blockade of myeloid-derived suppressor cell expansion with ... · 5 in vivo tumor models and...
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Blockade of myeloid-derived suppressor cell expansion with all-trans retinoic acid
increases the efficacy of anti-angiogenic therapy
Raimund Bauer *1,2,7, Florian Udonta *1,2, Mark Wroblewski 1,2, Isabel Ben-Batalla 1,2, Ines Miranda Santos 4,5, Federico Taverna 4,5, Meike Kuhlencord 3, Victoria Gensch 1,2, Sarina Päsler 1,2, Stefan Vinckier 4,5, Johanna M. Brandner 6, Klaus Pantel 2, Carsten Bokemeyer 1,
Thomas Vogl 3, Johannes Roth 3, Peter Carmeliet 4,5, Sonja Loges 1,2
1) Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumorzentrum, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. 2) Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg. 3) Institute of Immunology, University of Muenster, Muenster, Germany. 4) Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KULeuven, Leuven, B-3000, Belgium. 5) Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Leuven, B-3000, Belgium. 6) Department of Dermatology and Venerology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. 7) Current address: Department of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Währingerstraße 10, 1090 Vienna, Austria.
* Both authors contributed equally to this work
Running title: Targeting of MDSC increases efficiency of VEGFR-2 inhibitors
Keywords: Anti-angiogenic therapy, Myeloid-derived suppressor cells, vessel normalization, All-trans retinoic acid, S100A8
Further information: 4968 words, 6 Figures, 1 Table, 9 Supplementary Figures Editorial correspondence: Prof. Sonja Loges, M.D., Ph.D.
Department of Oncology, Haematology and BMT with section
Pneumology, Hubertus Wald Tumorzentrum & Department of
Tumor Biology, University Medical Center Hamburg-Eppendorf,
Martinistrasse 52, D-20246, Hamburg, Germany
tel: 49-40-7410-51962; fax: 49-40-7410-56546
e-mail: [email protected]
Conflicts of interest:
S. Loges receives research funding, advisory board honoraria, speaker honoraria and travel
support from Roche, Lilly, Sanofi Aventis and Boehringer Ingelheim. M. Wroblewski received
advisory board honoraria and travel support from Lilly.
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ABSTRACT
Intrinsic and adaptive resistance hamper the success of anti-angiogenic therapies (AAT),
especially in breast cancer where this treatment modality has proven largely ineffective.
Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such
as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells
(MDSC) which are key drivers of resistance to AAT. Therefore, we hypothesized that all-
trans retinoic acid (ATRA), which induces differentiation of MDSC into mature cells, could
improve the therapeutic effect of AAT. ATRA increased the efficacy of anti-VEGFR-2
antibodies alone and in combination with chemotherapy in preclinical breast cancer models.
ATRA reverted the anti-VEGFR-2-induced accumulation of intratumoral MDSC, alleviated
hypoxia, and counteracted the disorganization of tumor microvessels. Mechanistic studies
indicate that ATRA treatment blocked the AAT-induced expansion of MDSC secreting high
levels of vessel-destabilizing S100A8. Thus, concomitant treatment with ATRA holds the
potential to improve AAT in breast cancer and possibly other tumor types.
INTRODUCTION
The vascular endothelial growth factor receptor-2 (VEGFR-2)-blocking antibody ramucirumab
received regulatory approval for the treatment of patients with gastric, colorectal and non-
small cell lung cancer. VEGFR-2 is frequently overexpressed in breast cancer and might
therefore represent a therapeutic target in this tumor entity (1,2). However, responses of
patients to blockade of VEGFR- or VEGF-signaling turned out to be very limited. Therefore,
the treatment of breast cancer patients with anti-angiogenic agents represents a relevant
clinical challenge (3,4).
Previous studies identified bone marrow (BM) derived CD11b+GR1+ murine myeloid-derived
suppressor cells (MDSC) as resistance conferring, detrimental mediators accumulating in
tumors upon treatment with anti-angiogenic therapies (AAT) (5,6). MDSC comprise a
heterogeneous population of immature, myeloid cells including G-MDSC
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(CD11b+Ly6G+Ly6Clow) and M-MDSC (CD11b+Ly6G-Ly6C+) subsets capable of inducing
angiogenesis and immunosuppression (6,7).
Tumors induce mobilization and recruitment of MDSC from the BM by secreting mediators
including G-CSF or GM-CSF (8,9). In addition, therapy-induced physiological adaptations of
the tumor microenvironment have been associated with increased MDSC expansion and
recruitment. In particular, intratumoral hypoxia has been well recognized as one of the main
drivers triggering this process (10,11). Reduced oxygen tension, resulting from rapid tumor
growth or blood vessel eradication upon AAT, leads to the induction of tumor hypoxia.
Hypoxia in turn favours the expression of tumor-derived factors such as CCL2, CXCL5 and
CXCL12/SDF-1, VEGF, and PLGF leading to enhanced recruitment of MDSC into the tumor
bed (6,11,12).
Once residing in the tumor, MDSC suppress T-cell mediated anti-tumor responses (13,14)
and induce tumor angiogenesis by various mechanisms. For example, MDSC secrete
proteinases such as MMP-9 that induce the mobilization of pro-angiogenic molecules
residing in the extracellular matrix of the tumor microenvironment (6,11). Furthermore, MDSC
express VEGF and FGF-2 in a STAT3-dependent manner resulting in enhanced tumor
neovascularization and growth (6,15).
The active vitamin A metabolite all-trans retinoic acid (ATRA) is currently used to induce
differentiation of leukemic blasts into mature myeloid cells in acute promyelocytic leukemia
(16). Importantly, ATRA also enhances the differentiation of MDSC into macrophages and/or
dendritic cells in vitro. In addition, treatment of tumor-bearing mice with ATRA resulted in a
significant reduction of MDSC in vivo (17,18). Therefore, we hypothesized that combinatorial
treatment with ATRA could improve the efficacy of AAT via reducing resistance-conferring
MDSC.
Our data using two syngeneic murine breast cancer models show that ATRA increases the
anti-tumor activity of AAT by a concomitant reduction of MDSC levels. Moreover, our work
provides evidence that MDSC-secreted S100A8 represents a resistance-conferring factor
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induced by AAT. S100A8 acts by destabilization of the tumor vasculature, which can be
reverted by combining AAT with ATRA.
MATERIALS AND METHODS
Animals: Female 8 to 9-week-old BALB/c mice were purchased from Charles River
Laboratories International (Sulzfeld, Germany). All animal experiments were carried out in
concordance with the institutional guidelines for the welfare of animals and were approved by
the local licensing authority Hamburg (project number G36/13 and G126/15). Housing,
breeding and experiments were performed under standard laboratory conditions (22 ± 1 °C,
55% humidity, food and water ad libitum).
Cells and culture conditions: Murine mammary adenocarcinoma cell lines 4T1 and TS/A
were provided by Prof. Peter Carmeliet (VIB Vesalius Research Center, KU Leuven) and
cultured in RPMI 1640 or DMEM medium supplemented with 10% fetal calf serum (FCS), 2
mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, respectively. Human
umbilical vein endothelial cells (HUVEC, Lonza) were cultured in EBM-2 medium
supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin
and a complete set of EGM-2 growth factors (Lonza). Cells were maintained at 37°C and 5%
CO2 in a humidified atmosphere and routinely tested to be mycoplasma negative (Venor
GeM Classic, Minerva Biolabs). Cells were cultured no longer than 15 passages before
experimental use. No cell line used in this study is listed in the ICLAC database of commonly
misidentified cell lines and were authenticated according to their in vitro / in vivo growth
characteristics and histology. To analyze the effect of S100A8 on HUVEC, cells were
washed 2 times with PBS and seeded in 96-well plates (1.5 x 104 cells / well) in EBM-2
medium containing 2% FCS and indicated concentrations of S100A8 for 48 hours. Cell
viability was determine using WST-1 reagent (Roche).
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In vivo tumor models and treatments: 4T1 or TS/A cells (5 x 105) were orthotopically
injected into the second mammary fat pad of 8-9 week old syngeneic female BALB/c mice.
When tumors reached 100 mm3, mice were randomized and treated either with ATRA (7.5
mg/kg, daily), DC101 (20 or 10 mg/kg, three times per week) or a combination of both drugs
by i.p. administration. Doxorubicin (3 mg/kg, i.p.) was administered 2 times per week. Tumor
size was measured with a digital caliper and the volume was calculated using the formula V=
(length2 x width) / 2. For histological analyses, BrdU (1 mg, i.p), pimonidazole (1 mg, i.p.) and
a FITC-conjugated lectin (0.05 mg, i.v.) were injected 12 hours, 2 hours and 10 min before
sacrifice, respectively.
Tumor digestion and generation of a single cell suspension: Tumor tissue (300-500 mg)
was mechanically minced and digested in 15 ml RPMI 1640 medium containing 0.2 mg/ml
collagenase A (Roche) for 60 min at 37°C and shaking at 80 rpm. Next, 10 ml of a PBS
solution containing 0.015 mg/ml DNase I (Roche) was added and digestion was continued
for another 30 min at 37°C. Cell suspensions were filtered through a 70 µm cell strainer.
After centrifugation, cells were resuspended in FACS buffer (2% FCS, 1 mM EDTA, 0.1%
NaN3 in PBS) and immediately used for flow cytometry.
Flow cytometry: For flow cytometry analysis, cells (1 x 106 / staining) were Fc-blocked (anti-
mouse CD16/32 antibody, Biolegend) for 15 min at 4°C. Afterwards, cells were stained for 40
min with the following fluorochrome-conjugated primary anti-mouse antibodies: PE-Cy7
CD11b (clone M1/70 BD Bioscience), PE Ly6-G (clone 1A8 BD Bioscience), PerCP-Cy5.5
Ly6-C (clone Hk1.4, eBioscience), FITC F4/80 (clone BM8, Biolegend), APC-Cy7 Gr-1 (clone
RB6-8C5, Biolegend), APC CD3 (clone 17A2, eBioscience), eFluor 450 CD8a (clone 53-6.7,
eBioscience), PE CD49b (clone DX5, eBioscience), PE-Cy7 NKp46 (clone 29A1.4,
eBioscience). DAPI was used as a viability stain. Samples were acquired using a BD FACS
Canto II flow cytometer and data were analyzed using the BD FACS Diva software.
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Immunohistochemistry and histology: All methods for histology and immunostaining have
been described in detail in (19,20). Tumor samples were fixed overnight in 4%
paraformaldehyde at 4°C and embedded in paraffin or further incubated overnight in 40%
sucrose and embedded in OCT medium for cryo-sectioning. Paraffin sections (4 µm) were
stained with primary antibodies to detect vessel number and vessel proliferation (anti-CD105,
R&D Systems, AF1320 + anti-BrdU, Abd Serotec, MCA2060), tumor hypoxia (pimonidazole,
HP3-1000kit; anti-GLUT1, Abcam, ab115730), vessel number, perfusion and permeability
(anti-CD105, R&D Systems, AF1320 + FITC-lectin, Vector Laboratories, FL-1171), and
vessel associated ZO-1 (anti-CD105, R&D Systems, AF1320 + anti-ZO-1 clone ZO-1-1A12,
Invitrogen). Cryosections (8 µm) were stained and analyzed for pericyte coverage (anti-
CD31, Dianova, DIA-310 + anti-NG2, AB5320 Merck) of tumor microvessels. For the analysis
of tumor cell proliferation, tumor sections were stained with an anti-phosphohistone H3
antibody (clone D7N8E Cell Signaling). Sections were then incubated with the corresponding
HRP- or fluorescently conjugated secondary antibodies. Nuclei were counterstained with
DAPI. For morphometric analysis 8-10 optical fields per tumor section were acquired using a
Zeiss Axio Scope A1 for immunohistochemistry or a Leica DM1000 fluorescence microscope
for immunofluorescence analysis. Images were analyzed using the NIH Image J analysis
software.
Scanning electron microscopy: SEM imaging to assess the intratumoral microvessel
architecture was performed as previously described (20,21) (see also Supplementary
Information).
Generation of conditioned media: 1.5 x 106 4T1 cells were seeded in T-75 cell culture
flasks and incubated in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-
glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin for 3 days to generate tumor cell
conditioned media (TCM). TCM was sterile filtered using 0.2 µm filters, supplemented with
10 mM HEPES and 20 µM ß-mercaptoethanol and stored at -80 °C until further use.
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Generation of in vitro MDSC and co-culture with HUVEC: Bone marrow was isolated
under sterile conditions from WT BALB/c mice and subjected to erythrocyte lysis (155 mM
NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH7.4) for 2 min at 4°C. Primary bone marrow
mononucleated cells (BMMC) were adjusted to a density of 0.5 x 106 cells / ml in 75% 4T1
conditioned medium + 25% RPMI medium supplemented with 10% FCS, 10 ng/ml GM-CSF,
20 µM ß-Mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100
µg/ml streptomycin. Cells were treated with or without 1.5 µM ATRA for 4 days at 37°C / 5%
CO2 following purification of MDSC using the myeloid-derived suppressor cell isolation kit
(Miltenyi). For co-culture assays, 6 x 104 HUVEC were seeded in the lower compartment of a
24 transwell chamber using inserts with 0.4 µm pore size. 2 x 105 MACS-purified MDSC
were seeded in the upper compartment and co-culture was performed for 48 hours. HUVEC
viability was assessed using WST-1 reagent (Roche).
Endothelial permeability assay: Permeability across endothelial cell monolayers was
measured using matrigel-coated transwell filters (3 µm pore size, Greiner). HUVEC cells
were seeded on two consecutive days at a density of 4 x 104 cells per well (upper chamber)
and were further cultured for 24 h. Afterwards, cells were incubated for 6 hours in the
presence of human S100A8 (10µg/mL) or human VEGFA (200ng/mL). FITC-dextran
(1 mg/ml, 3 kDa; Molecular Probes) was added to the lower compartment of the transwell
system and permeability was measured by its diffusion into the upper compartment (485nm
excitation 535 nm emission, Tecan infinite F200 Pro).
Statistics: Data represent mean ± SEM of representative experiments, unless otherwise
stated. To compare the means of two groups, an unpaired, two-tailed student’s t-test was
used. Pairwise comparison testing in experiments with more than two groups was performed
using one-way ANOVA. Pairwise comparisons of tumor growth kinetics were performed
using two-way ANOVA. Statistical significance was assumed when p<0.05.
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RESULTS
ATRA increases the anti-tumor effect of DC101.
In order to investigate our hypothesis that treatment with ATRA increases the efficacy of anti-
angiogenic drugs by reducing MDSC numbers, we combined ATRA with DC101, a
monoclonal antibody targeting murine VEGFR-2. For the combinatorial treatment approach
we utilized two well-characterized syngeneic models of breast cancer, 4T1 and TS/A (8,22).
We injected the cell lines orthotopically in the second mammary fat pad of BALB/c mice and
started treatment when the mean tumor burden reached 100 mm3 (Fig. 1A). We deliberately
chose a submaximal dose level of DC101 (20 mg/kg, 10 mg/kg) to detect potential additive
effects of ATRA treatment. The dose level of ATRA (7.5 mg/kg) was utilized because
previous studies with similar concentrations showed biological activity without the presence
of side effects (23,24). In both models, ATRA slightly but not significantly reduced tumor
growth (Fig. 1B-E) in concordance with literature (23). Interestingly, the combination of
DC101 and ATRA induced an additive reduction of tumor volume and weight when
compared to DC101 monotherapy in the 4T1 and in the TS/A model (Fig.1B and C;
Supplementary Note 1; Supplementary Fig. S1A and B). In the TS/A model, the additive
effect on tumor weight was only significant when 10 mg/kg DC101 was used in combination
with ATRA (Supplementary Fig. S1A and B). This additive therapeutic effect was maintained
during a treatment period of 18 days and after the discontinuation of treatment the mice
survived for 6 more days compared to placebo treatment (Supplementary Fig. S1A-C and
S2). Accordingly, tumor cell proliferation, measured by phospho-histone H3+ (pHH3+) nuclei,
was significantly reduced upon combination of ATRA and DC101 compared to control- and
DC101-treated tumors (Fig. 1F, G, H).
These results collectively indicate that the addition of ATRA increases the anti-tumor activity
of the VEGFR-2 targeting antibody DC101 in murine syngeneic breast cancer models.
ATRA alleviates DC101-induced hypoxia and abrogates the accumulation of MDSC.
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It is well-known that anti-angiogenic drugs increase hypoxia upon chronic treatment (11,25),
which represents an important driver of MDSC-recruitment and a resistance-conferring factor
(10,11). In concordance with previous data, treatment with DC101 significantly increased
pimonidazole positive areas, a surrogate for intratumoral hypoxia, while monotherapy with
ATRA did not modify hypoxia (Fig. 2A and B). Interestingly, the addition of ATRA to DC101
treatment almost completely alleviated intratumoral hypoxia (Fig. 2A and B; Supplementary
Fig. S1C). Notably, staining of the alternative hypoxia marker GLUT1 by
immunohistochemistry yielded similar results (Supplementary Fig. S3A and B).
Consequently, we investigated the effects of ATRA, previously shown to reduce MDSC in
tumor-bearing mice (17,18), on intratumoral MDSC populations using flow cytometry. These
analyses revealed that in accordance with the increase in hypoxia, both
CD11b+Ly6G+Ly6Clow G-MDSC and CD11b+Ly6G-Ly6C+ M-MDSC were significantly elevated
in tumors treated with DC101 (Fig. 2C-F). Concomitant treatment with ATRA normalized the
frequencies of both MDSC subsets in DC101-treated tumors to similar levels as detected in
control-treated tumors (Fig. 2C-F). However, ATRA had no impact on intratumoral
frequencies of CD8+ cytotoxic T cell and natural killer cell populations (Supplementary Fig.
S3C and D).
To characterize the angiogenic phenotype of intratumoral MDSC upon treatment, FACS-
isolated MDSC were subjected to qPCR expression analysis using a panel of pro-angiogenic
genes. These experiments revealed that in the G-MDSC subset, the expression of pro-
angiogenic Vegfa, Hgf, Mmp-9 and iNos was not significantly altered by any of the
therapeutic interventions (Supplementary Fig. S4A). However, in the M-MDSC fraction,
Mmp-9 mRNA levels were increased in DC101 treated tumors, which was blunted by the
addition of ATRA (Supplementary Fig. S4B).
ATRA promotes tumor vessel normalization and maturation.
Changes in tumor oxygenation have been shown to be associated with modifications of the
tumor vasculature (26). Our findings that i) concomitant treatment with ATRA alleviated
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tumor hypoxia and ii) ATRA reduced the frequencies of MDSC, which can act pro-
angiogenic, prompted us to analyze the vessel phenotype and functionality in tumors treated
with DC101 with and without ATRA.
As expected, DC101 reduced the microvessel density of tumors, which was not further
decreased upon combination with ATRA (Fig. 3A and B). By injecting a FITC-labeled lectin,
we observed that DC101 reduced microvessel perfusion, which was reverted by addition of
ATRA, leading to an increase in the relative number of functional vessels (Fig. 3C). Next, we
quantified the fraction of proliferating vessels after injection of BrdU. These analyses
revealed an increase of BrdU+ vessels upon DC101 treatment compared to control treatment
as previously described (27), while ATRA monotherapy had no effect (Fig. 3D). Interestingly,
the combination of DC101 and ATRA blunted the re-induction of tumor vessel proliferation
(Fig. 3D). Moreover, leakage of FITC-lectin from tumor vessels was significantly increased in
DC101-treated tumors compared to controls, whereas the addition of ATRA reduced the
DC101-evoked vascular permeability (Fig. 3E).
The coverage of blood vessels with pericytes represents an important attribute of their
maturity and functionality (28). The quantification of NG2+ pericytes showed a significant
reduction of pericyte-covered vessels upon DC101 treatment (Fig. 3F and G). In contrast,
ATRA monotherapy resulted in an increase of NG2+ cells adjacent to CD31+ endothelial cells
(EC) in comparison to control-treated tumors, which was maintained upon combination with
DC101 (Fig. 3F and G). Together, our histomorphometric analyses revealed an increase of
mature, functional tumor microvessels upon combination of DC101 with ATRA.
In order to further investigate the blood vessel architecture at the ultrastructural level we
performed intratumoral scanning electron microscopic (SEM) imaging. These analyses
revealed a disorganized blood vessel architecture in the DC101-treated tumors as indicated
by irregular-shaped vessel walls and EC extensions protruding into the vessel lumen in
concordance with previous literature (20,21) (Fig. 4). In contrast, ATRA and the combination
of ATRA with DC101 induced a normalization of the tumor vessel morphology indicated by
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lower abundance of protrusions and a regular, flat cobble-stone morphology of the
endothelial monolayer (Fig. 4).
Vascular normalization and consecutive alleviation of tumor hypoxia might be correlated with
remodeling of the extracellular matrix and/or changes in the tumor metabolome. Analysis of
key extracellular matrix components including fibronectin 1 (Fn1), EDA-Fn1, EDB-Fn1, IIICS-
Fn1, collagens 1A-4A (Col1A - 4A), secreted protein and rich in cystein (Sparc) and periostin
(Postn) revealed a decrease in mRNA levels of Fn1 and its splice variants EDA-Fn1 and
IIICS-Fn1 upon DC101 administration, which was abrogated by the addition of ATRA, while
the other matrix components were essentially unchanged (Supplementary Fig. S5A and B).
Interestingly, LC-MS based metabolomic analysis revealed that, compared to controls,
tumors treated with DC101 showed (a trend of) elevated glycolytic intermediates, which was
partially abrogated by the addition of ATRA (Table 1).
Collectively these data indicate that ATRA reverts the DC101-induced vessel destabilization
phenotype ultimately leading to increased tumor vessel functionality, reduced hypoxia and a
decrease in the glycolytic capacity of the tumor, which could explain the observed decrease
in tumor cell proliferation (Fig. 1F, G and H).
ATRA reduces S100A8 levels by counteracting tumor-induced MDSC expansion.
The capacity of MDSC to secrete pro-angiogenic, vessel-destabilizing factors is well-
recognized (5,15,29). Therefore, we hypothesized that the reduced number of MDSC upon
treatment with ATRA might be one cause for the normalization of the DC101-induced vessel
phenotype. In order to elucidate which MDSC-derived angiogenic mediators are reduced
upon treatment with ATRA we utilized an in vitro MDSC culture system. Therefore, we
incubated primary mouse bone marrow mononucleated cells (BMMC) with 4T1 breast cancer
tumor cell-conditioned medium (TCM), which led to an efficient expansion of G-MDSC and
M-MDSC populations (Fig. 5A). Importantly, and to demonstrate their functionality, MDSC
generated with 4T1 TCM showed potent inhibitory capacity in T-cell proliferation assays as
shown for CD8+ and CD4+ T-cell subsets (Supplementary Fig. S6A and B).
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The treatment of TCM-stimulated BMMC with ATRA (1.5µM) decreased the frequencies of
G- and M-MDSC compared to DMSO-treated control mimicking our in vivo findings (Fig. 5B
and C). Conversely, treatment with ATRA increased a CD11b+Ly6G-Ly6C- non-MDSC
population, which was mainly comprised of CD11b+GR1-F4/80+ Macrophages (Fig. 5D and
E) with reduced T-cell suppressive activity compared to the MDSC-population
(Supplementary Fig. S6C and D).
We next asked whether the blockade of MDSC expansion with ATRA holds potential to
reduce MDSC-derived vessel-destabilizing mediators. Therefore, we compared S100A8,
S100A9, HGF, VEGFA, FGF-1 and FGF-2 levels in supernatants from magnetic-activated
cell sorting (MACS)-separated MDSC with those secreted from the CD11b+Ly6G-Ly6C- cell
population, which expands upon ATRA treatment (Fig. 5A and D). Here, we found that
S100A8 was secreted much more efficiently from the MDSC population when compared to
the CD11b+Ly6G-Ly6C- cells (Fig. 5F). In contrast, HGF and VEGFA were secreted to a
lower extent from MDSC compared to the CD11b+Ly6G-Ly6C- fraction. Secretion of S100A9
and FGF-2 did not show differences between both populations, whereas FGF-1 was
undetectable (Fig. 5F). Importantly, ATRA did not change S100A8 secretion levels neither in
CD11b+Ly6G-Ly6C- cells nor in MDSC (Fig. 5G). However, MDSC efficiently secreted
S100A8, whereas the protein was almost not secreted from the CD11b+Ly6G-Ly6C-
population (Fig. 5G). These data further underline our hypothesis that ATRA might indirectly
affect the bioavailability of S100A8 by reduction of MDSC frequencies. Accordingly, DC101-
treated animals showed a 3-fold increase in S100A8 protein levels in tumor lysates, which
was normalized upon administration of ATRA to similar values as observed in control-treated
animals (Fig. 5H). Analysis of the plasma concentration of S100A8 showed similar, but
smaller effects of DC101 and ATRA (Fig. 5I).
Based on these results, we focused on S100A8, a small calcium-binding protein well
described for its MDSC-chemoattractive properties (30,31). Moreover, S100A8 and its
heterodimeric partner S100A9 trigger the activation of EC for efficient phagocyte recruitment
at sites of inflammation (32). This includes the induction of adhesion molecule expression
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and the reduction of EC integrity by downregulating the expression of tight junction proteins
such as ZO-1 (33). S100A8 was previously described to be the active component of the
S100A8/S100A9 heterodimer and exerts effector capacity also in its monomeric form in vitro
(34,35). Accordingly, we hypothesized that S100A8 could mediate MDSC-induced vessel
destabilization observed in the DC101 monotherapy setting. Therefore, we next investigated
the effects of S100A8 on EC integrity and barrier function.
S100A8 reduces EC integrity and correlates with vessel leakiness.
In a first step, we incubated human umbilical vein endothelial cells (HUVEC) with increasing
concentrations of purified S100A8 protein. These experiments revealed that recombinant
S100A8 reduced the viability of HUVECs in a dose-dependent manner (Fig. 6A). Next, we
investigated the effects of MDSC-derived S100A8 on HUVEC and performed trans-well co-
culture assays with MDSCs differentiated from the bone marrow of WT- or S100A9 KO mice,
which essentially lack S100A8 (36). In these co-cultures, HUVEC viability was increased in
the presence of S100A8/A9-deficient MDSC in comparison to WT MDSC (Fig. 6B).
Permeability assays indicated that S100A8 increased the leakiness of a confluent HUVEC
monolayer to a similar extent as VEGF, measured by the diffusion of a 3 kDa FITC-labeled
dextran (Fig. 6C). Accordingly, intratumoral S100A8 levels were positively correlated with
FITC-lectin leakage from tumor microvessels (Supplementary Fig. S7A).
A reduction of EC viability and enhanced vascular leakiness is often associated with a loss of
tight junction proteins, which are essential for endothelial integrity (26,33). We therefore
quantified the EC-associated tight junction protein ZO-1 in tumor sections of mice treated
with DC101 alone or in combination with ATRA. Thereby, we observed a significant reduction
of ZO-1 protein in response to DC101 treatment, which could be normalized upon
combination with ATRA (Fig. 6D, E). Moreover, S100A8 protein levels and ZO-1
fluorescence intensities showed an inverse correlation among all treatment groups (Fig. 6F).
Of note, incubation of HUVEC with increasing concentrations of ATRA did not lead to an
increase in ZO-1 levels, thus a direct effect of ATRA on the endothelial phenotype is unlikely
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(Supplementary Fig. S7B). These observations identified S100A8 as a potential MDSC-
secreted candidate driving tumor microvessel destabilization.
To further substantiate our findings, we performed bone marrow (BM) transplantations with
S100A9 KO bone marrow to investigate DC101 efficiency in the absence of S100A8 in
MDSC (S100A8 KO mice are not viable and S100A9 KO mice are also described to lack
S100A8 (36), Supplementary Fig. S8A). S100A8 was absent before tumor inoculation in
S100A9 KO transplanted mice (Supplementary Fig. S8B and C). However, we observed a
complete recovery of S100A8 in blood plasma and the bone marrow of S100A9 deficient
tumor-bearing mice (Supplementary Fig. S8B, C, D, E and F). These results indicate a so far
unknown mechanism of S100A9-independent S100A8 secretion elicited by presence of
tumors. Therefore, we did not observe differences in the efficacy of DC101 in mice
transplanted with WT versus S100A9-deficient BM (Supplementary Fig. S8G).
To functionally validate the ATRA-mediated vascular normalization in vivo, we combined
treatment with DC101 and ATRA with the chemotherapeutic drug doxorubicin (Doxo). Here,
the monotherapies of DC101 and Doxo and the combination DC101/Doxo exerted similar
anti-tumor effects while the triple combination of DC101/Doxo/ATRA showed a more
pronounced reduction in tumor volume and weight, indicating enhanced cytotoxic activity of
Doxo in the presence of DC101 and ATRA (Fig. 6G and H).
Collectively, the data indicate that DC101 monotherapy gives rise to a hypoxic tumor
microenvironment that triggers the infiltration of S100A8-secreting MDSC, eventually causing
the loss of blood vessel stability and integrity. The combination of DC101 with ATRA reverts
the AAT-induced accumulation of MDSC, resulting in decreased intratumoral S100A8 levels.
Thereby, ATRA triggers the normalization of the tumor vasculature and alleviates tumor
hypoxia, which leads to an overall increase of the anti-tumor activity of AAT and
chemotherapy.
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DISCUSSION
Sustained treatment with AAT has been previously reported to increase tumor hypoxia by
pruning intratumoral vessels (37). Tumor hypoxia in turn is well recognized to trigger a
disorganized vessel phenotype and therapy resistance by inducing uncoordinated rescue
angiogenesis amongst other mechanisms (10,38). Furthermore, hypoxia and vessel
leakiness lead to enhanced recruitment of MDSC into tumors (10,11), which have been
described as one of the major resistance-conferring cell populations accumulating in tumors
upon treatment with AAT (7,11,12). However, therapeutic approaches targeting MDSC in
combination with AAT have not been reported so far.
The findings of this study show that ATRA blocks the DC101-induced increase of S100A8-
producing MDSC in experimental breast cancer, which translated into vascular normalization,
alleviation of intratumoral hypoxia and a reduction in the glycolytic activity of the tumor.
Consequently, we observed improved therapeutic efficacy of the VEGFR-2 blocking antibody
DC101 alone and in combination with chemotherapy. The withdrawal of the treatment
resulted, as expected, in faster tumor growth (39,40), but the survival of the mice was
prolonged by six days. This indicates a clinically relevant treatment effect and the necessity
for continuous treatment which reflects clinical practice in oncology (4,41,42).
Our data show a novel mechanism for counteracting AAT-induced vessel disorganization, an
approach that can be useful in many therapeutic settings when AAT is applied, especially in
combination with chemotherapeutic drugs. Therefore, our data, pending clinical validation,
are relevant both from the therapeutic but also mechanistic perspective.
In our breast cancer models, ATRA abrogated the accumulation of MDSC in tumor tissues
(Fig. 2C-F). Moreover, in vitro differentiation assays showed that ATRA blocks MDSC
development. In concordance, published data show that vitamin A-deficient mice exhibit a
substantial expansion of CD11b+GR1+ MDSC in bone marrow and spleen, which could be
reversed upon supplementation of the diet with vitamin A (43). This cytodifferentiating effect
of ATRA seems to be the predominant mechanism of S100A8 reduction, as ATRA had no
direct effect on S100A8 secretion from MDSC. As previous studies identified S100A8 as the
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16
signal-transducing component either in its monomeric form or as the active component of the
S100A8/S100A9 heterodimer (34,35), the capacity of MDSC to efficiently secrete S100A8
might represent an important mechanism of how these cells interact with the tumor
vasculature on a molecular level.
Accordingly, in our tumor models high S100A8 levels correlated with a loss of the tight
junction protein ZO-1 from tumor blood vessels, which represents an important component in
maintaining endothelial barrier function and vascular integrity (44). The AAT-induced
increase of S100A8 might therefore trigger a chronic activation of tumor EC that eventually
leads to vessel destabilization. Whereas a direct effect of ATRA on ZO-1 protein levels in
HUVEC is unlikely (Supplementary Fig. S7B), ATRA mediates vessel normalization mainly
via reducing MDSC, one of the predominant cell populations secreting S100A8 in the tumor
microenvironment.
MDSC are located in close proximity to tumor blood vessels, which renders an important
influence on EC very likely (6). We identified MMP-9 as another candidate factor upregulated
in M-MDSC upon treatment with AAT, capable of driving tumor (rescue) angiogenesis via its
capacity to liberate matrix-bound pro-angiogenic factors such as VEGF, amongst other
mechanisms ((6,10,11) and Supplementary Fig. S4B). Interestingly, ATRA was able to
counteract the DC101-induced expression of Mmp-9, which represents another potential
mechanism of how ATRA exerts its vessel normalizing effects.
Our data indicate that the ATRA-mediated reduction of MDSC frequencies and the
concomitant vascular normalization provides a microenvironment that causes slower tumor
growth, which could be explained by the following scenarios: First, vessel normalization and
improved vessel functionality might translate to a more efficient distribution of DC101 to sites
of active angiogenesis and tumor growth, thereby blocking the development of immature
vessels. Second, ATRA counteracts the DC101-mediated increase in the glycolytic activity of
the tumors. These findings are in concordance with the ability of ATRA to decrease DC101-
induced hypoxia (a stimulus of glycolysis, Fig. 2A and B) and expression of glucose
transporter 1 (GLUT1, Supplementary Fig. S3A and B), that allows enhanced glucose uptake
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17
to fulfil the high energetic demands of fast proliferating, anabolic tumor cells. Moreover,
glycolysis generates metabolic intermediates that are required for nucleotide, amino acid and
fatty acid biosynthesis that support the proliferation of cancer cells (45). Therefore, addition
of ATRA might diminish the proliferation of breast cancer cells by decreasing their glycolytic
capacity (45,46).
Beside the important role of MDSC, mast cells and cancer associated fibroblasts (CAF)
represent important mediators of resistance against AAT because they can secrete pro-
angiogenic mediators besides the VEGF axis (27,47,48). However, in the current study
neither DC101 nor ATRA or the combination had an impact on Vegfa or Fgf-1/2 expression
in CAF sorted from 4T1 breast cancer tissue. Whereas Fgf-1 and Fgf-2 were not detectable,
the combination of DC101 and ATRA increased Vegfa expression in mast cells
(Supplementary Fig. S9A and B). To get a more comprehensive understanding on the role of
mast cells and CAF in mediating potential resistance towards the DC101/ATRA treatment
regimen, future investigations are warranted.
Pericyte-deficiency has been shown to induce an increased transmigratory and infiltrative
potential of MDSC, which is accompanied by a defective tumor vasculature and an increased
hypoxic tumor microenvironment (49). In our study, we observed a pronounced effect of
ATRA treatment on the pericyte coverage of tumor vessels. The cellular origin of pericytes is
still incompletely understood, however, one possibility is that pericytes arise and/or share
functional plasticity with mesenchymal stem cells residing in the proximity of blood vessels
(28). Considering its differentiation-inducing capacity, interaction of ATRA with pericyte-
precursors such as mesenchymal stem cells might lead to the expansion of NG-2 positive
cells, which could subsequently act in a vessel maturating and protective manner.
Our observations that DC101 treatment in the long-term setting induces vessel
destabilization are in concordance with literature (19,27,50). In contrast, previous studies
indicate that AAT can also induce vessel normalization due to the neutralization of excessive
amounts of pro-angiogenic factors (26,50). This vascular normalization increased the
delivery and efficacy of cytotoxic chemotherapeutic agents and radiation therapy and is
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therefore highly desirable (50). However, preclinical data show that the vascular
normalization window is limited to a rather short interval of up to approximately 8 days.
Afterwards, this effect declined and the vessel-pruning effect of AAT again predominated
(50). Clinical data indicate that AAT enhances the efficacy of chemotherapy in concordance
with vessel normalization leading to improved delivery of cytotoxic therapy. However, even
though a significant fraction of patients initially benefits, these responses are rarely durable
indicating a therapeutic need to enhance the vessel normalization window. Of note, the
addition of ATRA increased the anti-tumor activity of doxorubicin when combined with DC101
(Fig. 6G, H), which might represent an approach for sustained vascular stabilization leading
to enhanced efficacy of chemotherapeutic drugs.
Our study shows that the addition of ATRA leads to enhanced vessel functionality over the
course of long-term AAT. Therefore, combinatorial treatment with ATRA holds promise to
increase efficacy of AAT alone and in combination with chemo- or radiotherapy, both of
which are less effective in hypoxic conditions.
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ACKNOWLEDGEMENTS
The Authors would like to thank Stefanie Prien and Ewa Wladykowski for excellent technical
assistance and the FACS Core Facility (UKE, Hamburg, Germany) for helping with flow
cytometry. S. Loges was supported by the Max-Eder group leader program from the German
Cancer Aid. She is the recipient of a Heisenberg professorship from the German Research
Council (DFG, LO1863/4-1) and is funded by the Margarethe Clemens Stiftung. R. Bauer
received an Erwin-Schrödinger postdoctoral fellowship from the Austrian Science Fund
(FWF, J3664-B19). F. Udonta received a Werner Otto fellowship from the Werner Otto
foundation. M. Wroblewski was supported by the Medical Faculty of the University of
Hamburg (FFM program). The work of P. Carmeliet is supported by the VIB TechWatch
program, a Federal Government Belgium grant (IUAP7/03), long-term structural Methusalem
funding by the Flemish Government, grants from the Research Foundation Flanders (FWO-
Vlaanderen), Foundation against Cancer (2012-175 and 2016-078), Kom op Tegen Kanker
(Stand up to Cancer, Flemish Cancer Society) and ERC Advanced Research Grant (EU-
ERC743074). K. Pantel was supported by European Research Council Investigator Grant
"DISSECT" (no. 269081). T. Vogl and J. Roth were supported by Grants of the German
Research Foundation (DFG) CRC 1009 B8 and B9 and by the Federal Ministry of Education
and Research (BMBF), project AID-NET and E-RARE, Treat-AID to J. Roth.
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Table 1. Metabolomic analysis of 4T1 tumor tissue using LC-MS. The concentration of
glycolytic and tricarboxylic acid (TCA) cycle intermediates is shown in fold change (log2)
values of (A) control vs DC101 (B) control vs DC101/ATRA and (C) DC101 vs DC101/ATRA.
Gray squares, metabolite increases compared to reference (e.g. in control vs DC101, the
metabolite would be increased in the DC101 treatment condition). White squares, metabolite
decreases compared to reference. *p<0.05. The fold change (log2) values were calculated
with the BIOMEX™ software suite using the limma R package.
A B C
CTRL vs DC101 CTRL vs DC101/ATRA DC101 vs DC101/ATRA
AMP -1,634 * -0,858 0,776
ATP 1,561 1,317 -0,244
glucose-6-phosphate 0,650 0,349 -0,301
fructose-6-phosphate 0,789 0,378 -0,411
dihydroxyacetonephosphate 1,349 * 0,582 -0,766 *
glyceraldehydephosphate 0,340 0,100 -0,240
phosphoglycerate 0,970 0,154 -0,816 *
phosphoenolpyruvate 1,754 0,917 -0,836 *
pyruvate -0,346 -0,769 * -0,423
lactate 0,150 -0,155 -0,305
oxoglutarate -0,103 0,278 0,381
glutamate -0,044 -0,180 -0,137
malate 0,044 -0,071 -0,115
oxaloacetate 0,963 1,232 0,269
aspartate -0,349 -0,389 -0,040
Gly
coly
sis
T
CA
Cycle
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FIGURE LEGENDS
Figure 1. ATRA increases the therapeutic efficiency of DC101 in 4T1 and TS/A breast
cancer bearing mice. (A) Graphical overview of the experimental design and treatment
schedule for the DC101/ATRA combination treatment approach. Animals in the control group
received placebo treatments for DC101 (rat IgG) and ATRA (peanut oil). DC101 (20mg/kg)
was administered 3 x / week, ATRA (7.5 mg/kg) was given 1 x daily. (B) Syngeneic 4T1
breast cancer cells (0.5x106) were orthotopically injected into the second mammary fat pad of
BALB/c mice. When the tumors reached a size of 100 mm3, mice were randomized and
treated with either control, DC101 (20 mg/kg, 3 x / week), ATRA (7.5 mg/kg, daily) or a
combination of both compounds. Tumor growth kinetics were determined using a digital
caliper (n=7/5/7/6; *p<0.05; two-way ANOVA). (C) 4T1 tumor weight at experimental end
stage (n=10/9/7/10; *p<0.05; unpaired t-test). (D) Growth kinetics of a syngeneic TS/A breast
cancer tumor model in BALB/c mice as described in A (n=8/10/8/10; *p<0.05; two-way
ANOVA). (E) TS/A tumor weight at experimental end stage (n=8/10/8/10; *p<0.05; one-way
ANOVA). (F) Representative images of control-, DC101- (20mg/kg), ATRA- (7.5mg/kg), and
DC101/ATRA-treated 4T1 tumor sections immunohistochemically stained for pHH3+
proliferating tumor cells. Images were acquired using a Zeiss Axioscope with a 10x objective.
Scale bar, 200µm. (G, H) Quantification of proliferating tumor cells per mm2 of sectional area
in 4T1 and TS/A tumors, respectively (F, n=8/9/8/7; G, n=8/10/8/10; *p<0.05; one-way
ANOVA).
Figure 2. ATRA decreases DC101-induced intratumoral hypoxia and MDSC
frequencies. (A) Representative images showing hypoxic, pimonidazole+ areas (brown
colour) of control-, DC101- (20mg/kg), ATRA- (7.5mg/kg) and DC101/ATRA-treated 4T1
tumor sections. Pictures were acquired with a Zeiss Axioscope using a 10x objective. Scale
bar, 200 µm. (B) Hypoxic areas were quantified and displayed as percentage of the total
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25
tumor area using the Zeiss Axiovision Software. (C, D) Frequencies of G- and M-MDSC
populations were determined using flow cytometry of an enzymatically digested single cell
suspension of 4T1 tumor tissue (n=8/8/8/6; *p<0.05; one-way ANOVA). (E, F) Flow
cytometric analysis of G- and M-MDSC frequencies in dissociated TS/A tumor cell
suspensions (n=8/10/8/10; *p<0.05; one-way ANOVA).
Figure 3. Combinatorial treatment of DC101 with ATRA induces the normalization of
tumor microvessels. (A) Representative immunofluorescence images from paraffin sections
of tumor tissues of all four treatment groups stained for the blood vessel marker endoglin
(CD105; white) and BrdU (red). Perfused, functional vessels are displayed as FITC-lectin+
tumor vessels (green, arrows). Scale bar, 100 µm. (B) Histomorphometric analysis of
microvessel density in control-, DC101- (20mg/kg), ATRA- (7.5mg/kg), and DC101/ATRA-
treated 4T1 tumors. (C) Quantification of FITC-lectin+ blood vessels. (D) Quantification of
BrdU+ proliferating tumor microvessels. (E) Quantification of the overall FITC-lectin+ area
normalized to the microvessel density as a readout for vascular leakage (B - D, n=7/5/7/6; E,
n=8/9/8/7; *p<0.05; one-way ANOVA). (F) Representative immunofluorescence images of
control-, DC101- (20mg/kg), ATRA- (7.5mg/kg), and DC101/ATRA- treated 4T1 tumor
sections stained for blood vessels (CD31, green) and the pericyte marker NG2 (red). Scale
bar, 100µm. (G) Histomorphometric quantification of pericyte-covered microvessels
presented as a percentage of total vessels of the overall sectional area (n=7/5/7/6; *p<0.05;
one-way ANOVA).
Figure 4. ATRA counteracts the DC101-mediated structural destabilization of tumor
microvessels. Scanning electron micrographs (SEM) showing luminal tumor blood vessel
architecture of control-, DC101- (20mg/kg), ATRA- (7.5mg/kg), and DC101/ATRA-treated
end stage 4T1 tumors. Scale bar upper panels, 10 µm; scale bar bottom panels, 5 µm.
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26
Figure 5. ATRA decreases the expansion of S100A8-producing MDSC in vitro and
reduces systemic and intratumoral S100A8 levels in vivo. BMMC were incubated for 4
days in 4T1 TCM in the presence of 1.5 µM ATRA or DMSO. (A) Representative flow
cytometric plot of in vitro differentiated MDSC. Black box, MDSC fraction comprising G-
MDSC and M-MDSC populations. Green box, Ly6G-Ly6C- non-MDSC population. (B)
Frequencies of G-MDSC, (C) M-MDSC, (D) Ly6G-Ly6C- and (E) macrophage populations
were determined using flow cytometry (n=3/3; *p<0.05; unpaired t-test). (F) ELISA detecting
proangiogenic factors in supernatants of MACS-isolated MDSC and Ly6G-Ly6C- cell fractions
incubated for 48 h in 4T1 TCM (n=3/3; *p<0.05; unpaired t-test). (G) MACS isolated MDSC
and Ly6G-Ly6C- cell fractions were treated with either ATRA (1.5 µM) or DMSO for 48 h and
secreted S100A8 was measured in supernatants using ELISA (n=3/3; *p<0.05; one-way
ANOVA). (H) Tumor and (I) plasma S100A8 levels were determined with ELISA from
endstage 4T1 breast cancer bearing mice treated with either control, DC101 (20mg/kg),
ATRA (7.5mg/kg) or a combination of DC101/ATRA (n=7/5/7/6; *p<0.05; one-way ANOVA).
Figure 6. S100A8 reduces endothelial cell viability, induces vascular permeability and
correlates with a loss of the vessel-associated tight junction protein ZO-1, which can
be reverted by ATRA. (A) S100A8 dose-dependently reduces the viability of HUVEC after
48 hours of incubation (n=3/3 per time point). (B) Indirect co-cultures of HUVEC with MDSC
isolated from either WT or S100A9 KO mice (n=3/3). (C) Endothelial cell permeability assay
using S100A8 (10µg/ml) or VEGF (200ng/ml; n=3/3/3; A, B, C, *p<0.05; unpaired T-test). (D)
Representative immunofluorescence images of control-, DC101- (20mg/kg), ATRA-
(7.5mg/kg), and DC101/ATRA-treated 4T1 tumor sections stained for blood vessels
(Endoglin / CD105, green) and ZO-1 (red). Scale bar, 100µm. (E) Histomorphometric
analysis of the fluorescence intensity of vessel associated ZO-1 protein in the respective
treatment groups (n= 8/9/8/7; *p<0.05; one-way ANOVA). (F) Correlation of S100A8 levels in
tumor lysates with fluorescence intensities of the vessel associated tight junction protein ZO-
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27
1 (Pearson correlation, r = -0.9617, p=0.0383). (G) Syngeneic 4T1 breast cancer cells
(0.5x106) were orthotopically injected into the second mammary fat pad of BALB/c mice.
When the tumors reached a size of 100 mm3, mice were randomized and treated with either
control, DC101 (20 mg/kg, 3 x / week), Doxorubicin (Doxo, 3 mg/kg, 2 x / week), a
combination of both compounds (DC101/Doxo) or a triple combination of DC101/Doxo/ATRA
(7.5 mg/kg, daily). Tumor growth kinetics were determined using a digital caliper
(n=7/6/8/8/7; *p<0.05; two-way ANOVA). (H) 4T1 tumor weight at experimental end stage
(n=7/6/8/8/7; *p<0.05; one-way ANOVA).
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0
10
20
30
40
50
0
20
40
60
0 3 6 9 12 150
500
1000
1500
Figure 1
B C
D E
Tumor volume Tumor weight
Tumor volume Tumor weight
Control
ATRA (7.5 mg/kg) DC101 (20 mg/kg)
DC101/ATRA
+ + - - + - - + DC101
ATRA
Tum
or
volu
me
[m
m3]
Days of treatment
Tum
or
weig
ht
(g)
0.0
0.5
1.0
1.5
* *
0 3 6 9 120
200
400
600
800
1000
Tum
or
volu
me
[m
m3]
* *
Days of treatment
0.0
0.5
1.0
1.5
Tum
or
weig
ht
(g)
* *
p=0.66
+ + - - + - - + DC101
ATRA
Control
ATRA (7.5 mg/kg)
DC101 (20 mg/kg)
DC101/ATRA
* *
* *
*
DC101
ATRA
Control
DC101/ATRA
G H 4T1
pH
H3
+ c
ells
/ m
m2
* *
*
*
TSA
* *
*
- + + - -
+ - + DC101 ATRA
* ns
pH
H3
+ c
ells
/ m
m2
F
- + + - -
+ - + DC101 ATRA
Tumor cell
injection
Rando-
mization
Days of treatment: 0 2 4 7 9 11 14
End of
experiment
8-9 d ATRA (1 x daily i.p.)
DC101 (3 x / week i.p.)
A
16
4T1
TS/A
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0.0
0.5
1.0
1.5
2.0*
*
*
0
2
4
6
8
C D E F Tumor G-MDSC Tumor M-MDSC Tumor G-MDSC Tumor M-MDSC
+ + - - + - - + DC101
ATRA + + - - + - - + DC101
ATRA
0
5
10
15*
*
% G
-MD
SC
% M
-MD
SC
+ + - - + - - + DC101
ATRA
% G
-MD
SC
* *
*
0.0
0.2
0.4
0.6
0.8
% M
-MD
SC
+ + - - + - - + DC101
ATRA
*
* *
Control DC101 ATRA DC101/ATRA
*
4T1
Figure 2
0
5
10
15
Control
ATRA
DC101
A
B
DC101/ATRA
+ + - - + - - + DC101
ATRA
Tumor hypoxia
Hypoxic
are
a [
%]
*
*
*
*
*
TS/A
*
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0
50
100
150
200
0
2
4
6
8
0
10
20
30
40
Figure 3
B C
D
A
Perfusion BrdU CD105
DC101
ATRA DC101/ATRA
Control
0
50
100
150
Microvessel density
Mic
rovessels
/ m
m2
* *
*
*
Perf
used
vessels
[%
]
Microvessel perfusion
*
* *
Microvessel proliferation
Pro
lifera
ting
vessels
[%
] *
*
*
*
ns
F
CD31 NG2
G
0
10
20
30
40
Pericyte coverage index
NG
2 c
overe
d
vessels
[%
/ m
m2]
*
*
*
* *
Control DC101 ATRA DC101/ATRA
DC101 Control
DC101/ATRA ATRA
DC101 - + + - -
+ - + ATRA
DC101 - + + - -
+ - + ATRA
DC101 - + + - -
+ - + ATRA
DC101 - + + - -
+ - + ATRA
*
* *
FIT
C-lectin
+ a
rea
(µm
2/m
icro
vessel)
+ + - - + - - + DC101
ATRA
E Microvessel permeability
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Figure 4
CONTROL ATRA DC101 DC101/ATRA
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0
1
2
3
4
5
0
20
40
60
Figure 5
in vitro MDSC
MDSC
Ly6C
Ly6G- Ly6C-
Ly6G
A
F
D
I
B
20
30
40
50
G-M
DS
C [
%]
DMSO ATRA
*
C
0.0
0.1
0.2
0.3
0.4
0.5
M-M
DS
C [%
]
*
DMSO ATRA
20
30
40
50
DMSO ATRA
*
Ly6G
- Ly6C
- [%
]
E
0
10
20
30
40
Macro
phag
es [
%]
DMSO ATRA
*
Fold
change
cyto
kin
e s
ecre
tion
Ly6G- Ly6C-
MDSC *
* *
S100A8 S100A9 FGF2 HGF VEGFA
Secretion of proangiogenic cytokines
Pla
sm
a S
100
A8 [
ng
/ml]
*
*
+ + - - + - - + DC101
ATRA
Plasma S100A8
0
10
20
30
40
H
Tum
or
S100
A8 [
ng
/ml]
Tumor S100A8
* *
*
+ + - - + - - + DC101
ATRA
Control DC101 ATRA DC101/ATRA
0
20
40
60
80
G
Secre
ted
S100
A8
[p
g /
0.8
x 1
06 c
ells
]
MDSC Ly6G- Ly6C-
DMSO ATRA
* *
Secretion of S100A8
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E
Figure 6
F
A B C
Control DC101 ATRA DC101/ATRA
D
CD105 ZO-1
6000
8000
10000
12000
Via
ble
HU
VE
C /
well
HUVEC viability
S100A8
(µg/ml)
1 2 5 10
Control
S100A8
* * *
40000
45000
50000
55000
60000
65000
Via
ble
HU
VE
C /
well
MDSC co-culture
*
WT MDSC S100A9
KO MDSC
20000
25000
30000
35000
Arb
itra
ry lig
ht
units
* *
Control S100A8 VEGF
HUVEC Permeability
0
5000
10000
15000
Flu
ore
scence
inte
nsity /
RO
I
+ + - - + - - + DC101
ATRA
ZO1 Fluorescence
Control DC101 ATRA DC101/ATRA
8000 10000 12000 140000
10000
20000
30000S100A8 : ZO1
Tum
or
S100
A8 [
pg
/ml]
ZO1 [Fluorescence intensity]
DC101
Control
ATRA
DC101/ATRA
r = -0.9617
*
* *
0.0
0.5
1.0
1.5
0 3 6 9 12 15 180
500
1000
1500
Tum
or
volu
me
[m
m3]
Tum
or
weig
ht
[g]
Days of treatment
Control
DC101 (20 mg/kg; 3x/w)
DC101/Doxo Doxo (3mg/kg; 2x/w)
DC101/Doxo/ATRA
+
- - - + - - + DC101
ATRA + +
Doxo - -
-
+ +
*
*
*
G H
* *
*
Tumor volume Tumor weight
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