Origins of tumor-associated macrophages and
neutrophils
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Citation
Cortez-Retamozo, V., M. Etzrodt, A. Newton, P. J. Rauch, A.
Chudnovskiy, C. Berger, R. J. H. Ryan, et al. “Origins of TumorAssociated Macrophages and Neutrophils.” Proceedings of the
National Academy of Sciences 109, no. 7 (January 30, 2012):
2491–2496.
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http://dx.doi.org/10.1073/pnas.1113744109
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National Academy of Sciences (U.S.)
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Final published version
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http://hdl.handle.net/1721.1/91050
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Detailed Terms
Origins of tumor-associated macrophages
and neutrophils
Virna Cortez-Retamozoa,1, Martin Etzrodta,1, Andita Newtona, Philipp J. Raucha, Aleksey Chudnovskiya, Cedric Bergera,
Russell J. H. Ryanb, Yoshiko Iwamotoa, Brett Marinellia, Rostic Gorbatova, Reza Forghania, Tatiana I. Novobrantsevac,
Victor Kotelianskyc, Jose-Luiz Figueiredoa, John W. Chena, Daniel G. Andersond, Matthias Nahrendorfa, Filip K. Swirskia,
Ralph Weissleder a,e, and Mikael J. Pitteta,2
a
Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; bDepartment of Pathology, Massachusetts
General Hospital, Boston, MA 02114; cAlnylam Pharmaceuticals, Cambridge, MA 02142; dDavid H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA 02139; and eDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115
Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost
all solid neoplasms. The cells are known to descend from immature
monocytic and granulocytic cells, respectively, which are produced
in the bone marrow. However, the spleen is also a recently
identified reservoir of monocytes, which can play a significant
role in the inflammatory response that follows acute injury. Here,
we evaluated the role of the splenic reservoir in a genetic mouse
model of lung adenocarcinoma driven by activation of oncogenic
Kras and inactivation of p53. We found that high numbers of TAM
and TAN precursors physically relocated from the spleen to the
tumor stroma, and that recruitment of tumor-promoting spleenderived TAMs required signaling of the chemokine receptor CCR2.
Also, removal of the spleen, either before or after tumor initiation,
reduced TAM and TAN responses significantly and delayed tumor
growth. The mechanism by which the spleen was able to maintain
its reservoir capacity throughout tumor progression involved,
in part, local accumulation in the splenic red pulp of typically
rare extramedullary hematopoietic stem and progenitor cells,
notably granulocyte and macrophage progenitors, which produced CD11b+ Ly-6Chi monocytic and CD11b+ Ly-6Ghi granulocytic
cells locally. Splenic granulocyte and macrophage progenitors and
their descendants were likewise identified in clinical specimens.
The present study sheds light on the origins of TAMs and TANs,
and positions the spleen as an important extramedullary site,
which can continuously supply growing tumors with these cells.
cancer immunity
| tumor microenvironment
M
acrophages and neutrophils participate in defense mechanisms that protect the host against injury and infection.
However, extensive animal and clinical studies now indicate that
these cells also infiltrate most solid human cancers. Tumor-associated macrophages (TAMs) can stimulate tumor growth (1–
4), and their density is associated with adverse outcomes and
shorter survival in several cancer types, including breast cancer,
Hodgkin lymphoma, and lung adenocarcinoma (5–8). Tumorassociated neutrophils (TANs) can also promote the progression
of primary tumors; however, blockade of TGF-β signaling induces a population of antitumor TANs (3). Also, neutrophils in
tumor-bearing subjects can act to eliminate disseminated tumor
cells, and thus provide antimetastatic protection (9). Although the
interactions of TAMs and TANs with neoplastic cells are being
unraveled, the origin of TAMs and TANs, and the impact of
cancer on these cells’ precursors in vivo, remains less well explored.
Our understanding of the origins of tissue macrophages and
neutrophils, going back to self-renewing hematopoietic stem
cells (HSCs), is largely based on studies not involving cancer (10–
13). These studies have outlined how HSCs located in specialized niches of the bone marrow give rise to progeny that progressively lose self-renewal capacity and commit to certain lineages.
Granulocyte/macrophage progenitors (GMPs), for example, are
www.pnas.org/cgi/doi/10.1073/pnas.1113744109
clonogenic bone marrow cells that descend from HSCs and
commit to either neutrophils or monocytes. The latter cells are
released into circulation and can extravasate in distant tissue (1,
14, 15). The extravasation process is typically concurrent with
activation of an irreversible cell differentiation program (5, 13,
16): Circulating monocytes become tissue macrophages (or
dendritic cells), whereas circulating neutrophils become activated tissue neutrophils. Extending this to the tumor microenvironment, recent studies have shown that circulating Ly-6Chi
“inflammatory” monocytes accumulate in tumors and renew
nonproliferating TAM populations (15). TAMs and TANs are also
sometimes described as descendants of myeloid-derived suppressor cells (MDSCs). MDSCs in mice are defined as CD11b+ Gr-1+
bone marrow-derived immature cells and are composed of (Ly6Chi) monocytic and (Ly-6Ghi) granulocytic cells. Ly-6Chi inflammatory monocytes may contribute to monocytic MDSCs associated with tumors (13). In sum, the prevailing paradigm is that
TAM and TAN populations derive from monocytic and granulocytic progenitors, which are made in the bone marrow.
However, the spleen has recently been shown to constitute a
unique extramedullary reservoir of myeloid cells, specifically
monocytes (17), which can be mobilized in response to distant
acute inflammation and significantly contribute to the host response. Here, we thought to study the role of the myeloid reservoir in the context of cancer. We performed our animal studies
in a conditional genetic mouse model of lung adenocarcinoma
(KrasLSL-G12D/+;p53fl/fl; hereafter referred to as KP), which enables generation of autochthonous tumors from a few somatic
cells via activation of oncogenic Kras and inactivation of p53
(18). Such genetically engineered mice have shown promise in
guiding clinical research because they permit the study of cancers
as they develop de novo and as they evolve within their natural
environments (18, 19). The lesions of the mice recapitulate the
genetic alterations found in the human disease, progress to highgrade tumors, and are infiltrated by TAMs and TANs.
The findings in the KP model indicate that the spleen mobilizes
immature myeloid cells and that these cells amplify TAM and
TAN responses on recruitment to tumors. Also, HSCs and GMPs
accumulate in large numbers within the splenic red pulp of tumorbearing mice. These cells establish niches of proliferation, produce
Author contributions: V.C.-R., M.E., P.J.R., M.N., F.K.S., R.W., and M.J.P. designed research;
V.C.-R., M.E., A.N., P.J.R., A.C., C.B., Y.I., B.M., R.G., and J.-L.F. performed research; R.J.H.R.,
T.I.N., V.K., D.G.A., M.N., and R.W. contributed new reagents/analytic tools; V.C.-R., M.E.,
A.N., P.J.R., C.B., B.M., R.F., J.W.C., and M.J.P. analyzed data; and V.C.-R., M.E., and M.J.P.
wrote the paper.
Conflict of interest statement: T.I.N. and V.K. are Alnylam Pharmaceuticals employees,
and D.G.A. receives funding from, and is a consultant with, Alnylam Pharmaceuticals.
*This Direct Submission article had a prearranged editor.
1
V.C.-R. and M.E. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: mpittet@mgh.harvard.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1113744109/-/DCSupplemental.
PNAS | February 14, 2012 | vol. 109 | no. 7 | 2491–2496
IMMUNOLOGY
Edited* by Richard O. Hynes, Massachusetts Institute of Technology, Cambridge, MA, and approved January 3, 2012 (received for review August 23, 2011)
new CD11b+ Ly-6Chi monocytic and CD11b+ Ly-6Ghi granulocytic cells locally, and participate in the continuous replenishment
of the myeloid cell reservoir. The identification of these processes
should contribute to our understanding of myeloid responses in
cancer and provide new vantage points for therapy.
Results
Spleen Contributes TAMs and TANs. The lung contains different
populations of macrophages, which can express CD11b at variable levels (20) and include CD11b− CD11c+ F4/80+ alveolar
macrophages. To test whether the spleen participates in macrophage responses during tumor progression, we splenectomized
KP mice at week 0 (at tumor initiation) (Fig. 1A) via a procedure
that preserves bone marrow and blood cell pools (17). The
procedure reduced both CD11b+ and CD11b− lung macrophages
(TAMs) by week 11 in a large fraction of animals (Fig. 1B and
Fig. S1). Splenectomy also significantly reduced the numbers of
CD11b+ Gr1+ Ly-6G+ lung tissue neutrophils (TANs) (Fig. 1B).
The experiment was repeated with mice that were splenectomized at week 8 instead of week 0 (Fig. 1A) (i.e., mice with
preestablished cancer). These mice likewise showed impaired
TAM and TAN responses by week 11 (Fig. 1B). This finding
supports the previous notion that TAMs and TANs are continuously replaced during tumor progression (14, 15) and suggests
that the spleen acts to contribute these cells at least after week 8.
The spleen was also found to be required for full accumulation of
TAMs and TANs in two grafted tumor models (Fig. S2).
TAMs produce proteases that degrade ECM proteins and
enhance tumor cell invasion and intravasation (21). Ex vivo flow
cytometry analysis of KP mice that received a cysteine Cathepsin
B-targeted optical sensor revealed high cathepsin activity in
TAMs, as expected, but also in splenic monocytes (Fig. S3). We
next compared proteolytic activity in vivo by combining the optical sensor with fluorescent-mediated tomography/computed
tomography (CT) fusion imaging (2). The lungs of spleen-deficient animals showed significantly reduced proteolytic activity
(Fig. 1C), in line with the above findings that splenectomy reduced the TAM and TAN responses. Other tumor-promoting
functions have been identified for TAMs (5) and may also be
relevant in KP mice.
Spleen Contributes to Tumor Growth. Given that removal of the
spleen prevented full-fledged TAM accumulation and TAM-associated tumor-promoting activity, we investigated whether it
also affected tumor progression. To do this, spleen-sufficient and
spleen-deficient animals were monitored by CT at weeks 7, 9, and
11 after tumor initiation. The majority of KP mice from which
the spleen was removed showed decreased numbers of detectable
tumor nodules and reduced total tumor volumes over time (Fig. 1
D and E). Histopathological analysis at week 11 (Fig. S4) confirmed reduced tumor progression in the majority of spleen-deficient mice. This same phenotype was observed regardless of
whether mice were splenectomized at week 0 or at week 8. In
both cases, splenectomized mice often presented with fewer and
smaller tumor nodules, which were either grade 1 adenomatous
hyperplasias or grade 2 adenomas compared with grade 2 or
grade 3 adenocarcinomas in age-matched control animals.
Splenectomy decreased the TAM response before changes in tumor progression could occur (Fig. S5A), which is in accordance with
a causal association between TAMs and tumor progression. Also, in
support of the notion that the spleen contributed cells to tumors
directly, the number of blood CD11b+ Ly-6Chi monocytic cells decreased shortly after splenectomy in tumor-bearing KP animals (Fig.
S5 B and C). Splenectomy likely did not affect bone marrow hematopoiesis because bone marrow leukocyte counts remain normal in
steady state (17) and in the presence of growing tumors (Fig. S5D).
Histopathological analysis indicated that the tumor burden at
week 11 in spleen-deficient KP mice was similar (i.e., not lower)
to that at week 8 in spleen-sufficient mice (Fig. S4); thus, splenectomy delayed tumor progression but did not induce tumor
rejection. Antitumor cytotoxic T lymphocytes (CTLs) seemed
uninvolved because they remained rare in the tumor stroma of
KP mice even in spleen-deficient animals (Fig. S5E). Likewise,
the CTLs that did accumulate in tumors did not exhibit enhanced effector functions (Fig. S5F). Splenectomy also did not
affect the number of tumor-infiltrating FoxP3+ regulatory T cells
(Fig. S5G). In contrast, impaired tumor progression in spleendeficient KP mice was associated with decreased numbers of
TAMs and TANs (Fig. S5 H and I).
Inflammatory Ly-6Chi monocytes express the chemokine receptor CCR2, which is involved in the amplification of TAM
Fig. 1. Spleen removal impairs TAM and TAN responses
and tumor growth. (A) Experimental procedures. (B) Total
CD11b+ and CD11b− macrophage and neutrophil counts in
lungs of mice 11 wk after tumor initiation [+ adenovirus
expressing Cre recombinase (AdCre), n = 17] and in mice
from which the spleen was removed either immediately
before tumor initiation [+AdCre SPx (wk 0), n = 19] or after
tumors became apparent [+AdCre SPx (wk 8), n = 7]. Control
mice (−AdCre, n = 13) were age-matched. Histograms show
median values, and dots represent single mice. (C) In vivo
fluorescent-mediated tomography/CT fusion images and
quantification of Cathepsin B activity at week 11 after
AdCre infection in KP mice with (+AdCre +SP) or without
[+AdCre −SP (wk 0)] a spleen (n = 3). Data are presented as
the mean ± SEM. (D and E) High-resolution CT analysis of
tumor progression at weeks 7, 9, and 11 in mice with
(+AdCre, n = 7) or without [+AdCre −SP (wk 0), n = 12]
a spleen. (D) Number and volume of tumor nodules in each
group. Histograms show median values, and dots represent
single mice. (E) Representative 3D volume renderings of
tumors for each group. Tumors are shown in red, and lungs
are shown in green. *P < 0.05; **P < 0.01; ***P < 0.001; ns,
not significant.
2492 | www.pnas.org/cgi/doi/10.1073/pnas.1113744109
Cortez-Retamozo et al.
Splenic Monocytic and Granulocytic Cells Relocate to Cancer Sites.
Based on these results, we attempted to define whether reservoir
splenocytes physically relocate to tumor sites. We used a method
involving transplantation of an anastomosed spleen and tracking
of the cells originating from the transplanted spleen (17) (Fig.
3A). Perfusion of the transplant (Fig. 3B) allowed donor splenocytes to enter the recipient’s bloodstream directly, and thus to
relocate elsewhere. Spleens transplanted into tumor-free animals
did not trigger a measurable cell release (17) (Fig. 3C). However,
spleens transplanted into tumor-bearing KP mice (11 wk after
tumor initiation) triggered relocation of numerous cells to
tumors. Namely, 1.9 ± 1.2 × 105 TANs and 5.1 ± 3.2 × 105 TAMs
redistributed within 24 h from the donor spleen to the recipient’s
tumor stroma. A total of 66 ± 4% and 61 ± 17% of splenicderived TAMs down-regulated Ly-6C and up-regulated F4/80,
respectively, in line with the initiation of a differentiation program of these cells on recruitment to tissue. This approach did
not permit us to assess the relative contribution of spleen- vs.
bone marrow-derived cells; however, it showed that within 24 h,
the spleen replaced as many as 4% and 8–10% of the preexisting
endogenous TAM and TAN repertoires, respectively (Fig. 3D).
Because these repertoires increased, on average, only 2% daily,
it is likely that a small fraction of older cells either died locally or
exited the tumor stroma, in part, counteracting the contribution
of newly arrived splenic cells. Future studies will have to determine whether TAMs and TANs are short-lived or exit the
tumor stroma. Either way, these experiments positioned the
spleen as a significant contributor of TAM and TAN precursors.
Spleen Produces CD11b+ Ly-6Chi Monocytic and CD11b+ Ly-6Ghi
Granulocytic Cells Locally During Cancer Progression. The spleen
may enhance TAM and TAN responses by controlling the quality
and/or quantity of immature myeloid precursor/progenitor cells
Fig. 2. CCR2 silencing in splenic monocytes impairs TAM responses and
tumor growth. (A) Experimental procedures. AdCre, adenovirus expressing
Cre recombinase. (B) Number of macrophages and neutrophils in the lungs
of KP mice treated with siCCR2 (n = 4) or control-silencing (siCON; n = 4)
nanoparticles or in tumor-free mice (no cancer). (C) Volume of tumor nodules in the same KP mice identified by high-resolution CT. *P < 0.05; n.s., not
significant.
Cortez-Retamozo et al.
Fig. 3. Spleen contributes TAMs and TANs directly. (A) Experimental procedure for transplantation of a BrdU-labeled spleen into a splenectomized
(unlabeled) KP mouse. Procedural details are provided in SI Methods. (B)
Transplanted mice received an i.v. injection of the blood pool agent
Angiosense-680 (ViSen Medical–PerkinElmer) to reveal perfusion of the donor organ (spleen outlined by yellow dashed line). Scale bar: 5 mm. (C) The
number of BrdU+ cells was detected by flow cytometry from donor spleen
mobilized to the lung 24 h after transplantation. The recipient mice were
either tumor-bearing (KP mice 11 wk after tumor initiation) or tumor-free
(control mice). (D) Dot plots show endogenous cells (gray) and spleen donorderived cells (red) 24 h posttransplantation in a tumor-bearing mouse (from
n = 2). Percentages indicate the contribution of newly arrived splenic cells to
the preexisting lung myeloid cell pool. Lin, lineage.
released to tumors. For example, splenic monocytic and granulocytic cells may acquire unique functions locally by receiving
microenvironmental instruction signals, which differ from those
in bone marrow. Although this mechanism cannot be formally
excluded, a comparative analysis of (CD11b+ Ly-6Chi) monocytic and (CD11b+ Ly-6Ghi) granulocytic cells in bone marrow
vs. spleen of tumor-bearing hosts (Fig. S7A) and of splenic
monocytic and granulocytic cells in tumor-bearing vs. control
mice (Fig. S7 B and C) did not reveal notable differences. Phenotypic changes were detected mostly only on cell recruitment to
the tumor stroma (Fig. S7C and Table S1).
The spleen may also control TAM and TAN responses quantitatively by supplying new monocytic and granulocytic cells continuously during tumor progression. Continuous support of the
spleen, however, requires its constant replenishment with new
reservoir cells to compensate for the loss of those that are mobilized. Interestingly, the spleen of tumor-bearing mice contained
a significantly larger number of cells in the S/G2 phase of the cell
cycle, which indicated active cell proliferation within the organ.
The proliferating splenocytes exhibited a CD11b− Lin− phenotype
(Fig. 4A). Thus, cancer induced the accumulation of proliferating
splenocytes that were neither monocytic nor granulocytic but
could include their lineage progenitors. Accordingly, in vitro cultures of splenocytes from tumor-bearing mice produced more
granulocyte/macrophage colonies than from controls (Fig. 4B).
The established paradigm states the following paths of lineage
differentiation for monocytes and neutrophils: HSC→CMP (common myeloid progenitor)→GMP→MDP (macrophage/dendritic
cell progenitor)→monocyte (24) and HSC→CMP→GMP→neutrophil (10). The presence of these populations was tested by
flow cytometry with a combination of markers used for cell
phenotyping in bone marrow (10–12). HSCs, CMPs, GMPs, and
MDPs increased largely in the spleen (Fig. 4 C and D and Fig.
S8A) in comparison to bone marrow (Fig. S8 B and C) in KP
mice at 11 wk after tumor initiation. Similar splenic responses
were detected in two other grafted tumor models (Fig. S8D).
Subsequent experiments were restricted to the analysis of
HSCs and GMPs. These studies indicated that their expansion in
the spleen of tumor-bearing mice was likely the consequence of
both their continuous recruitment and their local proliferation.
To analyze recruitment, parabiosis was established between two
tumor-bearing mice for 16 d until HSC and progenitor cell
equilibration. The spleens contained a significant fraction of
HSCs (28.5 ± 2%) and GMPs (24 ± 4%) that originated from
the other parabiont; thus, circulating HSCs and progenitor cells
PNAS | February 14, 2012 | vol. 109 | no. 7 | 2493
IMMUNOLOGY
responses (15, 22). To test whether CCR2 controlled spleenderived TAM accumulation, we used a lipid nanoparticle that
contains short interfering (si) CCR2 RNA sequences and that
selectively silences CCR2 expression in splenic Ly-6Chi (CCR2+)
monocytes (23). KP mice received injections of CCR2-silencing
siRNA (siCCR2) or control-silencing (siCON) nanoparticles
starting on week 12 after tumor initiation and over 12 d (Fig.
2A). The siCCR2-treated mice showed an approximately twofold
reduction of TAMs (Fig. 2B). Splenic granulocytic cells do not
express CCR2 and, accordingly, siCCR2 treatment did not alter
TAN responses (Fig. 2B). Further evaluation of lung tissue
showed reduced tumor progression in mice that received siCCR2
nanoparticles (Fig. 2C and Fig. S6). These data indicate that
splenic-derived TAMs were recruited via CCR2 and were causally associated with tumor progression. The apparently unaltered
TAN response in siCCR2-treated animals was insufficient to
promote tumor growth; thus, further investigation will be needed
to define the precise impact of spleen-derived TANs.
Fig. 4. Tumors induce splenic accumulation of HSCs and
progenitor cells. (A) Percentage of monocytic [CD11b+ Lin− Ly6G− (F4/80/CD11c/I-Ab)lo Ly-6C+], granulocytic (CD11b+ Gr-1+
Ly-6G+), Lin+ CD11b− and Lin− CD11b− populations in S/G2
phase in the spleen of control and tumor-bearing mice (n = 3)
at week 11 after adenovirus expressing Cre recombinase
(AdCre) infection. Lineage (Lin) refers to the following combination: B220/NK1.1/DX5/Ly-6G/CD90.2. (B) (Upper) Number
of granulocyte/macrophage colonies in CFU assays (GM-CFU)
from splenocytes of tumor-bearing (+AdCre) and control
(−AdCre) mice. (Lower) Image of granulocyte/macrophage
colonies. Scale bar: 2 mm. (C) Identification of splenic HSCs
and GMPs by flow cytometry in KP mice on week 11.
(D) Quantification of splenic HSCs and GMPs in tumor-bearing
(+AdCre) KP animals (n = 17) and age-matched controls
(−AdCre, n = 12). Data are presented as the mean ± SEM.
***P < 0.001.
contributed to the splenic repertoire. These observations are in
line with previous evidence that a small fraction of HSCs and
progenitor cells exit the bone marrow and traffic through different extramedullary tissues, including the spleen (25, 26), and
that in response to inflammatory stimuli, these cells produce
tissue-resident cell populations (27–29). To analyze local proliferation, splenic HSCs and GMPs were labeled with a cell cycle
marker. Flow cytometry analysis showed that a high fraction of
these cells were in the S/G2 phase of the cell cycle, similar to
their bone marrow counterparts (Fig. S8E). Splenic HSCs and
GMPs were functionally active in vitro, because they formed
colonies as efficiently as their bone marrow counterparts.
We performed additional experiments to evaluate the fate of
GMPs in vivo. Intravital microscopy 5 d after injection of fluorescently tagged GMPs indicated that the cells gave rise to large
numbers of colonies in the splenic red pulp parenchyma next to,
but outside, collecting venous sinuses (Fig. 5A). Characterization
of the cells by flow cytometry identified that GMP descendants
were virtually all monocytic or granulocytic (Fig. 5B). The ability
of transferred GMPs to produce myeloid progeny was highest
within the spleen of KP animals with cancer. The activity
remained relatively low within the bone marrow as well as the
liver (another extramedullary site that can exhibit myelopoietic
activity) even in splenectomized mice (Fig. 5B and Fig. S8F). We
made similar observations on adoptive transfer of HSCs instead
of GMPs, although, as expected, the appearance of HSC-derived
monocytic and granulocytic cells was delayed by several days.
Finally, GMPs had a similar fate in a grafted mouse tumor model
(Fig. S8G). The data indicate that splenic HSCs and progenitor
cells actively produce Ly-6Chi monocytic and Ly-6Ghi granulocytic cells locally and participate, at least in part, in replenishing
the splenic reservoir in tumor-bearing mice.
Splenic GMPs Contribute to the TAM and TAN Responses. Tracking of
the GMP progeny in the lungs of KP mice also identified that
these cells produced tissue macrophages (TAMs) and neutrophils (TANs) (Fig. 5C). The capacity of the transferred GMPs
to generate these cells markedly increased in mice with cancer
(11 wk after tumor initiation). To test whether the GMP-derived
TAMs and TANs required the spleen, we repeated the GMP
adoptive experiments (11 wk after tumor initiation); however, we
used spleen-sufficient or spleen-deficient mice as recipients this
time. Five days after transfer, GMPs failed to produce full TAM
and TAN responses in the spleen-deficient mice (Fig. 5C). This
result supports the role of splenic GMPs as TAM and TAN
progenitors and positions the spleen as an important site for
amplification of these cells.
Human Patients with Cancer Expand Splenic Myeloid Progenitor Cells.
Mouse and human spleens are physiologically very similar (30),
and the spleen of patients with chronic inflammatory disorders is
capable of producing leukocytes (31–33). However, whether the
extramedullary myeloid response seen in animal models also
occurs in human patients has not been addressed.
Fresh splenic tissue was collected from seven control patients
and seven patients with pathological evidence of invasive cancer.
Splenic GMP-like cells were sorted based on the expression of
cell surface markers used previously to identify human bone
marrow GMPs (11) (i.e., CD117+ CD34+ CD38+ IL-3Ra+
CD45RA+ Lin−/int; Fig. 6A).
PCR analysis identified that the splenic GMP-like cells produced the transcription factor PU.1 and CCAAT/enhancer
binding protein ε (C/EBPε), the granulocyte colony-stimulating
factor (CSF), granulocyte-macrophage (GM)-CSF receptors,
and the enzyme myeloperoxidase, in line with the study of Manz
et al. (11). The cells did not express GATA binding protein 1,
Fig. 5. Fate mapping of adoptively transferred GMP cells. (A)
(Upper) In situ confocal image of the splenic red pulp from
a tumor-bearing mouse 5 d after i.v. injection of (ACTB-eYFP)
7AC5Nagy/J (EYFP+) GMPs (green). EYFP+ cell clusters are
highlighted in circles, and venus sinuses are shown in red. Scale
bar: 1 mm. (Lower) Higher magnification images. Scale bar:
100 μm. (B) Phenotyping and quantification of the progeny of
EYFP+ GMP cells in the spleen (SP) and bone marrow (BM) of
tumor-bearing [+ adenovirus expressing Cre recombinase
(AdCre), n = 12] and tumor-free mice (−AdCre, n = 6). i.n., intranasal. (C) Tracking of EYFP+ GMP cells injected into tumorfree mice (−AdCre, n = 6) or tumor-bearing KP mice with
(+AdCre, n = 12) or without [+AdCre −SP (wk 0), n = 4]
a spleen. Data are presented as the mean ± SEM. *P < 0.05;
**P < 0.01; ***P < 0.001; n.s., not significant.
2494 | www.pnas.org/cgi/doi/10.1073/pnas.1113744109
Cortez-Retamozo et al.
GATA binding protein 3, von Willebrand factor, or IL-7 receptor α (IL-7Ra), all of which are involved in developmental
lineages disparate from neutrophils and monocytes (Fig. 6B). In
vitro, purified splenic GMP-like cells produced colonies and
generated cells that were mostly CD14+ CD115+ CD68+
CD11b+/− (Fig. S9A). Because we could not detect granulocytes,
it is possible that the culture conditions skewed maturation toward the mononuclear phagocyte lineage or that the splenic
population was already committed to this lineage (and thus may
also qualify as human MDPs).
Patients with invasive cancer had significantly higher numbers
of splenic GMPs ex vivo compared with controls (P < 0.05; Fig.
6C), and in vitro cultures of splenocytes of patients with cancer
(n = 2) produced higher numbers of granulocyte/macrophage
colonies than splenocytes of a control patient (Fig. S9B). Also,
a flow cytometry protocol established to detect endogenous
human splenic monocytes and neutrophils ex vivo (Fig. S9C)
showed increased numbers of these cells in patients with cancer
(Fig. 6D). These findings suggest that patients with cancer can
mount active GMP responses and produce monocytic and granulocytic cells locally.
To address directly whether human splenic GMPs produce
monocytic and granulocytic cells in vivo, we purified human
splenic progenitor cells obtained ex vivo from a patient with invasive cancer and injected the cells into a nonobese diabeticSCID mouse bearing an NCI-H226 human non-small cell lung
carcinoma xenograft. Within 5 d of the transfer, the GMP progeny accumulated and differentiated mainly into CD11b+ CD68−
CD11c− monocyte-like cells in the spleen. Human cells also accumulated at the tumor site, where they acquired a CD68+
macrophage-like phenotype (Fig. 6E).
Together, the human data appear to mirror our observations
in the mouse, and they are consistent with the notion that the
spleen can support production of monocytic and granulocytic
cells, at least in patients with cancer. Aside from the fact that
splenic GMP responses develop in both species, it is noteworthy
that the magnitude of these responses was comparable, if not
more pronounced, in humans (taking scale differences between
species into account; Fig. 6F).
Discussion
Here, we show that the spleen significantly amplifies tumorpromoting TAM responses in KP mice and can do so over time
during cancer progression. Our findings in favor of this notion
are as follows: (i) the impaired TAM response and tumor
growth in splenectomized animals, (ii) the impaired TAM response and tumor growth on CCR2 silencing in splenic
monocytic cells, (iii) the redistribution of splenic TAM precursors to tumors in the spleen transplant experiments, (iv) the
expansion of splenic myeloid progenitors in tumor-bearing
mice, and (v) the reduced capacity of these progenitors to
produce TAM when transferred into spleen-deficient mice.
Future studies should address whether manipulation of the
Cortez-Retamozo et al.
splenic reservoir not only suppresses primary tumor growth but
delays metastasis and increases survival. The functions of
splenic-derived TANs also remain to be determined.
The constant “demand” for TAMs is met, at least in part, by
active and chronic proliferation of splenic HSCs and progenitor
cells, which continuously replenish the reservoir with new
monocytic cells. The latter cells, produced within the spleen,
do not necessarily become local resident cells but are capable
of relocating to tumor sites. These findings shed light on the
mechanisms of immune cell production and relocation in cancer.
They also prompt a number of questions, such as the following: (i)
“Why does the bone marrow ‘farm out’ the production of TAM
(and TAN) precursors?” (ii) “How do cancers induce the extramedullary response?” (iii) “Does a splenectomy in cancer help or
hurt?” (iv) “How does chemotherapy affect splenic myeloid cell
production?” and (v) “How can the HSC→TAM(/TAN) axis be
modulated to create more efficient anticancer therapies?”
The data presented here agree with the established notion that
the bone marrow in the steady state autonomously replaces and
maintains the pools of relatively short-lived monocytes and neutrophils (13, 17, 24, 34). However, during chronic inflammation,
our findings suggest that the spleen can also continuously contribute Ly-6Chi monocytic and Ly-6Ghi granulocytic cells. Others
have shown the liver as a site for extramedullary myelopoiesis in
cancer (35) and other inflammatory processes (29). It is likely that
the outsourcing process occurs when demand for macrophages/
neutrophils is high. It may also be that medullary and extramedullary environments differ significantly enough from one
another that they generate cells with divergent functions. Currently, our phenotyping studies suggest that bone marrow- and
spleen-derived monocytic cells are similar. However, it is worth
noting that CD11b+ Gr-1+ MDSCs, which often accumulate
within the spleen of tumor-bearing animals, exhibit a distinct
immunosuppressive phenotype (36). GMP-derived splenic monocytic and granulocytic cells are also CD11b+ Gr-1+; thus, at least
a fraction of bona fide MDSCs likely derive from splenic
(CD11b−) GMPs. Future work should identify the splenic signals
that instruct HSCs and progenitor cells and their lineage
descendants and also whether these signals differ from those in
the bone marrow.
The extramedullary hematopoietic response triggered in cancer could be host-controlled and/or tumor-controlled: The loss of
splenic cells on mobilization to tumors may activate host-controlled feedback homeostatic mechanisms to replenish the reservoir, although tumors may also actively secrete long-range signals
that trigger/amplify the response. Growth factors, such as GM-CSF
(37) and the glycoprotein osteopontin (38), have already been
implicated as long-range signals controlling hematopoietic cells
in the bone marrow. GM-CSF and other factors control the
mobilization of bone marrow HSCs to the periphery and should
next be investigated for their roles in extramedullary hematopoietic responses. Interestingly, adenocarcinomas driven by oncogenic Kras only, as opposed to KP cancers, fail to evoke a
PNAS | February 14, 2012 | vol. 109 | no. 7 | 2495
IMMUNOLOGY
Fig. 6. Splenic myeloid response in human
patients with invasive cancer. (A) Identification of GMP-like cells (blue) ex vivo by flow
cytometry from single-cell suspensions of
spleen tissues obtained from a human patient
with invasive cancer. (B) Gene expression signature of purified human splenic GMP-like
cells and of bone marrow GMPs, as previously
reported (10). (C) Total counts of GMP-like
cells in the spleen of patients with (n = 7) or
without (n = 7) invasive cancer. (D) Total
number of monocytes and neutrophils retrieved from spleens of the same patients. (E) Fate of human Lin− cKit+ splenic progenitor cells 5 d after injection into
a nonobese diabetic SCID mouse bearing a lung carcinoma xenograft. Lin, lineage. (F) Table showing spleen weight and total numbers of splenic GMPs and
monocyte- and neutrophil-like cells in tumor-bearing mice and in patients with cancer. The fold difference between species is also shown. Data are presented
as the mean ± SEM. *P < 0.05.
splenic monocytic/granulocytic response through all stages of tumor progression. Thus, a comparative analysis of KrasLSL-G12D/+
and KP mice may be useful to identify remote cross-talk
instigators.
Although this study has indicated that splenectomy can delay
tumor growth in KP mice, the consequences of such a procedure
in humans remain unclear. Retrospective population-based
studies (39–41) have shown that patients whose spleens were
removed because of traumatic rupture do not show a reduced
risk for developing cancer. However, the spleen is a multicellular
organ, capable of accommodating many major functions for the
host, including immune protection against pathogens as well as
orchestration of healing responses (17). Thus, although removal
of the entire spleen may carry positive effects in some circumstances, it would be preferable to identify specific tumor-promoting
activity in the organ, against which therapeutic approaches could
be designed.
Many chemotherapeutic drugs not only target tumor cells but
eliminate proliferating HSCs in the bone marrow. Because
TAMs and TANs derive from HSCs, one could imagine that the
drugs act to suppress the tumor-promoting immune response.
However, chemotherapeutic drugs can also mobilize bone marrow HSCs to the periphery, resulting in an increase in the circulating HSC repertoire (42). It would therefore be useful to
determine whether chemotherapies suppress or enhance tumorpromoting extramedullary hematopoietic responses. With accumulating evidence indicating the immune system as critical for
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2496 | www.pnas.org/cgi/doi/10.1073/pnas.1113744109
tumorigenesis, an emerging concept for fighting established
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Methods
Detailed methods are available in SI Methods. KP mice in a 129 background
were obtained from Tyler Jacks (Massachusetts Institute of Technology, Cambridge, MA). To induce lung adenocarcinoma, KP mice were infected with an
adenovirus expressing Cre recombinase by intranasal instillation (18). Spleen
transplants were performed using KP donor and recipient mice 11 wk after
AdCre infection.
ACKNOWLEDGMENTS. We thank T. Jacks (Massachusetts Institute of Technology), R. Weinberg (Whitehead Institute, Cambridge, MA), and E. Meylan
and M. Winslow (Massachusetts Institute of Technology) for helpful discussions; K. Hassard, S. Holder, and the Massachusetts General Hospital
surgical pathology accessioners for assistance with human tissue collection;
J. Sullivan and R. Kohler (Massachusetts General Hospital) for imaging;
M. Maier, H. Epstein-Barash, and W. Cantley for medium-scale siRNA synthesis
and formulation (Alnylam); and M. Waring (Ragon Institute, Massachusetts
General Hospital) for sorting cells. This work was supported, in part, by
National Institutes of Health Grants P50-CA86355, R01-AI084880, and
R56-AI084880 (to M.J.P.) and National Institutes of Health Grant U54CA126515 (to R.W.). M.E. was supported by the American Association for
Cancer Research Centennial Predoctoral Fellowship and the Boehringer
Ingelheim Fonds.
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Cortez-Retamozo et al.
Supporting Information
Cortez-Retamozo et al. 10.1073/pnas.1113744109
SI Methods
Animals. KrasLSL-G12D/+;p53fl/fl (referred to as KP) and KrasLSL-G12D/+
(referred to as K) mice in a 129 background were used for
conditional mouse models of non-small cell lung cancer and
were obtained from Tyler Jacks (Massachusetts Institute of
Technology, Cambridge, MA). (ACTB-eYFP)7AC5Nagy/J (referred to as EYFP+) mice in a 129 background were a gift from
Andras Nagy (Mount Sinai Hospital, Toronto, ON, Canada).
Atgr1a−/− B6.129P2-Agtr1atm1Unc/J (referred to as AT-1−/−)
mice, C57BL/6-Tg (UBC-GFP)30Scha/J (referred to as GFP)
mice, and B6.SJL-Ptprca Pepcb/BoyJ (referred to as CD45.1)
mice were purchased from the Jackson Laboratory. BALB/c and
C57BL/6 mice were obtained from Taconic Farms, Inc. All animal experiments were approved by the Massachusetts General
Hospital Subcommittee on Research Animal Care.
Tumor Models. To induce lung adenocarcinoma, KP or K mice
were infected with an adenovirus expressing Cre recombinase
(AdCre) by intranasal instillation, as described previously (1).
AdCre was purchased from the University of Iowa Gene
Transfer Vector Core. Where indicated, the murine CT26 colon
carcinoma, B16 melanoma, and EL-4 lymphoma tumor cell lines
(all from American Type Culture Collection) were used for s.c.
injection.
Recovery of Cells from Lung Tissue. Lungs from tumor-bearing
or control animals were perfused abundantly with PBS, cut up,
and incubated in RPMI 1640 (GIBCO; Invitrogen) containing
0.2 mg/mL collagenase type IV (Sigma–Aldrich) for 1 h at 37 °C.
Digested lungs were filtered, and single-cell suspensions were
washed and resuspended in PBS with 0.5% BSA. The same
procedure was adapted for recovery of cells from stroma of tumor cell lines that were grafted by s.c. injection.
Histology. For histopathological analysis, lung tissue was har-
vested, fixed in 10% (vol/vol) formalin solution, and embedded in
paraffin. Serial paraffin-embedded sections were prepared and
then stained with H&E. For immunohistochemistry, all sections
used were incubated in 1% hydrogen peroxide solution to block
endogenous peroxidase activity before antibody staining. To detect macrophages, tissue sections were deparaffinized and rehydrated for heat-induced epitope retrieval using a retrieval solution
(pH 6.0; BD Biosciences) and then incubated with a rat antimouse Mac-3 mAb (BD Biosciences). To detect neutrophils,
tissue sections were treated for 5 min in 1% SDS (Boston BioProducts) in PBS and then stained with the rat anti-mouse
NIMP-R14 mAb (Santa Cruz Biotechnology). A biotinylated antirat IgG antibody (Vector Laboratories) was used to detect primary
antibodies, and the Vectastain ABC kit (Vector Laboratories) was
used to detect the secondary antibody. Finally, color development
was performed with a 3-amino-9-ethylcarbazole substrate (Dako
Cytomation) to reveal the staining, and all sections were counterstained with Harris Hematoxylin solution (Sigma-Aldrich).
Images of slides were captured and digitized automatically at
a magnification of 40× (NanoZoomer 2.0RS; Hamamatsu).
mAbs and Flow Cytometry. The following antibodies were used:
phycoerythrin (PE)-conjugated anti-CD90 (clone 53-2.1), PEconjugated anti-B220 (clone RA3-6B2), PE-conjugated antiCD49b (clone DX5), PE-conjugated anti-NK1.1 (clone PK136),
PE-conjugated anti-Ly-6G (clone 1A8), PE-conjugated antiCD11c (clone N418), Allophycocyanin (APC)-conjugated antiCortez-Retamozo et al. www.pnas.org/cgi/content/short/1113744109
CD11b (clone M1/70), APC-Cy7–conjugated anti-CD11b (clone
M1/70), PE-Cy7–conjugated anti-F4/80 (clone BM8), Alexa Fluor
700-conjugated anti-CD11c (clone HL3), APC-conjugated antiCD117 (clone 2B8), APC-Cy7–conjugated anti-CD16/32 (clone
2.4G2), PE-Cy7–conjugated anti-Sca1 (clone D7), Alexa Fluor
700-conjugated anti-CD34 (clone RAM34), FITC-conjugated
anti-CD34 (clone RAM34), PE-conjugated anti-CD127 [IL-7 receptor α (IL-7Ra); clone A7R34], FITC-conjugated anti-BrdU,
APC-conjugated anti-BrdU, APC-conjugated anti-CD45.1 (clone
A20), peridinin chlorophyll protein (PerCP)-Cy5.5–conjugated
anti-CD45.2 (clone 104), biotin-conjugated anti-CD115 (clone
AFS98), and PerCP-conjugated streptavidin (all from BD Biosciences); PE-conjugated anti-CD19 (clone 1D3), PE-conjugated
anti-CD11b (clone M1/70), APC-conjugated anti-mouse TNF-α,
and FITC-conjugated anti-mouse CD62L (clone MEL-14) (all
from BD Pharmingen); FITC-conjugated anti-mouse Granzyme B
(clone 16G6; eBioscience); and APC-conjugated anti-mouse
IFN-γ (R&D Systems). For progenitor staining, the lineage (Lin)
antibody mix included the following PE-conjugated antibodies:
anti-CD90, anti-B220, anti-CD19, anti-CD49b, anti-NK1.1, anti–
Ly-6G, anti-CD11b, anti-CD11c, and anti-CD127. For cell labeling, single-cell suspensions were labeled for 30 min at 4 °C with
appropriate antibodies in PBS supplemented with 1% FBS. For
cell cycle analysis, FxCycle violet stain (Invitrogen) was used. The
number of monocytes/macrophages, dendritic cells, neutrophils,
progenitors, and other cells was defined as the total number of
cells per organ multiplied by the percentage of each cell type
identified by flow cytometry (LSRII; BD Biosciences). Data were
analyzed with FlowJo v.8.8.7 (Tree Star, Inc.). For adoptive
transfer experiments, cell suspensions obtained from spleen or
bone marrow were stained with appropriate antibodies; cells of
interest were sorted (FACS Aria; BD Biosciences) and injected
i.v. without delay. Typically, 6.5 × 104 GMPs or HSCs were injected. For cytokine detection, cells were stimulated in RPMI +
10% FBS (vol/vol) for 4 h at 37 °C with 0.05 μM or 0.1 μM
phorbol 12-myristate 13-acetone (BD Biosciences). Golgi-plug
(1 μL/mL; BD Biosciences) was added after 1 h into the reaction.
Surgical Removal of the Spleen. Skin covering the spleen was
shaved and cleaned with 70% ethyl alcohol (vol/vol). A splenectomy was performed under isoflurane anesthesia by making an
incision in the abdominal left hypochondrium. The spleen was
gently prodded away from the connective tissue, while cauterizing
associated vessels. The incision was thereafter sutured.
Spleen Transplantation. Spleen transplants were performed using
KP donor and recipient mice 11 wk after AdCre infection. The
surgical procedure of spleen transplantation and the enumeration
of cells deployed from the donor spleen to distant tissue were
performed as previously described (2). We did not have syngeneic KP mice expressing different allelic markers, which could be
used for tracking donor cells in the recipient. Instead, donor
splenocytes were “tagged” by administering BrdU into the donor
mice before transplantation (daily injections of 1 mg of BrdU in
the peritoneal cavity for 3 d). BrdU was incorporated into the
DNA during proliferation, and control experiments using flow
cytometry indicated that 60% of splenic monocytes and neutrophils became BrdUhi. To define the number of cells that migrated from the spleen to the lung, the number of BrdUhi TAMs
and TANs (these cells originated from the donor spleen) were
counted, and this number was multiplied by 1.67 to correct for
the fact that not all splenic monocytes and neutrophils were
1 of 8
BrdUhi. Control experiments included spleen transplantation in
tumor-free animals.
population was calculated as %GFP+/(%GFP+ + %GFP−) in
C57BL/6 mice and as %GFP−/(%GFP+ + %GFP−) in GFP mice.
Colony-Forming Cell Assay. To determine the presence and number
of myeloid colony-forming units (cfus) in whole tissue or in
FACS-sorted cell populations, human or murine cells were cultured in appropriate complete methylcellulose-based medium
(for human cells, MethoCult GF H4034 Optimum; for mouse
cells, MethoCult GF M3434; both from STEMCELL Technologies) in six-well plates. Colony numbers and morphology were
assessed after 10–14 d of culture.
Human Spleen Processing and Flow Cytometry. De-identified human
spleen samples were obtained from the Massachusetts General
Hospital Department of Pathology following an approved institutional review board protocol. Specimens were processed
under Biosafety Level 2 conditions in compliance with the
Harvard Medical School Committee on Microbiological Safety.
All samples were processed identically, and patients were subsequently divided into two groups based on pathological and/or
clinical findings. Group 1 included samples from patients with no
evidence of malignant cancer (e.g., biopsied patients with benign
pathological findings, patients who required a splenectomy following a physical trauma), and group 2 included patients with
invasive cancer (e.g., pancreatic ductal adenocarcinoma, colon
carcinoma). Spleen sections weighing 1–3 g were cut into small
pieces and incubated in RPMI 1640 containing 20 units/mL hyaluronidase (Sigma–Aldrich), 41.6 units/mL collagenase type XI
(Sigma–Aldrich), 150 units/mL collagenase type I (Sigma–Aldrich), DNase I (Sigma–Aldrich), 6.67 μL/mL of Hepes Buffer
(Mediatech), and 305.43 μL/mL of PBS without calcium or
magnesium (Lonza) for 1 h at 37 °C. Digested spleens were filtered, and cells were washed in RPMI and resuspended in red
blood cell lysis buffer for 2 min. For flow cytometry analysis, cell
suspensions were washed and fixed/permeabilized following the
BD Cytofix/Cytoperm (BD Biosciences) protocol for intracellular antibody labeling. The following mAbs were used: FITCconjugated antibodies specific for lineage markers anti-CD3e
(clone ucht1) and NKp46 (clone 195314) (both from R&D
Systems); anti-CD15 (clone hi98), anti-CD19 (clone hib19), and
anti-CD56 (clone mem188) (all from BD Biosciences); PEconjugated anti-CD11c (clone b-ly6); anti-CD68 (clone y1/82a);
PerCP-conjugated anti-CD14 (clone mfp9); APC-conjugated
anti-CCR2 (clone 48607); anti-HLADR (clone 1243); PE-Cy7–
conjugated anti-CD16 (clone 3g8); APC-conjugated anti-CD11b
(clone icrf44); biotinylated anti-CD120a (clone mabtnfr1-b1);
and anti-CD115 (clone 12-3a3-1b10). For analysis and sorting of
myeloid progenitors, cells were stained with PE-Cy5–conjugated
antibodies specific for the following lineage markers: CD2 (clone
RPA-2.10), CD11b (clone ICRF44), CD20 (clone 2H7), and
CD56 (clone B159) (all from BD Pharmingen); CD3 (clone S4),
CD4 (clone S3.5), CD8 (clone 3B5), and CD19 (clone SJ25-C1)
(all from Invitrogen); CD7 (clone CD7-6B7) and CD10 (clone
5-1B4) (both from Biolegend); and CD14 (clone TU.K.4) (from
eBioscience). The cells were also stained with FITC-conjugated
anti-CD45RA (clone MEM56; Abcam), PE-conjugated anti–IL3Ra (clone 9F5; Biolegend), biotinylated anti-CD38 (clone HIT2;
Biolegend), PE-Cy–conjugated anti-CD117 (clone A3C6E2; Biolegend), and APC-conjugated anti-CD34 (clone HPCA-2; Biolegend). Alexa Fluor 700 conjugated to streptavidin (Invitrogen)
was used for visualization of biotinylated antibodies.
Detection of Protease Activity by Flow Cytometry. To assess the
proteolytic activity of monocytes and neutrophils in the spleen
and lung, mice were injected i.v. with 2 nmol of ProSense-680
(VisEn Medical–PerkinElmer), a cathepsin activatable probe
(3). Mice were killed 24 h after administration of the agent and
analyzed by flow cytometry as described above.
Fluorescent Mediated Tomography/CT Fusion Imaging. Fluorescentmediated tomography (FMT)/CT fusion imaging was done on an
FMT-2500 instrument (VisEn Medical–PerkinElmer) as described previously (3). Mice were placed on a highly purified
reduced-manganese diet (Harlan) to reduce autofluorescence
caused by normal mouse chow diet when imaging. For efficient
image fusion and reconstruction, mice were placed in a multimodal imaging cartridge containing fiducial markers detectable by
FMT and CT (4, 5). For FMT imaging, mice received 2 nmol of
ProSense-680 as described above. Mice were shaved before imaging to reduce the attenuation effects of fur on the optical signal
and anesthetized with isoflurane inhalation during imaging. A 3D
FMT dataset was reconstructed, where fluorescence per voxel was
expressed as a nanomolar measure. For CT imaging, Isovue-370
(Bracco Diagnostics) was infused i.v. at 55 μL/min through a tail
vein catheter to increase vascular contrast. Tumor size was
quantified using OsiriX imaging software (OsiriX Foundation).
Noninvasive Evaluation of Tumor Burden by High-Resolution CT. Mice
infected with AdCre, and splenectomized or not, were anonymized and subjected to high-resolution CT at different time
points. For imaging, mice were anesthetized with isoflurane gas
inhalation and had a respiratory pillow placed underneath the
abdomen to provide gating of X-ray exposures on expiration of
the respiratory cycle (to prevent motion artifacts). The CT data
were acquired on an Inveon PET-CT system (50-μm isotropic
spatial resolution; Siemens) and analyzed blindly by a radiologist. Multiplanar and 3D reconstructions were obtained using
OsiriX and AMIRA (Visage Imaging, Inc.).
Intravital Microscopy. EYFP+ GMP cells were adoptively trans-
ferred and visualized in the spleen 5 d later using an Olympus
FV1000 confocal laser scanning system on an upright BX61-WI
microscope (Olympus) running Fluoview 1000 version 2.1 software
(Olympus). For imaging, the mice were placed onto a temperature-regulated heating plate (Olympus Corporation) set to 37 °C.
The spleen was exposed surgically and kept moist during imaging
as previously described (2), and it was imaged with an Olympus
objective (0.5 N.A.) with a magnification of 20×. EYFP+ cells and
blood vessels (labeled with AngioSense-680; ViSen Medical–
PerkinElmer) were visualized through excitation at 488 nm and
635 nm, respectively, also as previously described (2).
Parabiosis. For parabiosis experiments, weight-matched WT and
GFP C57BL/6 mice (or CD45.1 and CD45.2 C57BL/6 mice or
WT 129 and EYFP+ mice) were used. Mice were anesthetized
with isoflurane and joined by a technique adapted from Bunster
et al. (6). Mice were then surgically separated by a reversal of the
procedure. The percentage of chimerism for each studied cell
Cortez-Retamozo et al. www.pnas.org/cgi/content/short/1113744109
Gene Expression Profile Signature of Human Progenitor Cells. RTPCR analysis on spleen-derived human progenitors of a representative set of genes expressed in progenitor cells was performed
as described by Manz et al. (7). Briefly, human progenitor populations were isolated by FACS from cell suspensions of human
spleens. Approximately 1,000 cells were directly sorted into 350
μL of RNeasy Lysis Buffer (RLT) (Qiagen) supplemented with
1% 2-mercaptoethanol. RNA was extracted using the RNAeasy
micro Kit (Qiagen). The entire sample was used for cDNA
generation using the High Capacity cDNA Reverse Transcription
Kit (Applied Biosystems). RT-PCR was performed with predesigned and validated primer sets (Applied Biosystems) (Table S1).
siRNA Silencing. KP mice at week 12 after AdCre administration were
treated i.v. four times each day for 4 d with a dose of 0.5 mg/kg of
2 of 8
Statistics. Results were expressed as the median or mean ± SEM.
Statistical tests included an unpaired two-tailed Student t test
and one-way ANOVA, followed by Tukey’s multiple comparison
test (for more than 2 groups). Otherwise, for data not normally
distributed, we applied the nonparametric Kruskal–Wallis test,
followed by Dunn’s multiple comparison test. P values of 0.05 or
less were considered to denote significance.
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siCCR2 or control (siCON; siRNA targeting luciferase as a control)
per injection (8). Animals were killed 1 d after the last injection.
Fig. S1. Gating scheme for identification of CD11b+ and CD11b− macrophages and of neutrophils in mouse lungs by flow cytometry. (Insets) Gated neutrophils are Ly-6G+, whereas CD11b+ macrophages are F4/80+. FSC, Forward Scatter; SSC, Side Scatter.
Fig. S2. Effect of splenectomy on the TAM and TAN responses in grafted EL-4 and CT26 tumors. EL-4 T-cell lymphoma (1 × 106 cells) and CT26 colon carcinoma
(1 × 106 cells) cell lines were injected s.c. into C57BL/6 mice and BALB/c mice, respectively, from which the spleen was either kept (+SP) or removed (−SP). The
data show total counts of TAMs and TANs in tumors on day 14 after implantation of EL-4 tumors (n = 4 for each condition) and on day 7 after implantation of
CT26 tumors (n = 7 for each condition). Data shown in the graph are representative of two independent experiments. Data are presented as the mean ± SEM.
*P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S3. Quantification of Cathepsin B activity in diverse cell populations obtained ex vivo from tumor-bearing KP mice. Single-cell suspensions were either
obtained from the lung (Upper) or spleen (Lower). Monocytes were defined as CD11b+ Lin− Ly-6G− (F4/80/CD11c/I-Ab)lo Ly-6C+. Neutrophils were defined as
CD11b+ Gr-1+Ly-6G+. Lineage (Lin) refers to the following combination: B220/DX5/NK1.1/CD90.2/Ly6G. Experiments used KP mice at week 11 after tumor
initiation (n = 4). Data shown in the graph are representative of two independent experiments. Data are presented as the mean ± SEM. MFI, mean fluorescent
intensity.
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Fig. S4. Histological identification of tumor burden in the lungs of KP mice. The images show H&E staining of lungs obtained from multiple animals, which
belong to five different cohorts. The five cohorts are as follows: (i) +AdCre: positive controls, KP animals infected with AdCre at week 0 and analyzed at week 8
(n = 4); (ii) −AdCre: negative controls, KP animals not infected with AdCre (n = 3); (iii) +AdCre: positive controls, KP animals infected with AdCre at week 0 and
analyzed at week 11 (n = 8); (iv) +AdCre −SP (wk 0): KP animals splenectomized and infected with AdCre at week 0 and analyzed at week 11 (n = 8); and
(v) +AdCre −SP (wk 8): KP animals infected with AdCre at week 0, splenectomized at week 8, and analyzed at week 11 (n = 4). Scale bar: 5 mm.
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Fig. S5. Splenectomy in KP mice has an impact on TAM and TAN responses but does not improve cytotoxic CD8 T-cell responses or suppress regulatory CD4 Tcell responses. (A) Accumulation of TAMs and TANs in mice shortly after splenectomy. KP mice were infected with AdCre to induce lung tumors; 11 wk later,
they were either splenectomized (−SP, n = 4) or sham operated (+SP, n = 4). All animals received BrdU i.p. and were killed 24 h later for analysis by flow
cytometry. Histograms show the total number of BrdU+ TAMs and BrdU+ TANs in the lungs. (B and C) Number of blood monocytes in KP mice shortly after
splenectomy. (B) Number of blood monocytes in control mice (ø, n = 4), KP mice 11 wk after tumor initiation (KP +SP, n = 4), and KP mice 11 wk after tumor
initiation and shortly after splenectomy (KP −SP, n = 4). (C) Number of BrdU+ blood monocytes in KP mice 11 wk after tumor initiation; on week 11, the KP mice
were either sham operated (KP +SP, n = 4) or splenectomized (KP −SP, n = 4). Animals received BrdU i.p. and were killed 24 h later for analysis by flow cytometry (same as for A). (D) Number of bone marrow monocytes and neutrophils in KP mice with or without a spleen during tumor development. Monocytes
(Left) and neutrophils (Right) are shown in the bone marrow of KP animals that were splenectomized (−SP, n = 19) or not splenectomized (+SP, n = 17) at the
time of tumor initiation (week 0). Mice were killed on week 11. (E) Total number of CD8+ T cells retrieved in the lungs of control mice (−AdCre, n = 8), mice
infected with AdCre (+AdCre, n = 8), and mice infected and splenectomized at week 0 [+AdCre −SP (wk 0), n = 8]). (F) Determination of the production of IFN-γ
(IFN-g) and Granzyme b (GZMB) by flow cytometry on CD8 T cells in the animal cohorts described in E. The graphs show the mean fluorescent intensity (MFI) as
a measure of cytokine production. (G) Total number of FoxP3+ CD4+ regulatory T cells retrieved in the lungs of control mice (−AdCre, n = 8), mice infected with
AdCre (+AdCre, n = 8), and mice infected and splenectomized at week 0 [+AdCre −SP (wk 0), n = 8]. (H) Lung weight as a measure of tumor burden. Lung
weight correlates positively with the number of tumors detected by high-resolution CT in animals analyzed at week 11 after AdCre infection. Data shown are
representative of two independent experiments. Data are presented as the mean ± SEM. (I) Relationship between tumor burden (here, based on lung weight;
Fig. S6) and number of TAMs and TANs on week 11. Mice with a spleen (+AdCre +SP, brown diamonds, n = 17), mice splenectomized on week 0 (+AdCre −SP,
blue diamonds, n = 19), and mice splenectomized on week 8 (+AdCre −SP, green diamonds, n = 3) are shown. Dotted lines represent maximal lung weight and
macrophage/neutrophil numbers observed in age-matched animals without cancer (n = 12).
Fig. S6. CCR2-dependent tumor growth. KP mice were infected with AdCre to induce lung tumors; 12 wk later, they received nanoparticles containing either
siCCR2 (CCR2-silencing siRNA) or siCON (control nanoparticles), as described by Leuschner et al. (8). KP mice received nanoparticle injections every 4 d for 12 d.
(A) Weight of lungs from tumor-free mice (Control) or tumor-bearing mice treated with siCCR2 (n = 4) or siCON (n = 4). (B) Representative pictures of lungs
from mice treated with siCON or siCCR2. Scale bar: 5 mm.
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Fig. S7. Phenotypic characterization of splenic HSCs, GMPs, monocytes, and neutrophils. (A) Comparison of splenic and bone marrow cells isolated from KP
mice (+AdCre, week 11, n = 3). The markers characterize different progenitor populations (CD117, Sca-1, CD16/32, CD34, and CD115) or monocytes and
neutrophils (CD11b, Ly-6C, F4/80, CD11c, and CD62L). Lineage-positive cells (shown in gray) were investigated in parallel as internal controls. The populations
analyzed did not show notable phenotypic differences. (B) Comparison of splenic cells (monocytes and neutrophils) in tumor-free (−AdCre, n = 3) and tumorbearing (+AdCre, week 11, n = 3) mice. The markers were chosen to provide information on enzymatic activity, phagocytic capacity, cytokine production, and
activation state. For the determination of enzymatic activity, a pan-cathepsin activatable probe (Prosense-680) was injected 24 h before flow cytometry
analysis. In a similar fashion, phagocytosis was determined by injection of a fluorescently labeled nanoparticle (CLIO-VT680; CLIO nanoparticles are synthesized
in house and are not commercially available, the particles are conjugated to the dye VT 680 purchased from Visen Medical/ Perkin Elmer) 24 h before analysis.
For intracellular detection of TNF-α production, splenic cell suspensions were restimulated for 4 h with phorbol 12-myristate 13-acetone. Lineage-positive cells
(shown in gray) were investigated in parallel as internal controls. We did not observe notable differences between the two cohorts of mice. (C) Further
comparison of splenic monocytes in tumor-free (group 1) and tumor-bearing (group 2) mice and of TAMs (group 3) (n = 4 for each group). The nine genes were
chosen based on their reported role in monocyte/macrophage function and/or differentiation. Relative gene expression was measure by Taqman real-time PCR
and normalized to 18s rRNA. Analysis of the gene expression profile showed that the presence of tumors did not significantly alter the phenotype of monocytes in the spleen [except for IFN-γ (IFN-g)] but that splenic monocytes differ from TAMs. Data shown in the graph are representative of two independent
experiments. Data are presented as the mean ± SEM. ***P < 0.001.
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Fig. S8. Quantification of HSC and progenitor responses. (A) Quantification of CMPs and MDPs in the spleen of tumor-bearing (+AdCre) KP animals (n = 17) in
comparison to age-matched tumor-free mice (−AdCre, n = 12) on week 11. (B) Quantification of HSCs, CMPs, GMPs, and MDPs in the bone marrow of tumorbearing (+AdCre) KP animals (n = 17) in comparison to age-matched tumor-free mice (−AdCre, n = 12) on week 11. (C) Fold increase in the total number of
hematopoietic progenitor cells (HSCs, CMPs, GMPs, and MDPs) in the bone marrow and spleen of KP mice infected with AdCre (KP +AdCre) at week 11. The
data are compared with those in syngeneic tumor-free control mice. (D) Fold increase accumulation of GMPs in bone marrow and spleen in three tumor mouse
models. (Left to Right) KP mice infected with AdCre (KP +AdCre, n = 12) analyzed at week 11, C57BL/6 mice grafted with EL-4 tumors (B6 +EL-4, n = 10) analyzed
at day 14, and BALB/c mice grafted with CT26 tumors (BALB/c +CT26, n = 4) analyzed at day 8. The data are compared with those on syngeneic tumor-free mice.
(E) Percentage of HSCs and GMPs in the S/G2 phase in the bone marrow and spleen of tumor-bearing KP mice at week 11 (n = 4). (F) Total numbers of HSCs
(Left), GMPs (Center), and the progeny of adoptively transferred GMPs (Right) in the liver of tumor-free animals (−AdCre; n = 6) and in animals infected with
AdCre and from which the spleen was either retained (+AdCre; n = 6) or not retained [+AdCre −Spleen (wk 0), n = 5]. Analyses were performed 11 wk after
AdCre infection. (G) Fate mapping of GMPs transferred into C57BL/6 mice grafted with EL-4 tumors (+EL-4, n = 10) and tumor-free mice (−EL-4, n = 6). (Left)
Experimental procedure. (Right) Quantification of GMP-derived monocytes and neutrophils in the bone marrow (BM) and spleen (SP). Data shown in the graph
are representative of four independent experiments (A–D) or two independent experiments (E–G). Data are presented as the mean ± SEM. *P < 0.05; **P <
0.01; ***P < 0.001; ns, not significant.
Fig. S9. Spleen of human patients with invasive cancer accumulates myeloid progenitor cells and lineage descendants. (A) (Upper) Photograph of a colony
derived from human GMP-like cells. Colonies were harvested, and the cells were analyzed by H&E staining on cytospins (Lower Left) and by flow cytometry
(Lower Right) (n = 3). Scale bars: Top, 50 μm; Bottom, 10 μm. Data are presented as the mean ± SD. (B) Total number of GM-CFU colonies estimated to derive
from the spleen of a control patient and of two patients with invasive cancer. (C) (Upper) Identification by flow cytometry of monocytes (red) and neutrophils
(green) in human spleen samples. (Lower) Histograms show the expression of eight cell surface markers for the same cells. FSC, Forward Scatter; SSC,
Side Scatter.
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Table S1.
Taqman probes used for gene expression analysis in human and mouse
Gene symbol
c/EBPe
CSF2A
EPOR
GAPDH
GATA1
GATA3
KIT
MPO
PU.1
vWF
Arg1
Cd276
Ifn-g
Il-18
Il-6
Il4ra
Itgam
Nos2
Tgfb
Gene description
Assay identification
CCAAT/enhancer binding protein ε (C/EBPε)
Colony stimulating factor 2
Erythropoietin receptor
Glyceraldehyde-3-phosphate dehydrogenase
GATA binding protein 1
GATA binding protein 3
CD117
Myeloperoxidase
Transcription factor PU.1
von Willebrand factor
Arginase 1
Costimulatory molecule B7H3
IFN-γ
IL-18
IL-6
IL-4 receptor α
Integrin α-M, CD11b
Nitric oxide synthase 2
TGF-β1
Hs00357657_m1
Hs00531296_g1
Hs00959427_m1
Hs00266705_g1
Hs00231112_m1
Hs00231122_m1
Hs00174029_m1
Hs0092496_m1
Hs02786711_m1
Hs00169795_m1
Mm00475988_m1
Mm00506020_m1
Mm00801778_m1
Mm00434225_m1
Mm00446190_m1
Mm00439634_m1
Mm00434455_m1
Mm00440485_m1
Mm00441724_m1
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