Blockade of myeloid-derived suppressor cell expansion with ... · 5 In vivo tumor models and treatments: 4T1 or TS/A cells (5 x 105) were orthotopically injected into the second mammary

1

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.

Research. on September 11, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 19, 2018; DOI: 10.1158/0008-5472.CAN-17-3415

http://cancerres.aacrjournals.org/

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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




11

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




13

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




14

(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.




15

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




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




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




18

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.




19

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.




20

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23

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




24

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




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.




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-




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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Figure 1

B C

D E

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




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

*




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]

*

*

*

* *


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




Figure 4

CONTROL ATRA DC101 DC101/ATRA




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


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




E

Figure 6

F

A B C


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


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

* *

*





Published OnlineFirst April 19, 2018.Cancer Res Raimund Bauer, Florian Udonta, Mark Wroblewski, et al. therapy

anti-angiogenicall-trans retinoic acid increases the efficacy of Blockade of myeloid-derived suppressor cell expansion with

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