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J. Exp. Med. Vol. 206 No. 6 1317-1326
Cancer pathogenesis involves not only the cell-
autonomous defects that arise from alterations
in oncogenes and tumor suppressors but also the
impact of host antitumor responses (1). Cancer
cells that have escaped immune control are se-
lected for the ability to exploit factors present in
the tumor microenvironment to further disease
progression (2–4). Among this array of soluble
moieties, inflammatory cytokines including
TNF-, IL-6, and IL-1 play key roles through
triggering NF-B–, STAT-3–, and MyD88-
dependent pathways (5–8).
GM-CSF is another cytokine frequently pro-
duced in the tumor microenvironment, where
it may contribute to either tumor protection or
promotion (9). Through studies of GM-CSF–
deficient mice, we identified milk fat globule epi-
dermal growth factor–8 (MFG-E8) as a critical
determinant of the pro- and antiinflammatory ac-
tivities of the cytokine (10). MFG-E8 is a secreted
phosphatidylserine-binding protein that signals
through v3 and v5 integrins (9–12). Under
steady-state conditions, GM-CSF induces MFG-
E8 expression in mononuclear phagocytes, en-
abling the efficient uptake of apoptotic cells, the
production of TGF- and CCL22, and the main-
tenance of FoxP3+ T reg cells (10). Under condi-
tions of cellular stress, however, the ligation of
Toll-like receptors dampens MFG-E8 expression,
whereupon GM-CSF elicits CD4+ and CD8+ ef-
fector T cells through an MFG-E8–independent
pathway. Thus, the levels of MFG-E8 present in
the tumor microenvironment might modulate the
functions of GM-CSF during carcinogenesis.
In malignant melanoma, MFG-E8 expres-
sion is increased in tumor cells and/or infiltrating
Abbreviations used: 5-FU,
5-fluorouracil; BMDC, bone
marrow–derived dendritic cell;
MFG-E8, milk fat globule epi-
dermal growth factor–8; NOD-
SCID, nonobese diabetic–severe
VEGFR-2, antivascular endo-
thelial growth factor receptor–2.
Milk fat globule epidermal growth factor–8
blockade triggers tumor destruction
through coordinated cell-autonomous
and immune-mediated mechanisms
Masahisa Jinushi,1 Marimo Sato,1 Akira Kanamoto,1 Akihiko Itoh,1
Shigenori Nagai,2,3 Shigeo Koyasu,2 Glenn Dranoff,4 and Hideaki Tahara1
1Department of Surgery and Bioengineering, Advanced Clinical Research Center, Institute of Medical Science, University
of Tokyo, Tokyo 108-8639, Japan
2Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan
3Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075, Japan
4Department of Medical Oncology and Cancer Vaccine Center, Dana-Farber Cancer Institute and Department of Medicine,
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
Carcinogenesis reflects the dynamic interplay of transformed cells and normal host elements,
but cancer treatments typically target each compartment separately. Within the tumor micro-
environment, the secreted protein milk fat globule epidermal growth factor–8 (MFG-E8)
stimulates disease progression through coordinated v3 integrin signaling in tumor and host
cells. MFG-E8 enhances tumor cell survival, invasion, and angiogenesis, and contributes to
local immune suppression. We show that systemic MFG-E8 blockade cooperates with cyto-
toxic chemotherapy, molecularly targeted therapy, and radiation therapy to induce destruction
of various types of established mouse tumors. The combination treatments evoke extensive
tumor cell apoptosis that is coupled to efficient dendritic cell cross-presentation of dying
tumor cells. This linkage engenders potent antitumor effector T cells but inhibits FoxP3+ T reg
cells, thereby achieving long-term protective immunity. Collectively, these findings suggest
that systemic MFG-E8 blockade might intensify the antitumor activities of existing therapeu-
tic regimens through coordinated cell-autonomous and immune-mediated mechanisms.
© 2009 Jinushi et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
SYSTEMIC MFG-E8 BLOCKADE | Jinushi et al.
trol, in contrast to the limited impact of individual agents.
MFG-E8 blockade also intensified the activity of an epidermal
growth factor receptor tyrosine kinase inhibitor and an antivas-
cular endothelial growth factor receptor–2 (VEGFR-2) mAb
(Fig. 1 D). Additionally, a short course of radiation therapy
directed toward subcutaneous MC38 lesions was rendered
more efficacious when combined with anti–MFG-E8 anti-
bodies (Fig. 1 E). Synergistic effects of anti–MFG-E8 anti-
bodies and chemotherapy were similarly observed in the poorly
immunogenic B16 melanoma model. In this system, combina-
tions of MFG-E8 blockade with doxorubicin, etoposide, or
dacarbazine achieved significant tumor control, although gem-
citabine proved inactive (Fig. 1 F and not depicted). More-
over, the combined administration of anti–MFG-E8 antibodies
and doxorubicin also triggered the destruction of established
EL-4 thymomas (Fig. S2). Collectively, these experiments re-
veal the ability of systemic MFG-E8 blockade to intensify the
antitumor effects of conventional oncologic therapies in di-
verse tumor models.
MFG-E8 blockade enhances drug-induced apoptosis
Because standard cancer treatments displayed only modest
single-agent activity against various types of tumors, we
wondered whether the anti–MFG-E8 antibodies might
modulate tumor cell killing. In this context, we previously
showed that MFG-E8 triggered Akt activation in tumor cells
through v3 integrin signaling, which resulted in an attenu-
ation of etoposide-induced death (12). In accordance with
these findings, MFG-E8 was not detectable in MC38 and
B16 cells at baseline, whereas cells that survived an overnight
exposure to diverse chemotherapeutic drugs manifested sig-
nificant MFG-E8 expression, which was evident intracellu-
larly, at the surface membrane, and in culture supernatants
(Fig. 2, A and B; and not depicted). irradiation also in-
duced MFG-E8 surface expression on EL-4 thymoma (EG.7-
OVA) cells (Fig. 2 C). Furthermore, stable drug-resistant
variants of MC38 cells, which were generated through pro-
longed exposure to escalating concentrations of gemcitabine,
CPT-11, or 5-FU in vitro, and similarly derived doxorubi-
cin-resistant B16 cells demonstrated much higher levels of
MFG-E8 compared with the parental tumor cells (Fig. 2 D
and not depicted).
Consistent with an antiapoptotic function for surface and/
or secreted MFG-E8, the addition of anti–MFG-E8 mAbs but
not irrelevant control antibodies potentiated the killing of
MC38 cells with gemcitabine and 5-FU, as revealed by the
enhanced expression of annexin V (Fig. 2 E). The addition of
rabbit polyclonal anti–MFG-E8 serum manifested comparable
effects (unpublished data). The induction of apoptosis with
the combined therapy also resulted in a loss of mitochondrial
membrane potential (unpublished data). Moreover, the
combination therapies triggered increased levels of tumor
apoptosis in vivo. MC38 tumors harvested from mice that re-
ceived gemcitabine plus anti–MFG-E8 antibodies showed en-
hanced caspase 3 activation as compared with tumors isolated
from mice treated with either agent alone (Fig. 2 F). Similarly,
myeloid elements upon progression to the vertical growth
phase, the stage in which melanoma cells acquire the com-
petence for invasion and dissemination (12, 13). In a mouse
melanoma model, MFG-E8 augmented tumorigenicity and
metastatic capability through Akt- and Twist-dependent
mechanisms (12). MFG-E8 enhanced melanoma cell resis-
tance to apoptosis, induced an epithelial-to-mesenchymal
transition, and stimulated invasion and angiogenesis. MFG-E8
also contributed to local immune suppression by evoking
FoxP3+ T reg cell infiltrates and suppressing Th1 reactions and
NK and CD8+ T cell cytotoxicity.
Because MFG-E8 is expressed at high levels in diverse tu-
mor types (14, 15), including melanoma, this soluble protein
might serve as a general target for cancer therapy. In contrast
to most oncologic treatments, which primarily address either
the tumor or host separately, MFG-E8 antagonists might
affect both compartments. Indeed, shRNA knockdowns of
MFG-E8 sensitized tumor cells to cytotoxic agents and small
molecule inhibitors of receptor tyrosine kinases in vitro,
whereas MFG-E8 blockade with a dominant-negative mutant
potentiated tumor immunity generated with irradiated, GM-
CSF–secreting tumor cell vaccines (10, 12). Based on these
results, we hypothesized that systemic targeting of MFG-E8
might contribute to tumor destruction in several complemen-
tary ways. In this paper, we show that antibodies to MFG-E8
cooperate with conventional cancer therapies to effectuate
sustained control of established mouse tumors through the
coupling of cell-autonomous and host-mediated pathways.
Combinatorial therapy with systemic MFG-E8 blockade
To explore the therapeutic potential of anti–MFG-E8 anti-
bodies, we first characterized the moderately immunogenic
MC38 colon carcinoma model that is syngeneic to C57BL/6
mice. At 10 d after intradermal inoculation, when tumors were
well established (25 mm2), the systemic administration of
gemcitabine, a cytotoxic agent with modest activity in patients
with colon carcinoma (16), afforded a small delay in MC38
tumor growth in a dose-dependent fashion (Fig. 1 A). Al-
though a blocking anti–MFG-E8 mAb (17) displayed minimal
antitumor activity when infused alone, combination treatment
with gemcitabine resulted in tumor regressions that were sus-
tained throughout the duration of the study (4 mo). A rabbit
polyclonal anti–MFG-E8 serum (18) showed comparable ef-
ficacy (Fig. 1 B), whereas an isotype control mAb was inac-
tive (not depicted), establishing the specificity of the response.
Synergistic antitumor effects were also obtained when the
anti–MFG-E8 antibodies were administered beginning 3 or 7 d
after gemcitabine but not when infused before chemother-
apy, indicating that the sequence of the combination was im-
portant for activity (Fig. S1).
Systemic anti–MFG-E8 antibodies similarly enhanced the
therapeutic potency of 5-fluorouracil (5-FU) and CPT-11
(Fig. 1 C), two agents frequently used in the treatment of ad-
vanced colon carcinoma patients (16). As with gemcitabine,
these combination therapies achieved prolonged tumor con-
JEM VOL. 206, June 8, 2009
Combinatorial therapy with MFG-E8 blockade stimulates
T cell immunity
Because MFG-E8 plays a key role in T reg cell homeostasis
(10), we investigated whether the antitumor activities of
combined chemotherapy and MFG-E8 blockade might also
involve host immunity. In accordance with this idea, anti–
MFG-E8 antibodies failed to increase the minimal killing of
MC38 cells achieved with gemcitabine in immunodeficient
nonobese diabetic–severe combined immunodeficiency
(NOD-SCID) mice (Fig. 3 A). Moreover, antibody deple-
tion experiments established that CD8+ and to a lesser extent
CD4+ T cells, but not NK cells, were required in wild-type
B16 melanomas manifested elevated caspase 3 activation after
combined dacarbazine and MFG-E8 blockade. These results
support the idea that MFG-E8 blockade compromises tumor
cell viability in a cell-autonomous fashion, although the in-
creased caspase activation in vivo might partially reflect the
death of stromal elements within the tumor microenviron-
ment. The anti–MFG-E8 antibodies may also attenuate tumor
angiogenesis (18), which might contribute to tumor cell death
as well. Collectively, these results reveal a role for MFG-E8 in
mediating resistance to cytotoxic therapy and suggest that anti–
MFG-E8 antibodies might serve as a complementary strategy
to intensify drug-induced tumor cell killing.
Figure 1. MFG-E8 antibody blockade synergizes with cytotoxic therapies to mediate tumor destruction. (A) Established MC38 carcinomas (25 mm2)
were treated with systemic gemcitabine (GEM) and/or an anti–MFG-E8 mAb, as indicated. (B) Same conditions as in A, but with rabbit anti–MFG-E8
sera. (C) Established MC38 carcinomas were treated with 5-FU or CPT-11 with or without anti–MFG-E8 mAb. (D) Established MC38 carcinomas were
treated with anti–VEGFR-2 mAb DC101 or EGFR-TKI AG490 with or without anti–MFG-E8 mAb. (E) Established MC38 tumors were treated with local irra-
diation (XRT) with or without systemic anti–MFG-E8 mAb. (F) Established B16 melanomas (25 mm2) were treated with systemic doxorubicin, etoposide, or
GEM with or without anti–MFG-E8 mAb. Each experiment was performed with five mice per group, and similar results were observed for each panel in five
independent experiments. Shown are the means ± SEM per cohort for a representative experiment. *, P < 0.05 between a treatment group and control.
SYSTEMIC MFG-E8 BLOCKADE | Jinushi et al.
MFG-E8 blockade, however, did not manifest impaired func-
tion on a per cell basis, as assessed in standard in vitro suppres-
sion assays (unpublished data). These findings extend our earlier
demonstration that MFG-E8 is an important determinant of an-
titumor T reg cell numbers (10, 12).
The combination treatment, but not anti–MFG-E8 anti-
bodies alone, markedly increased CD4+ and CD8+ effector T cell
activation and function. This stimulation resulted in high levels of
surface CD44, IFN- production, and tumor-specific cytotoxic-
ity (Fig. 4, B–D). The combination therapy also augmented the
total levels of circulating IgG2a and IgG2b antibodies, which
might contribute to tumor destruction through Fc-dependent cy-
totoxicity (Fig. S3). These results highlight the synergy between
systemic MFG-E8 blockade and chemotherapy for boosting
multiple antitumor effector mechanisms.
Anti–MFG-E8 antibodies enhance dendritic cell
cross-presentation of tumor antigens
Because MFG-E8 promotes the uptake of apoptotic cells by
mononuclear phagocytes (10, 19–21), we next character-
ized the impact of chemotherapy and MFG-E8 blockade on
mice for tumor destruction with the combination therapy, al-
though the antitumor effects became significant compared
with gemcitabine alone 40 d after combined therapy even
with CD8+ T cell depletion (Fig. 3 B). These findings suggest
that the cell autonomous modes of tumor cell killing with
MFG-E8 blockade are not sufficient to maintain durable con-
trol of the established cancers. Consistent with a key role for
T cells in the generation of long-lived and specific protective
immunity, treated mice rejected a subsequent lethal challenge
with MC38 tumor cells but not B16 melanoma or MCA-205
fibrosarcoma cells (Fig. 3 C).
To characterize the T cell responses stimulated with treat-
ment, we isolated tumor-infiltrating lymphocytes from re-
gressing MC38 lesions. Although the proportions of total
CD4+ and CD8+ T cells did not vary as a function of therapy,
anti–MFG-E8 antibodies alone or in combination with CPT-11
significantly reduced the numbers of intratumoral Foxp3+
T reg cells (Fig. 4 A). Although chemotherapy alone had no
impact on T reg cell numbers, combined treatment with anti–
MFG-E8 antibodies decreased T reg cell numbers in the spleen
as well (Fig. 4 A). The T reg cells that were recovered after
Figure 2. Drug-resistant tumor cells express MFG-E8. (A) MC38 carcinoma and B16 melanoma cells were treated with various cytotoxic agents
under serum-free condition for 24 h, and intracellular MFG-E8 expression in viable cells (annexin V/propidium iodide) was determined with flow cy-
tometry. The shaded and open histograms represent the levels of expression on untreated and treated cells, respectively. Gemcitabine (GEM) and 5-FU
accomplished minimal killing of B16 cells (not depicted). (B) MFG-E8 levels in culture supernatants from A were measured with ELISA. (C) EL-4 thymoma
cells were treated with irradiation (100 Gy), and MFG-E8 expression in viable cells (annexin V/propidium iodide) was determined with flow cytometry.
(D) Stable drug-resistant variants of MC38 were generated and tested for MFG-E8 expression with flow cytometry (shaded histogram). The staining with
isotype control antibodies is also shown. (E) MC38 carcinoma cells were exposed to GEM or 5-FU in the presence of anti–MFG-E8 mAb or isotype control
as in A, and cell viability was determined with flow cytometry (percentages are shown). (F) Established MC38 and B16 tumors (25 mm2) were treated with
GEM or dacarbazine, respectively, with or with systemic anti–MFG-E8 mAb as in Fig. 1. 4 d after completion of therapy, tumor homogenates were pre-
pared and assayed for caspase 3 activation with ELISA. Presented data are representative of three independent experiments with similar results. Means ±
SEM are shown in B and F. *, P < 0.05 between the treatment and control.
JEM VOL. 206, June 8, 2009
our analysis of the tumor infiltrates raised the possibility that
dendritic cell capture of tumor cells in situ might be important
for T cell priming.
To study the impact of anti–MFG-E8 antibodies on den-
dritic cell cross-presentation in more detail, we used EL-4 thy-
moma cells engineered to express OVA (EG.7-OVA) (22) and
the syngeneic C57BL/6 mice harboring a transgenic TCR spe-
cific for an MHC class II–presented OVA peptide (OT-II) (23).
BMDCs efficiently ingested irradiated EG.7-OVA cells, and this
was only minimally blocked by an antibody to v integrins or
MFG-E8, indicating that this pathway was not required for
tumor cell uptake in this system (Fig. 5 B and not depicted).
Although anti–MFG-E8 antibodies did not affect the overall
phagocytosis of irradiated EG.7-OVA cells, a blocking antibody
to Fc receptors but not to v integrins partially attenuated up-
take. These results reveal that anti–MFG-E8 antibodies switch
the receptor for MFG-E8–mediated tumor cell ingestion from
v3/v5 integrins to Fc receptors.
Consistent with the ability of activating Fc receptors to trig-
ger immune stimulation (24), the opsonization of EG.7-OVA
cells with anti–MFG-E8 antibodies enhanced dendritic cell stim-
ulation of OT-II TCR transgenic CD4+ T cells. This activation
resulted in increased production of IFN- (Fig. 5 C) but not IL-4
or IL-17 (not depicted). In contrast, anti-Fc receptor antibodies
inhibited cross-priming, whereas anti–v integrin antibodies
augmented T cell responses when either irradiated EG.7-OVA
cells alone or anti–MFG-E8 antibody opsonized, irradiated
EG.7-OVA cells were fed to dendritic cells (Fig. 5 C). The anti–
MFG-E8 antibodies also increased cross-presentation to MHC
class I–restricted OT-I CD8+ T cells, resulting in increased
IFN- production (Fig. S6). Collectively, these findings suggest
that MFG-E8 blockade enhances Th1 antitumor responses.
To further examine the importance of MFG-E8 antibody–
mediated cross-priming in vivo, we injected irradiated EG.7-
OVA cells together with anti–MFG-E8 antibodies into the
footpads of OT-I mice and measured OVA-specific T cell re-
sponses in the draining lymph nodes 5 d later. In some experi-
ments, blocking anti-FcR antibodies were coadministered to
evaluate the role of FcR-mediated uptake for antigen presen-
tation. Although the injection of anti–MFG-E8 antibodies en-
hanced specific CD8+ T cell IFN- production, the concurrent
administration of anti-FcR antibodies substantially inhibited
this response (Fig. 5 D). These results indicate that MFG-E8
blockade promotes cross-priming of antigen-specific CD8+ T
cells primarily through FcR-mediated antigen uptake.
MFG-E8 modulates dendritic cell cytokine production
To further clarify the mechanisms underlying T cell stimulation,
we examined the impact of MFG-E8 on dendritic cell cytokine
production after the uptake of dying tumor cells. The addition
of rMFG-E8 protein increased dendritic cell IL-10 secretion,
whereas anti–MFG-E8 antibodies reduced IL-10 but enhanced
IL-12, IL-23, and TNF- production (Fig. 6 A). The effects of
MFG-E8 on cytokine profiles were blocked with the RMV-7
anti–v integrin antibody (unpublished data), confirming the
importance of v3 and v5 integrins in this response.
antigen-presenting cells. Although the combination treatment
did not influence the proportions of macrophages (CD11b+
Gr-1) or myeloid-derived suppressor cells (CD11b+ Gr-1+)
that were isolated from regressing MC38 colon tumors (Fig. S4),
the numbers of CD11b+, CD11c+ dendritic cells were signif-
icantly increased, and these cells expressed high levels of the
co-stimulatory molecule CD86 (Fig. 5 A). As bone marrow–
derived dendritic cells (BMDCs) efficiently phagocytosed
chemotherapy-exposed MC38 and B16 cells in vitro (Fig. S5),
Figure 3. The therapeutic activity of MFG-E8 antibody blockade
and chemotherapy involves host immunity. (A) NOD-SCID mice harboring
established MC38 carcinomas (25 mm2) were treated with systemic gem-
citabine (GEM) and anti–MFG-E8 mAb. (B) Established MC38 carcinomas
bearing wild-type C57BL/6 mice that were depleted of CD4+, CD8+, or
NK1.1+ cells with antibodies were treated with systemic GEM and anti–
MFG-E8 mAb. (C) Wild-type C57BL/6 mice that had rejected established MC38
carcinomas with systemic GEM and anti–MFG-E8 mAb showed specific long-
term protective immunity against subsequent challenge with MC38 cells
during the follow-up period (>200 d). Each experiment was performed with
five mice per group, and similar results were observed for each panel in three
independent experiments. Shown are the means ± SEM for each cohort in a
representative experiment. *, P < 0.05 between the treatment and control.
SYSTEMIC MFG-E8 BLOCKADE | Jinushi et al.
sine kinases and antibody blockade of VEGF serve as prototypes
of rationally designed agents that antagonize major pathogenic
mechanisms in cancer cells and the host, respectively (26, 27).
Although these treatments afford important clinical benefits,
most patients achieve only partial responses and eventually suc-
cumb to progressive disease caused by the emergence of drug-
resistant variants. However, analogous to the ways in which
antimicrobial agents cooperate with host reactions to effectuate
sterilizing immunity for some serious infections (28), cancer
treatments might be significantly improved through concur-
rently targeting the tumor and host. The results presented in
this paper illustrate the potential for systemic MFG-E8 anti-
body blockade in combination with conventional oncologic
therapies to accomplish this dual targeting.
MFG-E8 promotes cancer progression through coordi-
nated v3 integrin signaling in tumor cells, vascular elements,
and infiltrating myeloid cells (12). Although the administra-
tion of anti–MFG-E8 antibodies alone resulted in only mod-
est tumor destruction and immune stimulation, the coupling
To address the role of IL-12 in the immune stimulation
with anti–MFG-E8 antibodies, we used IL-12p35–deficient
mice in a series of in vivo and in vitro studies. The efficacy
of anti–MFG-E8 antibodies and CPT-11 treatment against
MC38 colon tumors was partially reduced in IL-12–deficient
mice compared with wild-type controls (Fig. 6 B). Moreover,
the anti–MFG-E8 antibody–mediated cross-presentation of
EG.7-OVA cells to OT-II CD4+ T cells was slightly decreased
with IL-12–deficient dendritic cells compared with wild-type
controls (Fig. 6 C). These results suggest that IL-12 contributes
to the anti–MFG-E8 antibody–triggered immunostimulation,
but other cytokines and/or cell-surface molecules also play im-
Although substantial evidence demonstrates that cross talk be-
tween tumor cells and normal host elements is critical to carci-
nogenesis (25), most cancer therapies primarily target individual
compartments. Small molecule inhibitors of oncogenic tyro-
Figure 4. Combination MFG-E8 antibody blockade and chemotherapy enhances antitumor effector T cells and inhibits FoxP3+ T reg cells.
Tumor-infiltrating lymphocytes (TILs) were harvested from mice bearing MC38 tumors 5 d after the indicated treatment. The TILs were gated as CD3+CD4+
or CD3+CD8+ T cells, and assayed for (A) FoxP3, (B) IFN-, and (C) CD44 expression with flow cytometry (percentages are shown). Representative stainings
are presented. The means ± SEM for six mice per group are shown in the adjacent panels. (D) Draining lymph nodes were harvested from MC38-bearing
mice after the indicated treatments and evaluated for cytotoxic activity against 51Cr-labeled MC38 and B16 targets in vitro. The percent specific lysis is
presented. Each experiment was independently performed four times. The means ± SEM at an effector/target ratio of 100:1 for six mice per group are
shown in the adjacent panels. *, P < 0.05 between the treatment and control.
JEM VOL. 206, June 8, 2009
of drug-induced tumor cell death to MFG-E8 blockade effec-
tuated the sustained regressions of established colon carcino-
mas, melanomas, and lymphomas. A key component of this
therapeutic synergy is the ability of anti–MFG-E8 antibodies
to attenuate tumor cell resistance to cytotoxic treatments, likely
because of the inhibition of Akt activation. Some degree of in-
trinsic tumor cell sensitivity to the cytotoxic agent appears nec-
essary for this enhancement, though, because gemcitabine, which
failed to provoke B16 cell death in vitro (unpublished data),
proved inactive in vivo in combination with MFG-E8 blockade.
An additional mechanism by which anti–MFG-E8 antibodies
might increase tumor cell killing, particularly in conjunction
with anti–VEGFR-2 antibodies, may involve a more robust in-
hibition of the tumor blood supply, as MFG-E8 is required for
VEGF-induced angiogenesis (14, 18). Moreover, knockdown of
MFG-E8 in MC38 carcinoma cells exposed to chemotherapy
also reduced VEGF production (unpublished data).
Upon the induction of tumor cell death in vivo with cyto-
toxic treatments, MFG-E8 blockade favored the establishment
of an immunogenic tumor microenvironment. This conversion
reflected the dual capacity of anti–MFG-E8 antibodies to antag-
onize v3 integrin–driven immune suppression and to promote
efficient Fc receptor–mediated dendritic cell cross-presentation.
In this context, recent analysis of mice harboring a myeloid cell–
specific KO of v integrins has underscored the importance of
this receptor in attenuating inflammation (29), perhaps through
the induction of Twist (12), an antagonist of the NF-B path-
way (30). Consistent with these results, we found that MFG-E8
blockade resulted in increased IL-12 production. This proin-
flammatory cytokine contributed to tumor protection, as re-
vealed through studies of p35-deficient mice, although other
factors such as IL-23, TNF-, and type I IFNs may play impor-
tant roles as well. Accumulating evidence has also highlighted the
ability of mAbs to opsonize tumor cells for efficient cross-presen-
tation by dendritic cells, thereby engendering potent antitumor
immunity (31). Collectively, these factors may promote the de-
velopment of dense intratumoral infiltrates composed of abun-
dant CD4+ and CD8+ effector T cells but only limited FoxP3+
T reg cells. It is tempting to speculate that this broad T cell re-
sponse may suppress the emergence of drug-resistant tumor cells
and mediate long-term protection against tumor recurrence. In
accordance with this idea, previous experimental and clinical
studies have shown that a high ratio of effector T cells to T reg
cells is tightly linked with sustained tumor destruction (32, 33).
Figure 5. Anti–MFG-E8 antibodies enhance dendritic cell cross-
presentation of dying tumor cells. (A) Tumor-infiltrating cells were
harvested from mice harboring MC38 carcinomas 4 d after the indicated
treatment. The CD3+ and B220+ lymphocytes were excluded by gating,
and the remaining forward/side scatter high cells were analyzed for
CD11c, CD11b, and CD86 with flow cytometry (percentages are shown).
Similar results were observed in three experiments. Shown to the right are
the means ± SEM for five mice per group. *, P < 0.05. (B) BMDCs were co-
cultured with PKH26-labeled EG.7-OVA cells (with or without opsoniza-
tion with anti–MFG-E8 mAbs) and evaluated for phagocytosis. The impact
of blocking antibodies to v integrins and Fc receptors was determined
(percentages are shown). Similar results were observed in three experi-
ments, and the means ± SEM are shown. *, P < 0.05. (C) BMDCs that were
loaded with EG.7-OVA cells as in B were co-cultured with OVA-specific
TCR transgenic CD4+ T cells, and IFN- production was evaluated with
flow cytometry (percentages are shown). The effects of anti–v integrin
and Fc receptor antibodies are shown. Similar results were observed in
three experiments, and the means ± SEM are shown to the right. *, P < 0.05.
(D) 106 irradiated EG.7-OVA cells per mouse were injected into the foot-
pads of OT-I mice with anti–MFG-E8, anti-FcR, and isotype control anti-
bodies as indicated. The draining lymph nodes were harvested after 5 d,
and CD8+ T cell IFN- production was determined with flow cytometry
(percentages are shown). Similar results were observed in three experi-
ments. Shown are the means ± SEM. *, P < 0.05.
SYSTEMIC MFG-E8 BLOCKADE | Jinushi et al.
In vivo tumor challenges. 6-wk-old C57BL/6, NOD-SCID, or IL-12 KO
female mice were injected intradermally with 105 MC38 colon carcinoma,
B16 melanoma, EL-4 thymoma, or MCA205 fibrosarcoma cells. After the
tumors grew to an approximate size of 25 mm2, the mice were treated with
various therapeutic regimens. For the MC38 tumors, mice received on days
10, 12, and 14 1 or 4 mg/ml gemcitabine, 5 mg/ml 5-FU, 1 mg/ml CPT-11,
40 mg/ml of the VEGFR-2 mAb DC101 (ImClone Systems), or the 40 mg/ml
of the EGFR tyrosine kinase inhibitor AG490 (MERCK). In some experi-
ments, tumors were treated with five daily doses of irradiation (3 Gy at a rate
of 4 Gy/min) using an isolator system. For the B16 tumors, mice were treated
with 5 mg/ml doxorubicin, 2 mg/ml etoposide, or gemcitabine as described.
For EL-4 thymoma cells, mice were treated with 5 mg/ml doxorubicin, as de-
scribed. A hamster mAb against MFG-E81 (at 2 mg/ml; MBL International),
an isotype control immunoglobulin IgG2a, or 1 mg/ml of rabbit polyclonal
anti–MFG-E8 sera (provided by C. Thèry, Institut National de la Santé et de
la Recherche Médicale, Paris, France) (17, 18) were administered concur-
rently with the cytotoxic therapies. In some studies, mice were also treated
with depleting antibodies against CD4 (clone GK1.5), CD8 (clone 53-6.72),
and NK1.1 (PK-136; American Type Culture Collection) at days 5, 3,
and 0 relative to the cytotoxic therapy (40, 42). Tumor growth was monitored
regularly and the products of the perpendicular diameters were recorded.
Apoptosis assays. Tumor cells were cultured in serum-free media and treated
with various cytotoxic agents overnight with or without anti–MFG-E8 anti-
bodies. To generate stable drug-resistant variants, MC38 cells were cultured in
gradually increasing concentrations of gemcitabine (from 1 to 20 µg/ml) or
CPT-11 (from 0.5 to 10 µg/ml) over 8 wk. MFG-E8 expression was mea-
sured by flow cytometry using the hamster anti–MFG-E8 mAb followed by
an anti–mouse IgG, as previously described (10). Secreted MFG-E8 levels
Recent work has illustrated that some cytotoxic therapies,
including anthracyclines and taxanes, may induce a form of
immunogenic cell death in which the release of calreticulin
and high mobility group box 1 from dying tumor cells serves
as an innate activating signal (34–36). Cytotoxic therapies may
additionally trigger the DNA damage response to up-regulate
tumor cell expression of NKG2D ligands, thereby eliciting
NK and CD8+ T cell responses (37). Moreover, agonistic an-
tibodies to TNF-related apoptosis-inducing ligand receptors,
which are frequently expressed on cancer cells, mediate potent
antitumor effects in association with other immunotherapies
(38, 39). Our results elucidate several novel antitumor mecha-
nisms through which systemic MFG-E8 blockade may poten-
tiate the efficacy of conventional oncologic therapies by
coordinately targeting tumor cells and components of the tumor
microenvironment. Perhaps the coupling of anti–MFG-E8 an-
tibodies to tumor cell death might be considered a new strat-
egy for in vivo cancer vaccination.
MATERIALS AND METHODS
Mice. C57BL/6, NOD-SCID, and OVA transgenic OT-II mice were ob-
tained from SRL or the Jackson Laboratory and housed under specific patho-
gen-free conditions. OT-I and IL-12p35 gene KO mice were used as previously
described (40, 41). All experiments were conducted according to a protocol ap-
proved by the review committee of animal research at the Institute of Medical
Sciences of the University of Tokyo or Keio University School of Medicine.
Figure 6. Anti–MFG-E8 antibodies modulate antigen-presenting cell cytokine profiles. (A) BMDCs were treated with recombinant MFG-E8 or
anti–MFG-E8 mAbs, and cytokine production was determined with ELISA. Shown are the means ± SEM for three experiments. *, P < 0.05. (B) C57BL/6
wild-type or IL-12p35–deficient mice harboring established MC38 colon carcinomas (25 mm2) were treated with systemic gemcitabine with or without
anti–MFG-E8 mAb, as shown in Fig. 1 A. Shown are the means ± SEM for five mice per group. Similar results were observed in a second experiment.
*, P < 0.05 between treated wild-type and IL-12 KO mice. (C) BMDCs from wild-type or IL-12–deficient mice were loaded with EG.7-OVA cells and
co-cultured with CD4+ T cells obtained from wild-type mice that were immunized with EG.7-OVA cells (five times). IFN- production was evaluated by
flow cytometry (percentages are shown). Similar results were observed in a second experiment.
JEM VOL. 206, June 8, 2009
responsible for experimental design; M. Jinushi, M. Sato, and S. Nagai were
responsible for the preparation and performance of experiments; data analysis and
interpretation were performed by M. Jinushi, M. Sato, A. Kanamoto, A. Itoh, S. Nagai,
S. Koyasu, G. Dranoff, and H. Tahara; and M. Jinushi, G. Dranoff, and H. Tahara
prepared the manuscript.
This study was supported in part by the Grants-in-Aid for Scientific Research
on Priority Areas (20015016 to H. Tahara) and for Young Scientists (Start-Up;
20890050 to M. Jinushi) from the Ministry of Education, Culture, Sports, Science
and Technology of Japan, as well as by the Melanoma Research Alliance and the
Research Foundation for the Treatment of Ovarian Cancer (G. Dranoff).
The authors have no conflicting financial interests.
Submitted: 19 November 2008
Accepted: 17 April 2009
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were measured with an ELISA according to the manufacture’s instruction
(R&D Systems). Cell death was determined with annexin V and propidium
iodide staining, or DiOC6 labeling to quantify the mitochondrial membrane
potential, according to the manufacturer’s instructions (BD). To measure tu-
mor apoptosis in vivo, we treated established MC38 or B16 tumors (25 mm2)
with 10 mg/ml gemcitabine or dacarbazine with or without anti–MFG-E8
antibodies, harvested the tumors 4 d after therapy, and measured caspase 3 ac-
tivation in tumor homogenates with a colorimetric assay kit (Invitrogen).
Immune assays. Tumor-infiltrating lymphocytes were harvested with a cell
gradient separation (Nocoprep; Axis-Shield), as previously described (10).
Cell populations were characterized using mAbs against CD3, CD4, CD8,
CD11b, CD11c, CD44, CD86, Gr-1, and Foxp3 (clone MF23). IFN- pro-
duction was measured by intracellular flow cytometry, as previously de-
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from draining lymph nodes with irradiated MC38 cells for 96 h and then
testing lytic activity against 51Cr-labeled MC38 and B16 targets. The percent
lysis was calculated as (experimental spontaneous)/(maximum sponta-
neous) × 100, using 5% Triton X and medium alone for determination of the
maximum and spontaneous counts.
BMDCs were cultured from bone marrow precursors using GM-CSF–
conditioned media for 7 d and were then treated with 100 ng/ml of recom-
binant MFG-E8 (R&D Systems), 20 µg/ml anti–MFG-E8 mAb (MBL
International), or 20 µg/ml of polyclonal MFG-E8 antiserum overnight.
Culture supernatants were evaluated for IL-12, IL-23, TNF-, and IL-10
levels with ELISAs.
Cross-priming assays. Day 7 BMDCs were also co-cultured with irradiated
EG.7-OVA cells (1:10 ratio) that were labeled with PKH26 (Sigma-Aldrich) in
12-well round-bottom plates, and phagocytosis was determined with flow cy-
tometry. In some experiments, the tumor cells were pretreated with 30 mg/ml
anti–MFG-E8 mAb for 30 min before the co-culture. The impact of blocking
antibodies to v integrins (clone RMV-7; Millipore) or Fc receptors (clone
2.4G2; BD) on tumor cell uptake was similarly evaluated. To antigen presenta-
tion by DCs, naive CD4+ T cells were isolated from the spleens of OT-I or
OT-II mice by magnetic cell sorting (Miltenyi Biotec) and added to the tumor
cell–loaded dendritic cells for 24 h. Intracellular IFN- expression in T cells
was then determined by flow cytometry.
For in vivo cross-priming assays, 106 irradiated EG.7-OVA cells per mouse
were injected into the footpads of OT-I mice with 1 mg/ml anti–MFG-E8
mAb, 1 mg/ml anti-FcR blocking antibody (clone 2.4G2), or isotype control.
5 d after the challenge, mice were sacrificed, and the draining lymph node cells
were isolated and cultured with 10 mg/ml MHC class I–restricted OVA pep-
tides overnight. IFN- production by CD8+ T cells was then determined by
flow cytometry or ELISA using the culture supernatants.
Statistical analysis. Statistical analysis was performed using the unpaired Stu-
dent’s t test or one-way analysis of variance through all of the experimental pro-
cedures. Differences were considered significant when the p-value was <0.05.
Online supplemental material. Fig. S1 associates temporal relationships of
anti–MFG-E8 antibody and chemotherapy administration in antitumor re-
sponses. Fig. S2 presents in vivo antitumor activities of anti–MFG-E8 antibody
and doxorubicin against established EL-4 thymomas. Fig. S3 shows the humeral
responses induced by in vivo treatment of anti–MFG-E8 antibody and chemo-
therapy. Fig. S4 shows the frequency of CD11b+ and CD11b+ Gr-1+myelid
cells at tumors with the treatments. Fig. S5 suggests the in vitro phagocytosis
of apoptotic tumor cells by BMDCs. In Fig. S6, the effect of anti–MFG-E8
antibody was examined in mediating in vitro cross-presentation of OVA anti-
gen to OT-I T cells by BMDCs. Online supplemental material is available at
We wish to thank Dr. Clotilde Thèry for valuable advice in the making of rabbit
polyclonal antibody against mouse MFG-E8; A. Kurose for her secretarial assistance;
and S. Nakayama, A. Asami and R. Miyake for assistance with the in vivo tumor
study, ELISA assay, and animal care. M. Jinushi, G. Dranoff, and H. Tahara were
1326 Download full-text
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