2. 1138 Cancer Immunol Immunother (2015) 64:1137–1149
1 3
TAM Tumor-associated macrophages
TAN Tumor-associated neutrophils
TGF-β Transforming growth factor-β
TLR Toll-like receptors
TME Tumor microenvironment
TNF-α Tumor necrosis factor-α
VEGF Vascular endothelial growth factor
Introduction
Advances in immunotherapy of cancer have given rise to
promising new therapies of which only a selected few have
been applied clinically. A major strategy focuses on the
activation of CD8 T lymphocytes, relying on the presenta-
tion of tumor-associated antigens (TAA) by cancer cells [1,
2]. However, anti-tumoral immune activation toward TAA
may be evaded by down-regulation or alteration in these
cancer cell antigens [3]. Another major obstacle to the
antitumor immune response is the presentation by cancer
cells of inhibitory regulators of immune checkpoints, e.g.,
programmed death ligand l, together with the induction of
immune tolerance by molecules secreted into the tumor
microenvironment (TME) [3–6]. The TME contains a mul-
titude of innate immune cells (IIC) which, in normal tissue,
play a crucial role in monitoring tissue homeostasis, pro-
tecting against pathogens and eliminating damaged cells;
thus, they seem qualified to eliminate cancer cells [7, 8].
However, IIC found in the TME, such as tumor-associated
macrophages (TAM), tumor-associated neutrophils (TAN)
and myeloid-derived suppressor cells (MDSC), have been
shown to contribute to tumor survival, propagation and
metastatic spread [5, 8–11]. These “alternatively” activated
tumor-associated IIC have been shown to promote angio-
genesis, tumor scaffolding and regeneration by secreting
factors such as vascular endothelial growth factor (VEGF)
and transforming growth factor-β (TGF-β) together with
immunosuppressive molecules that restrict antitumor acti-
vation, such as interleukin (IL)-10 [5, 7, 12–14].
In spite of the above findings, TAM may still regain
cytotoxicity toward tumor cells and switch to a “classi-
cally” activated phenotype following “re-education” [15].
Polymorphonuclear neutrophils have been found to con-
tribute to both tumor rejection and tumor promotion [11,
16]. Pro-cancerous “polarization” of these cells may be
switched to antitumor behavior by defined inducers [17],
and this may alter the nature of the interaction between the
tumor and the immune system in the TME, thereby ena-
bling the desired immunological responses.
Various molecules have been shown to enhance anti-
tumor responses by inducing immune cells via various
types of receptors, including Toll-like receptors (TLR), Fc
receptors (FcR) and complement anaphylatoxin receptor.
TLR, preferentially expressed on dendritic cells and mac-
rophages, function as molecular sensors that detect path-
ogen-associated molecular patterns, triggering secretion
of chemokines and cytokines to activate innate and adap-
tive immune cells [18]. The roles of diverse TLR in can-
cer immunotherapy have been described in many studies
(reviewed by Pradere et al. [19]). In human breast can-
cer cells, for example, apoptosis was induced by double-
stranded RNA in a TLR3-dependent manner [20]. In mouse
models, immunological antitumor activity was induced
following stimulation of TLR9 by synthetic oligode-
oxynucleotides with unmethylated CpG motifs [21, 22].
Tumor regression and long-term antitumor immunity were
achieved by treating mice with synthetic RNA that was
an agonist of TLR7 and TLR8 [23–25]. In another animal
model, TLR5 activation following flagellin administration
resulted in tumor growth inhibition [26]. Those studies sug-
gest that TLR-mediated antitumor activity involves several
mechanisms, including enabling of the immune response
of CD8(+) T cells by decreasing the number of regula-
tory T cells [27] and reversing the suppressive function
of various types of innate and acquired immune cells [28,
29]. Another mechanism might be based on triggering the
secretion of cytokines involved in the differentiation and
induction of potent adaptive immunity toward tumor anti-
gens [30].
FcR binds the Fc domain of antibodies that are attached
to antigens, enhancing cellular uptake of the complex.
This receptor is expressed on most hematopoietic cells,
e.g., monocytes, macrophages, interferon-γ-activated neu-
trophils, natural killer cells, mast cells and dendritic cells.
Complexes of Fc–FcR may induce an antitumor response
as summarized by Cassard et al. [31]. In some cancers,
following IgG recognition of cancer cells, Fcγ receptor
(FcγR) on IIC triggers an antitumor response [31].
Complement anaphylatoxin receptors are expressed by
several myeloid-derived IIC, including monocytes, mac-
rophages, dendritic cells (DC) neutrophils, basophils,
mast cells and eosinophils. They bind complement compo-
nents, such as C3a and C5a derived from complement cas-
cade activation, mediating chemotaxis and inflammatory
responses [32]. The controversial effects of C3a and C5a,
displaying both pro-tumoral and anti-tumoral responses,
have been reviewed by Sayegh et al. [32].
Recent studies have shown additional effects of com-
binations of ligands from several activation pathways on
immune activation, such as multiple TLR ligands and com-
binations with complement components, antibodies and
mannose [33–35].
The basic idea behind the present study was to activate
immune mechanisms similar to those directed at rejecting
foreign bodies (e.g., pathogens or transplants), with the aim
of “re-educating” tumor-residing IIC to attract cells from
3. 1139Cancer Immunol Immunother (2015) 64:1137–1149
1 3
both the innate and adaptive arms of the immune system
to the TME and activate them toward a tumoricidal effect.
We therefore stimulated TME-residing IIC with inducers
of innate cell activation loaded onto microparticles (MP).
Four distinct routes of activation, utilizing ligands of the
membranous TLR4 [36], TLR7 found on endocytic vesi-
cles [36], complement C5a receptor (C5aR) and FcγR, and
their combinations, were examined for immunological and
anti-tumoral responses.
Materials and methods
Primary splenocytes and cell lines
Single-cell suspensions of splenocytes were prepared from
male C57BL6 mice (8–12 weeks old). Harvested spleens were
dissociated in PBS using gentleMACS™ dissociator (Miltenyi
Biotec, Bergisch Gladbach, Germany). Following erythrocyte
lysis with distilled water, splenocytes were passed through a
70-µm cell strainer (BD Biosciences, Bedford, MA), then
washed with PBS and transferred into culture media.
Mouse melanoma cell line B16-F10, human monocyte
cell line THP-1 and hybridoma-produced mouse mono-
clonal IgG type-1 antibody 44-3A6 were purchased from
the American Type Culture Collection (Rockville, MD).
Mouse dendritic cell line DC2.4 was kindly provided by K.
Rock (Dana-Farber Cancer Institute, Boston, MA).
Splenocytes and 44-3A6 hybridomas were cultured in
RPMI 1640 media supplemented with 10 % fetal calf serum
(FCS: Gibco, Grand Island, NY), 1 % l-glutamate and 1 %
penicillin–streptomycin solution (Biological Industries,
Beit Haemek, Israel). The medium for DC2.4 cells was fur-
ther supplemented with 50 μM β-mercaptoethanol. THP-1
cells were grown in RPMI 1640 media supplemented with
2 % l-glutamate, 1 % sodium pyruvate and 5 % FCS, with-
out antibiotics. B16-F10 cells were grown in DMEM sup-
plemented with 10 % FCS, 2 % l-glutamate, 1 % sodium
pyruvate and 1 % penicillin–streptomycin solution.
Preparation of immune‑stimulating ligands and MP
assembly
Mouse IgG (mIgG) for the stimulation of FcγR was purified
from the supernatant of the 44-3A6 culture, using a protein
G Sepharose®
4B fast flow column (Sigma-Aldrich, Reho-
vot, Israel). Purified mIgG was biotinylated using EZ-Link
Biotin LC sulfo-NHS (Pierce, Appleton, WI), and lipopoly-
saccharide (LPS; L-2654, Sigma-Aldrich) was biotinylated
using EZ-Link biotin LC hydrazide (Pierce), as previously
described [34, 37]. The C5a receptor agonist hexapep-
tide (N-Methyl-Phe)-Lys-Pro-D-Cha-Cha-D-Arg-CO2H,
described in detail by Konteatis et al. [38], was synthesized
with the addition of biotin at the C-terminal position (Gen-
Script, Piscataway, NJ) and was designated C5a-pep. CL264
was purchased and biotinylated from InvivoGen (San Diego,
CA). BioMag®
magnetic particles (Bangslabs, Fishers, IN)
were purchased covalently coated with avidin. To produce
particle complexes, biotinylated inducers LPS, mIgG, C5a-
pep or CL264, individually or in combination, were mixed
with avidin-coated MP for 1 h and then stored at 4 °C. Each
single component was designated to bind at a ratio of ~1/3
of the particles’ biotin-binding capacity as stated by the
manufacturer. Prior to use, the designated MP were taken
out of storage and washed twice with PBS.
THP‑1 cell induction by C5a‑pep
THP-1 cells (105
) were suspended in 1-ml medium and
mixed with 100 µg C5a-pep, or PBS as a control. Escheri-
chia coli bacteria (104
in 100 µl) were then added with gentle
mixing at 37 °C. Samples of 100 µl were withdrawn imme-
diately (0 min), and after 30 and 60 min and chilled on ice.
Samples were centrifuged (300g for 5 min), and supernatant
was seeded on agar plates and incubated for 24 h at 37 °C. E.
coli colony-forming units (CFU) were then counted.
MP induction of cytokine expression determined
by semiquantitative RT‑PCR
DC2.4 cells or splenocytes were incubated overnight in
24-well plates (4 × 106
cell/well). The relevant MP were
added (10 µl from stock) to the wells and incubated for
4 h. Following incubation, medium was discarded and cells
were dissolved in TRI-Reagent®
(Sigma-Aldrich). B16-
F10 tumors were homogenized in TRI-Reagent®
(1 ml for
100 mg of tissue) using gentleMACS™ dissociator.
RNA was further extracted according to the TRI-Rea-
gent®
manufacturer’s protocol. Reverse transcription was
performed for total mRNA using the Verso™ kit protocol
(Fermentas, Vilnius, Lithuania) and oligo dT primers. PCR
was performed for cytokine and chemokine gene products
using DreamTaq Green PCR kit (Fermentas). The primers
used are listed in Supplementary Table 1.
PCR products were separated by electrophoresis on a
2.3 % agarose gel and stained with ethidium bromide. Induc-
tion levels cytokine and chemokine PCR products were ana-
lyzed relative to GAPDH using ChemiDoc XRS+
System
and Quantity One software (both by BioRad, Hercules, CA).
Detection of secreted cytokines and chemokines
in splenocyte culture by enzyme‑linked immunosorbent
assay (ELISA)
Splenocytes (5 × 106
per well) were incubated overnight
in 24-well plates. The relevant MP were then added (10 µl
4. 1140 Cancer Immunol Immunother (2015) 64:1137–1149
1 3
from stock) to the wells and incubated for an additional
16 or 48 h. The medium was collected and centrifuged
(500g for 5 min). The supernatant levels of the secreted
cytokines IL-6, tumor necrosis factor-α (TNF-α) and
IL-1β, and chemokine (C-X-C motif) ligand (CXCL) 1
were measured using a murine cytokine-detecting ELISA
kit according to the manufacturer’s protocol (PeproTech,
Rocky Hill, NJ).
Fluorescence‑activated cell sorting (FACS)
Tumors were dissociated using gentleMACS™ dissociator
as already described and then transferred into FACS buffer
made up of PBS supplemented with 0.1 % bovine serum
albumin and 0.05 % sodium azide. Cells (106
) were stained
with antibodies against CD45 (PE/Cy7), CD3 (APC)
combined with 33D1 (PE), CD49b (Pacific blue), GR-1
(PerCP) and F4/80 (APC/Cy7) or CD19 (Pacific blue),
CD4 (PerCP/Cy5) and CD8 (PE) (eBioscience, San Diego,
CA). FACS procedures were carried out by FACSAria
(Becton–Dickinson, San Jose, CA). The FACS results were
analyzed with FCS express 4 software (De Novo Software,
Los Angeles, CA).
Animal studies
All animal experiments were carried out in accordance
with the guidelines of the Israeli Ethics Committee. Female
C57BL6 mice, 8–10 weeks of age, were kept with free
access to food and water.
B16‑F10 tumor implantation and administration of MP
carrying immune‑stimulating ligands
B16-F10 mouse melanoma cancer cells were cul-
tured, trypsinized and washed twice with PBS. Cells
(4 × 104
) were suspended in 100 μl PBS and injected
subcutaneously into the lateral flank. The volumes of the
developing tumors were measured with a caliper [vol-
ume = (width2
* length)/2]. The relevant MP were diluted
twofold in PBS and injected (100 μl) intratumorally (i.t.).
Evaluation of immune activation in the TME
following single injection of MP carrying
immune‑stimulating ligands
Female C57BL6 mice carrying induced B16-F10 tumors
were injected with MP carrying various inducing ligands
when the tumor reached a volume of 200 ± 50 mm3
. The
mice were sacrificed 48 h post-MP injection. The tumors
were harvested and weighed, then immune cell populations
were determined by flow cytometry, and cytokine expres-
sion was analyzed by semiquantitative RT-PCR.
Evaluation of sequential i.t. injections of MP carrying
immune‑stimulating ligands
Animals carrying induced B16-F10 tumors were treated
with four i.t. injections of MP carrying various induc-
ing ligands. The first injection was administered when the
tumor reached a volume of 50 ± 15 mm3
, followed by three
consecutive injections given at 2- or 4-day intervals. Ani-
mals were killed when they showed distinct signs of mor-
bidity or when the tumor reached a volume of 500 mm3
.
Tumors were harvested and analyzed as already described.
Statistical analysis
The statistical significance of the RT-PCR, ELISA and
FACS results was evaluated by Student’s t test. Correla-
tion coefficients between immune cell population ratios
were analyzed using Pearson’s product-moment correlation
analysis. Kaplan–Meier analysis was performed on mice
from first injection until they showed signs of morbidity or
tumor volume reached 500 mm3
, and log-rank (Mantel–
Cox) test was used to determine the significance of differ-
ences in survival curves.
Results
To influence the immune response in the TME, four induc-
ers of different innate immune activation pathways, loaded
on MP, were tested: the TLR4 ligand LPS, TLR7 ligand
CL264, C5a receptor ligand C5a-pep agonist and FcγR
ligand mIgG.
Determination of cytokine expression induced
by loaded MP
Incubation of DC2.4 cells with CL264-loaded MP resulted
in elevated IL-1β levels, exceeding twofold that of soluble
CL264, with a parallel rise in IL-6 transcription (Supple-
mentary Fig. 1a). Exposure to C5a-pep did not significantly
induce cytokine expression following incubation with any
of the human or mouse immune cell lines tested (data not
shown). However, in THP-1 monocyte cultures incubated
with E. coli, addition of C5a-pep enhanced bacterial phago-
cytosis activity (Supplementary Fig. 1b).
Immune activation of cells by MP was further tested
in a primary culture of mice splenocytes. LPS-loaded MP
dramatically elevated inflammatory cytokines such as
IL-1β, IL-6 and TNF-α, with a rise in the levels of other
associated chemokines, such as CXCL1 (murine analog to
human IL-8), chemokine (C-C motif) ligand (CCL) 3 and
CXCL10 (Figs. 1, 2). In addition, LPS-loaded MP trig-
gered increased levels of the anti-inflammatory cytokine
5. 1141Cancer Immunol Immunother (2015) 64:1137–1149
1 3
IL-10 (Fig. 1c). MP loaded with mIgG as sole inducer only
induced a significant rise in TNF-α secretion, as shown
after 16 h of incubation, with a parallel increasing trend for
IL-1β and IL-6 levels (Fig. 2).
In splenocytes exposed to MP carrying a combination
of mIgG and LPS (IgG–LPS-loaded MP), a significant
increase in IL-6 transcription and secretion of CXCL1 and
TNF-α were recorded after 48 h, although this increase was
significantly lower than that induced by MP loaded only
with LPS (Figs. 1, 2). When splenocytes were incubated
with MP carrying C5a-pep alone or in combination with
mIgG (IgG–C5a-loaded MP), there were no significant
alterations in the transcription levels of any of the tested
cytokines. Nevertheless, exposure to MP loaded with C5a-
pep and LPS with (IgG–LPS–C5a-loaded MP) or without
(LPS–C5a-loaded MP) mIgG decreased the transcription
of the tested chemokines and totally abolished IL-10 tran-
scription relative to exposure to LPS- or IgG–LPS-loaded
MP (Fig. 1c).
Alterations in the immune cell populations in the TME
following i.t. administration of MP
To monitor immune activation by MP carrying different
ligands in vivo in the tumors, a low number of B16-F10
melanoma cells (4 × 104
) were injected s.c. into C57BL6
mice, allowing relatively slow tumor development and
more balanced leukocyte infiltration and activation. MP
were injected into tumors that had reached a volume of
200 ± 50 mm3
. In untreated or nonloaded MP (nMP; MP
with no inducer)-treated mice, a significant negative cor-
relation was observed between tumor developmental stage
(as reflected by tumor volume and weight) and total leuko-
cytes (CD45+
), T cells (CD3+
), cytotoxic T cells (CD8+
)
and natural killer cells (CD49b+
) residing in the tumor
(Fig. 3a). A similar trend was observed in untreated or
nMP-treated mice for the progression of tumor develop-
ment, which was accompanied by a decrease in all immune
cell lineages tested (data not shown). These results could
indicate either declining or stable proportions of immune
cell populations in the tumor as it progresses, resulting in
fewer such cells per unit volume of the tumor. Treatment
of the tumors with inducer-loaded MP completely abol-
ished the correlations between leukocyte number and tumor
progression (Fig. 3b). Notably, the positive correlation
between the percentage of tumor macrophages and neutro-
phils out of the total CD45+
cell population in untreated
and nMP-treated mice switched to a strong negative cor-
relation in mice with inducer-treated tumors (Fig. 3). An
overall change was observed in the balance of immune
Fig. 1 Cytokine transcription
levels in splenocytes promoted
by inducer-carrying MP. Semi-
quantitative RT-PCR of cytokine
levels following the incubation
of mouse splenocytes with (left
to right): nonloaded MP and
MP carrying mIgG, C5a-pep
(C5a), mIgG + C5a, LPS,
mIgG + LPS, C5a + LPS
or mIgG + C5a + LPS. The
normalized expression of IL-6
(a), CXCL10 (b), IL-10 (c)
and CCL3 (d) is presented
(Mean ± SD *P 0.05;
**P 0.01)
0
0.01
0.02
0.03
0.04
LPS
mIgG
C5a
- - - - +++ +
- - + + ++- -
- + - + +-- +
MP composition
IL
)
6-normalized)IL-10(normalized)
CXCL10(normalized)CCL3(normalized)
0
0.5
1.0
1.5
LPS
mIgG
C5a
- - - - +++ +
- - + + ++- -
- + - + +-- +
MP composition
LPS
mIgG
C5a
- - - - +++ +
- - + + ++- -
- + - + +-- +
*
**
0
0.2
0.4
0.6
MP composition
0
0.4
0.8
1.2
1.6
LPS
mIgG
C5a
- - - - +++ +
- - + + ++- -
- + - + +-- +
MP composition
(a) (b)
(c) (d)
7. 1143Cancer Immunol Immunother (2015) 64:1137–1149
1 3
Fig. 3 Immune cell populations
balance shift in tumors follow-
ing treatments with loaded MP.
Shown are correlation curves
demonstrating the relationship
between tumor volume meas-
urements on the day of killing
and (top to bottom): tumor
weight, % of CD45+
cells, % of
CD3+
T cells and % of CD49b+
natural killer cells from total
cell counts. (a) Untreated mice
or mice treated with nonloaded
MP. (b) Mice treated with
inducer-loaded MP. Immune
cells found in the microenviron-
ment were profiled by FACS.
(c) The ratio between F4/80+
macrophages and GR-1+
granu-
locytes, mainly neutrophils,
from the CD45+
cell population
(left and right correlate with a
and b columns)
%CD49boftotal
tumorevents
%CD3oftotal
tumorevents
Tumor
weight(mg)%CD45oftotal
tumorevents
%F4/80oftumor
CD45cells
Tumor volume (mm3)
%GR-1 of tumor CD45
0 200 400 600 800
0
500
1000
1500
r =0.837
P 0.0001
0 200 400 600 800
0
0.5
1.0
1.5
2.0
2.5
r =-0.449
P 0.01
0 200 400 600 800
0
0.2
0.4
0.6
0.8
r =-0.405
P 0.05
0 200 400 600 800
0
0.05
0.10
0.15
r =-0.435
P 0.05
0 5 10 15 20 25
0
20
40
60 r =0.418
P 0.05
0 20 40 60 80
0
20
40
60
r =-0.674
P 0.0001
0 100 200 300 400 500
0
0.05
0.10
0.15
n.s
0 100 200 300 400 500
0
0.2
0.4
0.6
n.s.
0 100 200 300 400 500
0
1
2
3
4
n.s.
0 100 200 300 400 500
0
200
400
600
800
r =0.708
P 0.0001
(a) (b)
(c)
8. 1144 Cancer Immunol Immunother (2015) 64:1137–1149
1 3
populations in the TME as a result of treatments with
inducer-loaded MP. A summary of all altered correlations
found between the tested immune cell populations with the
inducer-loaded MP treatment as compared to untreated and
nMP-treated tumors is presented in Supplementary Fig. 2.
LPS was the most potent mediator for % CD45+
cells in
treated tumors, inducing an over twofold increase in overall
immune cells relative to the nMP treatment (Fig. 4). Com-
bining LPS with C5a-pep, CL264 or mIgG loaded on a MP
did not significantly affect the influence of LPS on CD45+
levels (Fig. 4). The major cell populations that were elevated
in the tumors in response to LPS-loaded MP treatments
were GR-1+
neutrophils (2.8- to 5.6-fold) and F4/80+
mac-
rophages (1.6- to 2-fold) compared to the nMP treatment
(Fig. 4). Treating tumors with MP loaded with C5a, IgG and
C5a, or CL264 did not significantly change leukocyte pop-
ulation counts as compared to the nMP controls. However,
treatment with C5a-pep-loaded MP showed a consistent,
albeit insignificant decrease in T cell (CD3+
) counts (Fig. 4).
B (CD19+
), natural killer (CD49b) and dendritic
(33D1+
) cell populations were detected at extremely low
levels in the TME and were not significantly altered by MP
treatments (data not shown).
Alterations in cytokine and chemokine secretion
in the TME following i.t. MP treatment
The changes in the transcription patterns of cytokines and
chemokines in the TME were determined 48 h after treat-
ment. Increased transcription levels of pro-inflammatory
cytokines IL-1β and IL-6 were found. They seemed to be
primarily influenced by the presence of LPS on the MP
(Fig. 5a, b). LPS as sole inducer on MP caused no change in
IL-10 transcription levels, whereas the combination of C5a-
pep and LPS significantly decreased the transcription of this
cytokine. Injection of C5a-loaded MP mixed 1:1 (v:v) with
LPS-loaded MP resulted in significantly increased transcrip-
tion of IL-10 as compared to nMP (Fig. 5c).
MP composition
-- - -LPS
C5a-pep
CL264
mIgG
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
0
1
2
3
4
CD45
**
***
*
MP composition
-- - -
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
GR-1
0
0.5
1.0
1.5
2.0
*
***
* *
0.5
1.0
1.5
0
2.0
MP composition
-- - -
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
**
***
*
F4/80
MP composition
-- - -LPS
C5a-pep
CL264
mIgG
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
*
CD3
0
0.2
0.4
0.6
0.8
1.0
MP composition
-- - -
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
0
0.2
0.4
0.6
0.8
1.0
CD4
0
0.2
0.4
0.6
0.8
1.0
CD8
MP composition
-- - -
-
-
+
-
- +
+
-
-
+
+
+
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
-
%oftotaltumorevents
Fig. 4 Immune cell population changes in the TME after inducer-
loaded MP injections. FACS analysis for immune cell popula-
tions found in B16-F10 induced tumors after i.t. treatment with
(left to right): nonloaded MP or MP carrying CL264, C5a-
pep, mIgG + C5a-pep, LPS, mIgG + LPS, C5a-pep + LPS or
CL264 + C5a-pep + LPS. Nonloaded MP, CL264-MP, LPS–C5a-MP
and LPS–C5a–CL264-MP groups consisted of 6–9 mice, and other
groups consisted of three mice (Mean ± SD *P 0.05; **P 0.01;
***P 0.001)
9. 1145Cancer Immunol Immunother (2015) 64:1137–1149
1 3
Injections of inducer-loaded MP elevated the levels of
the inflammatory cytokines IL-23 and IL-7 quite signifi-
cantly (Fig. 5d, e). Both IL-23 and IL-7 showed slightly
higher transcription levels in response to LPS combined
with C5a-pep versus LPS alone (Fig. 5d, e). Increased
transcription was also evident for the chemokine CCL5,
though it had relatively high basal transcription levels
also in nMP-injected tumors (Fig. 5f). Transcription of
CXCL10 in tumors injected with inducer-carrying MP
decreased mainly in response to the LPS–C5a-pep combi-
nation. Additional tested cytokines or chemokines (IL-2,
IL-4, IL-12, IL-13 and IL-17) showed extremely low tran-
scription levels, and others (CCL2, CCL3 and CCL4) had
high basal transcription levels with no significant changes
induced by any MP–inducer combination (data not shown).
Altogether, induction of the pro-inflammatory cytokines
and chemokines resulted mainly from the presence of
LPS on the MP. However, of interest were changes, such
as decreased activation of the anti-inflammatory cytokine
IL-10, with the use of LPS–C5a-loaded MP.
Influence of i.t. MP administration on tumor growth
The effect of i.t. MP treatments on tumor development was
primarily evaluated 48 h post-injections (Supplementary
Fig. 3). Tumors injected with LPS-C5a-loaded MP were
significantly inhibited and showed promising molecular
and cellular immune activation (Supplementary Fig. 3,
Figs. 4, 5). To evaluate the potential effects of LPS-C5a-
loaded MP on tumor development, mice were treated with
four consecutive injections of MP every 2 or 4 days. Tumor
volume measurements showed a delay in tumor growth in
mice treated with LPS–C5a-loaded MP starting approxi-
mately 4 days after the first administration (Fig. 6a). Tumor
volume 11 days after the first treatment was 36 and 24 %
smaller when treated every 2 or 4 days, respectively, with
LPS–C5a-loaded MP, relative to tumors treated every
2 days with nMP (Fig. 6a). Distinct massive areas of dis-
coloration were seen on the excised tumors treated with
LPS–C5a-loaded MP under both treatment schedules, as
opposed to the uniform black melanin-rich appearance of
the B16-F10 tumors (Fig. 6b). This seems to evidence mas-
sive infiltration of leukocytes into the tumor mass. Analysis
of distinct immune cell populations showed a significantly
higher presence of leukocytes (CD45+
) in the TME fol-
lowing treatments with LPS–C5a-loaded MP as compared
to treatment with nMP. Leukocyte infiltration into the
TME was predominantly composed of GR-1+
neutrophils
(Fig. 6c–e). Mice treated under both treatment schedules
with LPS–C5a-loaded MP also exhibited significantly pro-
longed survival with the tumors, as compared to control
mice treated with either PBS or nMP under the two sched-
ules or mice with tumors treated with LPS-loaded MP or
IgG–LPS-loaded MP (Fig. 6f).
0.0
0.2
0.4
0.6
**
***
*
IL-10(normalized)
nMP
LPS-MP
LPS-C5a -MP
C5a-MP + LPS-MP
0.0
0.1
0.2
0.3
0.4
0.5
*
***
IL-7(normalized)
*
IL-6(normalized)
0.0
0.1
0.2
0.3
0.4
0.0
0.2
0.4
0.6
0.8
1.0
**
*
CCL5(normalized)
0.0
0.2
0.4
0.6
0.8
*
IL-1β(normalized)
0.00
0.02
0.04
0.06
0.08
0.10
*
IL-23(normalized)
0.0
0.2
0.4
0.6
0.8
1.0 *
CXCL10(normalized)
(a) (b) (c)
(f)(e)(d)
(g)
Fig. 5 Influence of i.t. MP treatments on cytokine expression. Lev-
els of cytokine mRNA were determined by semiquantitative RT-
PCR in tumors harvested 48 h after the injection of nonloaded MP
(nMP), LPS-MP, LPS–C5a-MP or a 1:1 volumetric mix of LPS-MP
and C5a-MP. The normalized expression of IL-1β (a), IL-6 (b), IL-10
(c), IL-23 (d), IL-7 (e), CCL5 (f) and CXCL10 (g) is presented. Each
group consisted of six mice (Mean ± SD *P 0.05; **P 0.01;
***P 0.001)
10. 1146 Cancer Immunol Immunother (2015) 64:1137–1149
1 3
Discussion
The use of specific TAA as targets in cancer immuno-
therapy is limited due to tumoral immune escape mecha-
nisms as a result of antigen loss [39, 40], down-regula-
tion of MHC class I presentation [40–43], suppression of
immune effector cell activation by the expression of sur-
face immune checkpoint ligands [4], secretion of down-
regulatory cytokines [44, 45] and recruitment of cells
and molecules that support tumor growth and suppress
the immune response [46]. To overcome these numerous
obstacles, the immune cells in the TME should be re-
directed to produce an antitumor response. Such effects
could derive from TLR activation of the TME-residing
IIC against the tumor [19]. An advantage of innate activ-
ity is that it does not rely on the recognition of TAA to
elicit the immune response, thus minimizing the possibil-
ity of tumor escape [47].
The basic approach presented in this study was to
induce an immunological environment at the tumor site
that mimics activation toward pathogen rejection and
thereby induce acute inflammatory response and destruc-
tive processes in the TME. To test this concept, various
combinations of four different ligands of distinct IIC acti-
vation pathways were loaded on MP, which served as an
optimal platform for their delivery in numerous possible
combinations [48].
MP carrying multiple copies of ligands combinations
were shown to promote multiple signal transduction events,
mediated by specific receptor cross-linking [49]. Consid-
erations in choosing MP size, shape and composition were
previously discussed [34]. In this study, single ligands or
their different combinations were attached to ~1.5-µm
iron oxide MP. The ligand’s ability to activate an immune
response in vitro after biotin and MP conjugation was
retained.
0 1 2 3 4
0.0
0.5
1.0
1.5
P 0.0001
r =0.9263
%CD45 (of Total events)
%GR-1(oftotalevents)
nMP LPS-C5a-MP
Every 2 days
Every 4 days
nMP every 2 days
LPS-C5a-MP every 2 days
LPS-C5a-MP every 4 days
%GR-1+(ofTotalevents)
%CD45+(ofTotalevents)
0 5 10 15 20
0
50
100
***
***
4SBP days
nMP 2days
nMP 4days
LPS-MP 4days
IgG-LPS-MP 4days
LPS-C5a-MP 2days
LPS-C5a-MP 4days
Days post first injection
Survival(%)
(a)
(d) (e) (f)
(b) (c)
Fig. 6 Effect of MP treatments on tumor growth rate. B16-F10
tumors induced s.c. were treated with four injections every 2 days
of nonloaded MP (nMP) or LPS–C5a-MP, or four injections every
4 days of PBS, nMP, LPS-MP, IgG–LPS-MP or LPS–C5a-MP.
(a–e) Comparison of i.t. treatments of nMP with LPS–C5a-MP. (a)
Tumor volume measurement calculated over 11 days. (b) Tumors har-
vested after reaching 500 mm3
(arrows demonstrate extensive white
regions of the tumor mass). (c) Correlation analysis demonstrating
relationships between CD45+
and GR-1+
granulocyte, mainly neu-
trophil, populations in tumors injected with LPS–C5a-MP. (d and e)
Flow cytometry results showing immune population analysis of (d)
%GR-1+
and (e) % of total CD45+
leukocytes population in tumors
(Mean ± SD **P 0.01). (f) Kaplan–Meier analysis of tested mice
induced with B16-F10 tumors and treated i.t. with MP. % Survival
is defined as percentage of mice within a group with tumor volume
smaller than 500 mm3
. Days were counted from first MP administra-
tion (at tumor volume of 50 mm3
). The nMP (2 days) and LPS–C5a-
MP groups consisted of eight mice, and other groups consisted of 3–4
mice (*P 0.05; ***P 0.001)
11. 1147Cancer Immunol Immunother (2015) 64:1137–1149
1 3
Two of the four ligands tested in this study, LPS and
CL264, which activate TLR4 and TLR7, respectively, both
recognize pathogen-associated molecular patterns [30, 50,
51]. The third ligand tested, IgG, is recognized by FcγR
which may induce phagocytosis, antibody-dependent cell
cytotoxicity or cytokine secretion [31]. The fourth ligand
tested, a synthetic C5a-pep, activates C5a receptor, which
may induce secretion of reactive oxygen species (ROS),
as well as chemotaxis and proliferation of immune cells.
Notably, C5aR can activate the NF-κB signal pathway in
monocytes or suppress this pathway in neutrophils [52, 53].
Evidence for the reciprocal influence of these receptors
activation, e.g., C5aR activation that regulates differential
expression of FcγR members [54], suggests that ligand
combinations may impact overall cellular activity.
In this study, immune activation by sole ligands, or
their combinations, loaded on MP was tested in vitro and
in vivo. In vitro induction of cytokine expression patterns
was tested both in cultured cell lines and in cultured iso-
lated splenocytes. LPS-loaded MP induced acute pro-
inflammatory cytokines and chemokines expression (e.g.,
IL-1β and TNF-α) accompanied by elevated expression of
the anti-inflammatory cytokine IL-10, which is considered
to play a major role in cancer evasion from immune-based
therapies [6]. However, MP co-loaded with C5a and LPS
induced expression of inflammatory cytokines, identical to
that promoted by LPS alone, but this combination blocked
the expression of the anti-inflammatory IL-10.
The effect of i.t. administration of ligands bound MP on
innate immune activation in vivo in the TME of melanoma
was tested 48 h after treatment. Cell populations, as well
as cytokine and chemokine expression patterns, were deter-
mined in parallel to the short-term follow-up of the tumor
growth. A prominent phenomenon observed was the change
from positive correlation between macrophages and neutro-
phils to a negative one in the treated tumors. The expres-
sion of the pro-inflammatory cytokines and chemokines,
accompanied by an increase in CD45+
immune cell popu-
lations, mainly composed of macrophages and neutrophils,
was mostly influenced by the presence of LPS on the MP.
Other combinations of inducers loaded on MP such as C5a
solely did not influence these immune parameters.
Elevated levels of IL-23 and IL-7, both inhibitors of the
regulatory T cell activation [55, 56], following i.t. treatment
with MP loaded with LPS and C5a, may have contributed
to the decrease in IL-10 levels, though barely detectable
transcription levels were assayed in vitro (data not shown).
This may imply that other mechanisms are also involved in
this response.
The high expression levels of the chemokine CXCL10,
as reported in numerous cancer types (e.g., melanoma,
ovarian carcinoma and multiple myeloma) [57], which
we observed in the tumors from the negative control of
nMP-treated mice, were significantly reduced following
treatment with LPS–C5a-loaded MP.
The combined activation of TLR4 and C5aR seemed to
induce a large-scale inflammatory response in the TME, as
seen by a sole activation of TLR4’s, while repressing paral-
lel induction of the tumor-beneficial anti-inflammatory reg-
ulating molecules, and possibly, tissue-repair factors (e.g.,
VEGF). Dual receptor induction, resulting in the repression
of the anti-inflammatory cytokine IL-10, while maintaining
all other inflammatory features, implies redirection toward
a “classical” immune activation, capable of tumor eradica-
tion. Notably, activation of these receptors in the TME, fol-
lowing injection of a mixture of MP, each bound to a dif-
ferent ligand, was associated with elevation of IL-10 levels,
indicating the importance of co-binding of these ligands to
the same MP. Additionally, short-term tumor volume meas-
urements indicated inhibition of tumors treated with LPS-
C5a-loaded MP, which was not seen following treatment
with the mixture of MP each bound to a different single
ligand. Thus, simultaneous activation of the same leuko-
cyte with both ligands seems to have an advantage over the
effect of mixed MP, each bound to separate inducers.
The unique pattern of cytokine expression following
repeated i.t. treatments with LPS–C5a-loaded MP, in par-
ticular the reduction in IL-10, was accompanied by the
observation of discolored sections on the excised tumors.
The loss of the tumor color, possibly as result of reduc-
tion in melanin levels, probably due to massive infiltra-
tion of leukocytes, may indicate inflamed or necrotic
areas in the TME. These observations are supported by
increased CD45+
cell populations, predominantly the
GR-1+
neutrophils, which were detected in these tumors’
microenvironments.
The described changes in cellular and molecular con-
tents in the TME following LPS–C5a-loaded MP treat-
ments resulted in significant inhibition of tumor growth as
compared to two LPS-MP treatments and controls (PBS
or nMP). This delay in tumor progression following LPS–
C5a-loaded MP treatment, accompanied by the induc-
tion of immune activation, may be explained by the inhi-
bition of the anti-inflammatory agents (e.g., IL-10) effect
on activated cells, disallowing their “misdirection” toward
immune tolerance. Other pro-tumoral factors, such as
VEGF and TGF-β, may also be down-regulated and should
be explored.
Our results show the possibility of changing tumor–
immune system homeostasis in the TME and influenc-
ing the composition and activation of residing immune
cell populations. The “polarization” of tumor-residing IIC
toward a “classical” pathogen-aggressive response may be
accomplished by appropriately activating the inflamma-
tory response while restraining the parallel tissue-repair
activity of the activated cells. The possibility of this effect
12. 1148 Cancer Immunol Immunother (2015) 64:1137–1149
1 3
expanding to the activation of IIC in distal metastases
deserves further investigation.
Our results support accumulated evidence suggesting
that treating tumors with a single IIC-activating agent,
e.g., TLR ligand, followed by an inflammatory response
is not sufficient for successful immunotherapy. On the
other hand, activation of IIC toward a tumoricidal immune
response may be achieved by combining ligands originat-
ing from several innate activation routes (e.g., complement
receptor together with TLR activation). Such combinations
displayed enhanced efficacy or an altered activation profile
as compared to single ligand activity [33–35, 58–60].
In conclusion, our study suggests that cells exist in the
TME with the ability to eliminate tumors needs to be re-
directed toward anti-tumoral responses. The current study
shows the feasibility of attracting immune cells and induc-
ing an acute inflammatory response in the TME, while
suppressing the pro-tumoral activity of the IIC, by using
a combination of different inducers ligands carried on the
same MP.
Conflict of interest The authors declare that they have no conflict
of interest in publishing this research.
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TAM Tumor-associated macrophages
TAN Tumor-associated neutrophils
TGF-β Transforming growth factor-β
TLR Toll-like receptors
TME Tumor microenvironment
TNF-α Tumor necrosis factor-α
VEGF Vascular endothelial growth factor
Introduction
Advances in immunotherapy of cancer have given rise to
promising new therapies of which only a selected few have
been applied clinically. A major strategy focuses on the
activation of CD8 T lymphocytes, relying on the presenta-
tion of tumor-associated antigens (TAA) by cancer cells [1,
2]. However, anti-tumoral immune activation toward TAA
may be evaded by down-regulation or alteration in these
cancer cell antigens [3]. Another major obstacle to the
antitumor immune response is the presentation by cancer
cells of inhibitory regulators of immune checkpoints, e.g.,
programmed death ligand l, together with the induction of
immune tolerance by molecules secreted into the tumor
microenvironment (TME) [3–6]. The TME contains a mul-
titude of innate immune cells (IIC) which, in normal tissue,
play a crucial role in monitoring tissue homeostasis, pro-
tecting against pathogens and eliminating damaged cells;
thus, they seem qualified to eliminate cancer cells [7, 8].
However, IIC found in the TME, such as tumor-associated
macrophages (TAM), tumor-associated neutrophils (TAN)
and myeloid-derived suppressor cells (MDSC), have been
shown to contribute to tumor survival, propagation and
metastatic spread [5, 8–11]. These “alternatively” activated
tumor-associated IIC have been shown to promote angio-
genesis, tumor scaffolding and regeneration by secreting
factors such as vascular endothelial growth factor (VEGF)
and transforming growth factor-β (TGF-β) together with
immunosuppressive molecules that restrict antitumor acti-
vation, such as interleukin (IL)-10 [5, 7, 12–14].
In spite of the above findings, TAM may still regain
cytotoxicity toward tumor cells and switch to a “classi-
cally” activated phenotype following “re-education” [15].
Polymorphonuclear neutrophils have been found to con-
tribute to both tumor rejection and tumor promotion [11,
16]. Pro-cancerous “polarization” of these cells may be
switched to antitumor behavior by defined inducers [17],
and this may alter the nature of the interaction between the
tumor and the immune system in the TME, thereby ena-
bling the desired immunological responses.
Various molecules have been shown to enhance anti-
tumor responses by inducing immune cells via various
types of receptors, including Toll-like receptors (TLR), Fc
receptors (FcR) and complement anaphylatoxin receptor.
TLR, preferentially expressed on dendritic cells and mac-
rophages, function as molecular sensors that detect path-
ogen-associated molecular patterns, triggering secretion
of chemokines and cytokines to activate innate and adap-
tive immune cells [18]. The roles of diverse TLR in can-
cer immunotherapy have been described in many studies
(reviewed by Pradere et al. [19]). In human breast can-
cer cells, for example, apoptosis was induced by double-
stranded RNA in a TLR3-dependent manner [20]. In mouse
models, immunological antitumor activity was induced
following stimulation of TLR9 by synthetic oligode-
oxynucleotides with unmethylated CpG motifs [21, 22].
Tumor regression and long-term antitumor immunity were
achieved by treating mice with synthetic RNA that was
an agonist of TLR7 and TLR8 [23–25]. In another animal
model, TLR5 activation following flagellin administration
resulted in tumor growth inhibition [26]. Those studies sug-
gest that TLR-mediated antitumor activity involves several
mechanisms, including enabling of the immune response
of CD8(+) T cells by decreasing the number of regula-
tory T cells [27] and reversing the suppressive function
of various types of innate and acquired immune cells [28,
29]. Another mechanism might be based on triggering the
secretion of cytokines involved in the differentiation and
induction of potent adaptive immunity toward tumor anti-
gens [30].
FcR binds the Fc domain of antibodies that are attached
to antigens, enhancing cellular uptake of the complex.
This receptor is expressed on most hematopoietic cells,
e.g., monocytes, macrophages, interferon-γ-activated neu-
trophils, natural killer cells, mast cells and dendritic cells.
Complexes of Fc–FcR may induce an antitumor response
as summarized by Cassard et al. [31]. In some cancers,
following IgG recognition of cancer cells, Fcγ receptor
(FcγR) on IIC triggers an antitumor response [31].
Complement anaphylatoxin receptors are expressed by
several myeloid-derived IIC, including monocytes, mac-
rophages, dendritic cells (DC) neutrophils, basophils,
mast cells and eosinophils. They bind complement compo-
nents, such as C3a and C5a derived from complement cas-
cade activation, mediating chemotaxis and inflammatory
responses [32]. The controversial effects of C3a and C5a,
displaying both pro-tumoral and anti-tumoral responses,
have been reviewed by Sayegh et al. [32].
Recent studies have shown additional effects of com-
binations of ligands from several activation pathways on
immune activation, such as multiple TLR ligands and com-
binations with complement components, antibodies and
mannose [33–35].
The basic idea behind the present study was to activate
immune mechanisms similar to those directed at rejecting
foreign bodies (e.g., pathogens or transplants), with the aim
of “re-educating” tumor-residing IIC to attract cells from](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)