 10, 371–380 (2008).
24. Brandt, B. et al. 3D-extravasation model — selection of highly motile and
metastatic cancer cells. Semin. Cancer Biol. 15, 387–395 (2005).
25. Stern,P.etal.A system forCre-regulated RNA interferencein vivo. Proc.NatlAcad.
Sci. USA 105, 13895–13900 (2008).
26. Im, J. H. et al. Coagulation facilitates tumor cell spreading in the pulmonary
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