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A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype
Accepted Manuscript A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype Xueping Lei, Minfeng Chen, Xiaobo Li, Maohua Huang, Qiulin Nie, Nan Ma, Heru Chen, Nanhui Xu, Wencai Ye, Dongmei Zhang PII:
S0304-3835(18)30038-7
DOI:
10.1016/j.canlet.2018.01.016
Reference:
CAN 13684
To appear in:
Cancer Letters
Received Date: 29 November 2017 Revised Date:
1 January 2018
Accepted Date: 8 January 2018
Please cite this article as: X. Lei, M. Chen, X. Li, M. Huang, Q. Nie, N. Ma, H. Chen, N. Xu, W. Ye, D. Zhang, A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.01.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A vascular disrupting agent overcomes tumor multidrug resistance
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by skewing macrophage polarity toward the M1 phenotype
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Xueping Lei1, 2,#, Minfeng Chen1, 2,#, Xiaobo Li1, 2, Maohua Huang1, 2, Qiulin Nie1, 2,
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Nan Ma1, 2, Heru Chen1, 2, Nanhui Xu1, 2, Wencai Ye1, 2,*, Dongmei Zhang1, 2,*
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Affiliations:
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1
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2
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College of Pharmacy, Jinan University, Guangzhou 510632, China
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University,
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Guangzhou 510632, China
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#: Lei Xueping and Chen Minfeng are co-first authors.
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Corresponding Authors: Dongmei Zhang, College of Pharmacy, Jinan University,
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601 Huangpu Avenue. Wes, Guangzhou 510632, China; Phone: +86-20-85222653;
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E-mail: [email protected]. or Wencai Ye, College of Pharmacy, Jinan
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University, 601 Huangpu Avenue. Wes, Guangzhou 510632, China; Phone:
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+86-20-85220004; E-mail: [email protected].
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Abstract Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC)
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transporters is the major obstacle for chemotherapeutic success. Although attempts
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have been made to circumvent ABC transporter-mediated MDR in past decades, there
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is still no effective agent in clinic. Here, we identified a vascular disrupting agent,
32
Z-GP-DAVLBH, that significantly inhibited the growth of multidrug-resistant human
33
hepatoma HepG2/ADM and human breast cancer MCF-7/ADR tumor xenografts,
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although these cells were insensitive to Z-GP-DAVLBH in vitro. Z-GP-DAVLBH
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increased the secretion of granulocyte-macrophage colony-stimulating factor in tumor
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tissues and serum of tumor-bearing mice to skew tumor-associated macrophages from
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the pro-tumor M2 phenotype to the antitumor M1 phenotype, thereby contributing to
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the induction of HepG2/ADM and MCF-7/ADR cell apoptosis. Our findings shed
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new light on the underlying mechanisms of VDAs in the treatment of drug-resistant
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tumors and provide strong evidence that Z-GP-DAVLBH should be a promising agent
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for overcoming MDR.
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Keywords: multidrug resistance; vascular disrupting agent; tumor-associated
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macrophages; granulocyte-macrophage colony-stimulating factor
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Abbreviations
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MDR: multidrug resistance; ABCB-1: ATP-binding cassette transporter B1; TAM:
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tumor-associated macrophage; VDA: vascular disrupting agent; iNOS: inducible
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nitric oxide synthase; Arg-1: arginase 1; Ly6G: lymphocyte antigen 6 complex locus
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G6D; IFN-β: interferon-β; CA4-P: combretastatin A4 phosphate. GM-CSF:
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granulocyte-macrophage
colony-stimulating
factor;
M-CSF:
macrophage 2
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colony-stimulating
factor;
IL-6:
interleukin-6;
IL-10:
interleukin-10;
LPS:
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lipopolysaccharide; INF-γ: interferon gamma; TNF-α: tumor necrosis factor alpha;
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HIF-1α: hypoxia-inducible factor.
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1. Introduction
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Multidrug resistance (MDR) is the phenomenon in which tumor cells are
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resistance to a wide range of chemotherapeutic drugs, and MDR has been considered
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one of the major reasons for chemotherapeutic failure [1]. The overexpression of
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ATP-binding cassette (ABC) transporters is a major cause of MDR and has a broad
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spectrum effect on the efflux of chemotherapy drugs that are frequently used in clinic,
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such as vinca alkaloids, doxorubicin (DOX) and taxanes [2]. Recently, the
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identification of effective ABC transporter inhibitors to reverse MDR is the major
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therapeutic approach. However, although four generations of ABC transporter
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inhibitors have been emerged and tested in preclinical or clinical trials, non-specific
66
toxicity limits their successful use in circumventing MDR in clinic [3-5]. In addition,
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nano-sized delivery systems, such as liposomal ABC transporter inhibitors, are
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proposed to reduce the non-selective inhibition of ABC transporters; however, these
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strategies also fail to reverse tumor MDR in clinic [6]. Therefore, it is necessary to
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explore new strategies or drugs for overcoming tumor MDR.
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Tumor-associated macrophages (TAMs) are derived from circulating monocytes
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or resident tissue macrophages [7]. TAMs display two main phenotypes, M1 and M2,
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in response to diverse microenvironmental stimuli; M1 macrophages have antitumor
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activity, and M2 macrophages exhibit tumor-promoting effects [8]. During
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chemotherapy, TAMs often polarize to M2 macrophages and then promote tumor
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angiogenesis, metastasis and suppress antitumor immunity, leading to tumor 3
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resistance to chemotherapy [9-13]. Strategies to deplete TAMs, block M2 macrophage
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programming and reprogram macrophages to the M1 phenotype have been proposed
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to augment the efficacy of chemotherapy [14-16]. The inhibition of tumor angiogenesis has been considered a potential strategy for
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treating tumors with MDR [17, 18], as its targets are endothelial cells (ECs), not
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cancer cells [19], and EC genome is relatively stable compared with that of cancer
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cells [20]. Tumor vessel disruption is the other important strategy for targeted
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treatment. Vascular disrupting agents (VDAs) selectively target tumor ECs to disrupt
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established tumor vasculature, resulting in widespread necrosis in the tumor core [21].
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VDAs have been shown to possess potential preclinical and clinical activity in
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multiple types of tumors that are sensitive to chemotherapy [22, 23]. However,
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whether VDAs can overcome tumor MDR and the underlying mechanisms remain to
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be investigated.
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In our previous study, we found that Z-GP-DAVLBH, a VDA that disrupts tumor
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vessels by targeting pericytes, has broad spectrum antitumor activity against
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chemotherapy-sensitive tumor xenografts [24], but its effect on tumors with MDR is
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still unclear. Here, we show that Z-GP-DAVLBH overcomes MDR by skewing TAMs
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toward
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colony-stimulating factor (GM-CSF) plays a key role in Z-GP-DAVLBH-induced M1
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macrophage repolarization.
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the
cytotoxic
M1
phenotype
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that
granulocyte-macrophage
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2. Materials and methods
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2.1 Reagents
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DAVLBH and Z-GP-DAVLBH (purity > 98%) were synthesized according to
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previously described methods [24]. They were dissolved into DMSO to prepare a 4
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stock solution at 20 mM and were stored at -20°C protected from light. The anti-
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ATP-binding cassette transporter B1 (ABCB1) antibody was from Santa Cruz
104
Biotechnology (Santa Cruz, CA). The antibodies against Ki67, poly ADP-ribose
105
polymerase
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(HRP)-conjugated secondary antibodies were obtained from Cell Signaling
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Technology (Danvers, MA). The antibody against CD31, the neutralizing antibody
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against GM-CSF and human recombinant GM-CSF (rhGM-CSF) were obtained from
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R&D Systems (Minneapolis, MN). Antibodies against F4/80, inducible nitric oxide
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synthase (iNOS), CD68, CD86, Arginase 1 (Arg-1), lymphocyte antigen 6 complex
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locus G6D (Ly6G), CD11b, interferon-β (IFN-β) and hypoxia-inducible factor
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(HIF-1α) were purchased from Abcam (Cambridge, UK). The DeadEnd Colorimetric
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TUNEL System was obtained from Promega (Madison, WI). The Annexin
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V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit
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was from Thermo Fisher Scientific (Waltham, MA). Enzyme-linked immunosorbent
116
assay (ELISA) kits for GM-CSF, macrophage colony-stimulating factor (M-CSF),
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interleukin-6 (IL-6), interleukin-10 (IL-10), lipopolysaccharide (LPS), interferon
118
gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) were purchased from Multi
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Sciences (Hangzhou, China). The 17β-estradiol slow release pellet (Cat. SE-121,
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60-day release, 0.36 mg/pellet) was obtained from Innovative Research of America
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(Sarasota, FL). DOX, vincristine, gadolinium chloride (GdCl3), phorbol 12-myristate
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13-acetate (PMA) and other agents were from Sigma-Aldrich (St. Louis, MO).
cleaved-PARP,
β-actin
and
horseradish
peroxidase
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(PARP),
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2.2 Cells and cell culture
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The human hepatocellular carcinoma HepG2 cell line, the human breast
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adenocarcinoma MCF-7 cell line and the human monocytic THP-1 cell line were from 5
ACCEPTED MANUSCRIPT American Type Culture Collection (ATCC). The multidrug-resistant HepG2/ADM
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cell line (DOX-selected ABCB1-overexpressing cells) was a kind gift from Prof.
129
Kwok-Pui Fung (Chinese University of Hong Kong, Hong Kong) [25]. The
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multidrug-resistant MCF-7/ADR cell line (DOX-selected ABCB1-overexpressing
131
cells) was generously provided by Prof. Li-Wu Fu (Sun Yat-Sen University, China)
132
[26]. All the cells were cultured in RPMI-1640 with 10% FBS (Life Technologies)
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and 1% penicillin-streptomycin at 37°C with 5% CO2. Macrophage-conditioned
134
medium was generated by incubating M1 macrophages in serum-free RPMI 1640
135
medium for 48 h. In addition, HepG2/ADM and MCF-7/ADR cells were cultured in
136
medium containing 1.2 µM DOX to maintain MDR. The cell lines used in this study
137
were authenticated no cross-contamination of other human cell lines using the STR
138
Multi-amplification Kit (Microreader TM21 ID System), and all cell lines were tested
139
negative for mycoplasma using the Mycoplasma Detection Set (M&C Gene
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Technology).
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2.3 Cell proliferation assay
1×104 cells per well were seeded in 96-well plates and cultured for 24 h.
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Adherent cells were treated with various concentrations of DAVLBH, DOX or
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vincristine,
146
5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) were added to
147
each well and incubated at 37°C for 4 h before reading the absorbance at 570 nm with
148
a plate reader.
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After
another
24
h
incubation,
30
µL
5
mg/mL
3-(4,
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2.4 In vivo assay Four- to six-week-old female and male BALB/c nu/nu mice were purchased from 6
ACCEPTED MANUSCRIPT HFK Bioscience Co., Ltd. (Beijing, China). All animal experiments were approved by
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the Experimental Animal Ethics Committee of Jinan University (Guangzhou, China).
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HepG2/ADM cells and MCF-7/ADR cells (2 × 106) suspended in 200 µL of a 50%
155
mixture of matrigel were inoculated subcutaneously into the backs of female
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(MCF-7/ADR cells) or male (HepG2/ADM cells) BALB/c nu/nu mice. Before
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MCF-7/ADR cells transplantation, the mice were anesthetized and subcutaneously
158
implanted with 17β-estradiol supplementation. When the tumor volume reached
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approximately 200 mm3, tumor-bearing mice were randomized to the appropriate
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groups (5 mice per group). The mice received an intravenous (i.v.) injection of test
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compounds once every other day. Mice in the vehicle group received only saline
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(containing 1% DMSO). GdCl3 (10 mg/kg diluted in saline) was injected into mice
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twice a week to selectively deplete M1 macrophages [27]. To neutralize GM-CSF, a
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neutralizing antibody against GM-CSF was administered via intraperitoneal (i.p.)
165
injection at a dose of 2 mg/kg in parallel with Z-GP-DAVLBH treatment. Tumor
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volume was measured with an electronic caliper (Mitutoyo, Tokyo, Japan) every two
167
days and calculated using the following formula: a × b2 × 0.5, where a is the longest
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diameter, and b is the diameter perpendicular to a. At the end of the experiment, the
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mice were anesthetized by an i.p. injection of 5 ml/kg of 1% pentobarbital sodium salt,
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and the tumors were removed, weighed, and photographed. Then, one section of each
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tumor was fixed in 4% paraformaldehyde for pathological examination; the other
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sections were stored at -80°C for ELISA analysis.
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2.5 Histology and immunohistochemical analysis
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Fixed tumors were embedded in paraffin and sectioned at a thickness of 5 µm.
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Hematoxylin-eosin (H&E) staining was performed according to standard procedures. 7
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178
antibodies overnight at 4°C. Next, the slides were incubated with HRP-conjugated
179
secondary antibodies, stained with a DAB kit, and counterstained with hematoxylin.
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TUNEL staining was detected by the DeadEnd Colorimetric TUNEL System kit
181
according to the manufacturer’s instructions. Three images from each slide were taken
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with an Olympus BX 53 microscope, and integrated optical density (IOD) values and
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the Ki67- or TUNEL-staining cells were analyzed with Image-Pro Plus 6.0 software
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(Media Cybernetics, Inc., Rockville, MD).
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2.6 Immunofluorescence analysis
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After successive soaks in 10%, 20% and 30% sucrose solution for 24 h, the fixed
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tumors were embedded in OCT and subsequently cut into 5-µm-thick slides. The
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slides were incubated with an anti-F4/80 antibody and antibodies against iNOS or
190
Arg-1 overnight at 4°C and then incubated with appropriate Alexa Fluor
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dye-conjugated secondary antibodies for 1 h at room temperature. DAPI was used for
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nuclear staining, and slices were observed with a Zeiss LSM 800 confocal
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microscope.
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2.7 Enzyme-linked immunosorbent assay The concentrations of GM-CSF, M-CSF, IL-6, IL-10, TNF-α, LPS and INF-γ
were measured using ELISA kits according to the manufacturers’ instructions.
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2.8 Generation and characterization of M1 macrophages
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M1 macrophages were generated from THP-1 cells according to a previous
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protocol with some modifications [28]. Briefly, THP-1 cells were treated with 100 nM 8
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PMA for 24 h to induce differentiation into macrophage-like cells. Then, the cells
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were stimulated with 50 ng/mL rhGM-CSF for 6 days. On the seventh day, the cells
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were harvested for further experiments.
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2.9 Western blotting
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Western blotting was performed as previously described [29]. Briefly, cells were
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collected and lysed with RIPA buffer, and total protein was analyzed by western
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blotting.
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2.10 Annexin V/PI assay
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HepG2/ADM and MCF-7/ADR cells seeded in 6-well plates were treated with
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DAVLBH (5 µM or 6 µM) alone or in combination with conditioned medium from
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GM-CSF-educated M1 macrophages for 24 h. The cells were then harvested and
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analyzed by flow cytometry (Guava Technologies, Millipore, Billerica, MA) using an
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Annexin V-FITC/PI apoptosis detection kit according to the manufacturer’s protocol.
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2.11 Statistical analysis
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The data are presented as mean ± SEM after analysis using GraphPad Prism 5.0
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(GraphPad Software, Inc., San Diego, CA). The two-tailed unpaired t-test was used to
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compare differences between two groups, and differences among more than two
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groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. P <
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0.05 indicated a significant difference.
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3. Results
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3.1 Z-GP-DAVLBH-mediated inhibition of HepG2/ADM tumor growth may be 9
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associated with the skewing of TAMs from the M2 to the M1 phenotype First, we evaluated the antitumor effect of Z-GP-DAVLBH on HepG2/ADM cells,
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which overexpress ABCB-1 and are resistant to DOX and vincristine compared with
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parental cells (Fig. S1A-C). We found that the anti-proliferative effect of
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Z-GP-DAVLBH on HepG2/ADM cells was much lower than that on HepG2 cells,
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with IC50 values of 16 µM and 0.55 µM, respectively (Fig. 1A). In addition,
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Z-GP-DAVLBH stimulates ABCB1 ATPase activity (Fig. S2). These results indicated
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that Z-GP-DAVLBH was the substrate of ABCB-1 thus exerting poor antitumor
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activity in the ABCB-1-overexpressing cancer cells in vitro. Consistent with previous
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reports
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Z-GP-DAVLBH effectively destroyed tumor vessels in HepG2/ADM xenografts
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within 4 h by selectively targeting tumor pericytes (Fig. S3A). As a result, 2.0 mg/kg
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Z-GP-DAVLBH dramatically inhibited tumor growth, with an inhibitory rate of
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approximately 75%. Tumor volume in the vehicle group increased from 159.06 ±
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33.35 to 635.91 ± 292.86 mm3, whereas that in the Z-GP-DAVLBH group merely
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increased from 145.29 ± 48.92 to 159.60 ± 44.48 mm3. In addition, the average tumor
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weight was 0.88 ± 0.26 g in the vehicle group and 0.38 ± 0.20 g in the
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Z-GP-DAVLBH-treated group (Fig. 1B-D). Further pathological examination
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revealed that Z-GP-DAVLBH caused widespread necrosis, decreased the Ki67
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proliferative index and CD31-positive microvessel density. However, Z-GP-DAVLBH
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had a negligible effect on tumor ABCB-1 expression, indicating that ABCB-1 is not
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involved in the antitumor effect of Z-GP-DAVLBH. In addition, we found that
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Z-GP-DAVLBH treatment increased the population of TUNEL-positive cells (Fig. 1E,
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F). These results indicate that Z-GP-DAVLBH inhibits HepG2/ADM tumor growth,
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and this effect may be associated with tumor vessel disruption and cancer cell
effects
on
chemotherapy-sensitive
tumor
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apoptosis. Given that M1 macrophages promote tumor cell apoptosis [8], we next explored
254
whether Z-GP-DAVLBH-mediated HepG2/ADM apoptosis is associated with the
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induction of macrophages toward the M1 phenotype. Immunofluorescence assays of
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macrophages in HepG2/ADM tumor xenografts with polarization state-specific
257
markers revealed that Z-GP-DAVLBH reprogramed TAMs from an M2- to an
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M1-dominant phenotype, as evidenced by the increase in iNOS+/F4/80+ M1
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macrophages and the reduction in Arg-1+/F4/80+ M2 macrophages (Fig. 1G).
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However, Z-GP-DAVLBH had a negligible effect on the Ly6G, CD11b and IFN-β
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staining tumor-associated N1 neutrophils [30, 31] (Fig. S4).
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3.2 Z-GP-DAVLBH-mediated inhibition of MCF-7/ADR tumor growth may be
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associated with the skewing of TAMs from the M2 to the M1 phenotype We next tested the effect of Z-GP-DAVLBH on another multidrug-resistant cell
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line, MCF-7/ADR. These cells overexpressed ABCB-1 and were resistant to DOX
267
and vincristine (Fig. S5). The anti-proliferative effect of Z-GP-DAVLBH on
268
MCF-7/ADR cells was much lower than that on MCF-7 cells (Fig. 2A), with IC50
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values of 19.92 µM and 0.91 µM, respectively. These results were consistence with
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the effect on HepG2/ADM cells (Fig. 1A). Moreover, Z-GP-DAVLBH (2 mg/kg)
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significantly suppressed MCF-7/ADR tumor xenograft growth, with an inhibitory rate
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of approximately 70% (Fig. 2B-D). We also found that Z-GP-DAVLBH effectively
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destroyed the vessels in MCF-7/ADR tumor xenografts within 4 h by selectively
274
targeting tumor pericytes (Fig. S3B). H&E staining showed that Z-GP-DAVLBH (2
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mg/kg) treatment led to extensive necrosis in both the core and periphery of
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MCF-7/ADR tumors. The CD31-positive microvessel density and the Ki67
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proliferative index, rather than ABCB-1 expression, were dramatically decreased in
278
tumors
279
immunofluorescence analyses showed that Z-GP-DAVLBH skewed TAMs from the
280
M2 phenotype to the M1 phenotype, as evidenced by the increased iNOS+/F4/80+ M1
281
macrophages and the decreased Arg-1+/F4/80+ M2 macrophages (Fig. 2G). Taken
282
together, these results indicate that Z-GP-DAVLBH has potent antitumor activity
283
against MCF-7/ADR tumor xenografts, similar to that against HepG2/ADM tumor
284
xenografts, and that this effect may be associated with the skewing of TAMs from the
285
M2 to the M1 phenotype.
the
Z-GP-DAVLBH-treated
group
(Fig.
2E-F).
Furthermore,
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3.3 M1 macrophage depletion attenuates the antitumor effect of Z-GP-DAVLBH
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on HepG2/ADM tumor xenografts
To further validate whether M1 macrophages are primarily responsible for the
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Z-GP-DAVLBH-induced suppression of tumor growth, GdCl3, a selective M1
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macrophage scavenger that induces the apoptosis of inflammatory macrophages via
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competitive inhibition of Ca2+ mobilization and damage to plasma membranes [28],
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was used to selectively deplete the M1 population. Mice bearing HepG2/ADM tumors
294
were pretreated with GdCl3 (10 mg/kg) for two weeks to deplete M1 macrophages.
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We found that GdCl3-induced M1 depletion significantly attenuated the antitumor
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effect
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GdCl3+Z-GP-DAVLBH group was 24%, which was much lower than that in the
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Z-GP-DAVLBH-alone group, in which the inhibitory rate was 47% (Fig. 3A-C).
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Tumors in the GdCl3-treated group were confirmed to lack iNOS+/F4/80+ M1
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macrophages, and the Z-GP-DAVLBH-mediated increase in M1 macrophages was
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blocked by GdCl3 treatment (Fig. 3D, E), whereas the Arg-1+/F4/80+ M2
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of
Z-GP-DAVLBH;
the
tumor
growth
inhibitory
rate
in
the
12
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GdCl3 alleviated the Z-GP-DAVLBH-induced HepG2/ADM tumor cell apoptosis
304
(based on the percentage of TUNEL-positive cells) and the decrease in the Ki67
305
proliferative index (Fig. 3F, G). These data indicate that M1 macrophages are critical
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for the antitumor effect of Z-GP-DAVLBH on HepG2/ADM tumor xenografts.
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3.4 GM-CSF contributes to Z-GP-DAVLBH-induced TAM reprograming toward
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the M1 phenotype
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We next investigated the underlying mechanism of Z-GP-DAVLBH-induced M1
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macrophage skewing. Given that LPS, INF-γ and GM-CSF are the three major
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stimulators of M1 macrophages [8, 32], we evaluated the changes in these growth
313
factors in serum and tumor tissue from mice bearing HepG2/ADM xenografts. The
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ELISA assay results showed that Z-GP-DAVLBH treatment for 24 h increased
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GM-CSF levels in both mouse serum and tumor tissue (Fig. 4A, B) but had a
316
negligible effect on the levels of LPS and INF-γ (Fig. S7). We also found that the
317
level of M-CSF, a stimulator of M2 macrophages [8], was decreased in mouse serum
318
and tumor tissue after Z-GP-DAVLBH treatment (Fig. 4C, D). In addition,
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Z-GP-DAVLBH increased IL-6 levels (M1 macrophage marker) and decreased IL-10
320
levels (M2 macrophage marker) in mouse serum and tumor tissue (Fig. 4A-D) [8]. To
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determine the role of GM-CSF in the antitumor effect of Z-GP-DAVLBH, a GM-CSF
322
neutralizing antibody was used to capture the secreted GM-CSF in HepG2/ADM
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tumor xenografts. Our results showed that GM-CSF neutralizing antibody (2 mg/kg)
324
retarded the antitumor effect of Z-GP-DAVLBH (2 mg/kg); the tumor growth
325
inhibitory rate in the group treated with the GM-CSF neutralizing antibody and
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Z-GP-DAVLBH
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was
11%,
which
was
much
lower
than
that
in
the 13
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Z-GP-DAVLBH-alone group (the inhibitory rate was 41%) (Fig. 5A-C). The
328
GM-CSF neutralizing antibody decreased the population of iNOS+/F4/80+ M1
329
macrophages and attenuated the Z-GP-DAVLBH-mediated increase in M1
330
macrophages (Fig. 5D, E). In addition, our results showed that the GM-CSF antibody
331
abated
332
(TUNEL-positive cells) and the decrease in the Ki67 proliferative index (Fig. 5F, G).
333
These data indicate that GM-CSF is a key regulator of Z-GP-DAVLBH-induced M1
334
macrophage skewing, which contributes to the antitumor effects of Z-GP-DAVLBH.
HepG2/ADM
tumor
cell
apoptosis
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3.5 M1 macrophage-conditioned medium promotes Z-GP-DAVLBH-induced
337
apoptosis of cancer cells with MDR in vitro
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Next, we further investigated whether GM-CSF-stimulated M1 macrophages
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contribute to Z-GP-DAVLBH-induced apoptosis of multidrug-resistant cancer cells.
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THP-1 cells in the monocyte state were stimulated with PMA (100 nM) and GM-CSF
341
(50 ng/mL) to differentiate into an M1 macrophage-like phenotype, as characterized
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by the increased secretion of IL-6 and TNF-α (Fig. 6A) and the overexpression of
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F4/80, iNOS, CD68 and CD86 compared with unstimulated THP-1 cells (Fig. 6B). As
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Z-GP-DAVLBH can be hydrolyzed into DAVLBH in tumor tissue [24], we combined
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M1 macrophage-conditioned medium with DAVLBH to test the cytotoxicity in
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HepG2/ADM and MCF-7/ADR cells. Annexin V-FITC/PI assays showed that
347
treatment with M1 macrophage-conditioned medium or DAVLBH (5 µM and 6 µM)
348
alone had a negligible effect on HepG2/ADM and MCF-7/ADR cells, whereas
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DAVLBH (5 µM and 6 µM) combined with M1 macrophage-conditioned medium
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induced the apoptosis of approximately 40% and 60% of HepG2/ADM cells (Fig. 5C)
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and of approximately 30% and 40% of MCF-7/ADR cells (Fig. 5D). Western blotting
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treatment with the combination of M1 macrophage-conditioned medium and
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DAVLBH, while almost no cleaved-PARP was observed in the groups treated with
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M1 macrophage-conditioned medium or DAVLBH alone (Fig. 5E). Taken together,
356
these data indicate that DAVLBH is not sufficient to induce the apoptosis of
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HepG2/ADM and MCF-7/ADR cells and that M1 macrophages promote such cell
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apoptosis.
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4. Discussion
For decades, MDR has hampered the success of cancer chemotherapy. Attempts
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have been made to identify selective ABCB-1 inhibitors that overcome MDR without
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non-specific toxicity, but there is still no such effective and safe agent in clinic. Here,
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we show that Z-GP-DAVLBH, a pericyte-targeting VDA, selectively disrupted the
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tumor vasculature, increased the secretion of GM-CSF, skewed TAMs toward the
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antitumor M1 phenotype, induced tumor cell apoptosis, and ultimately inhibited the
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growth of multidrug-resistant HepG2/ADM and MCF-7/ADR tumor xenografts.
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VDAs are considered promising agents for cancer treatment [21]. However,
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TAMs have been shown to hamper the efficacy of VDAs [33]. For example, a
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frequently used VDA, CA4-P, triggers TIE-2+ M2 macrophage recruitment [34],
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which promotes tumor angiogenesis, metastasis and tumor regrowth and these are
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some of the significant reasons for the failure of this treatment. In contrast to CA4-P,
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Z-GP-DAVLBH did not increase M2 macrophages but rather skewed TAMs toward
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the antitumor M1 phenotype by increasing GM-CSF secretion and then induced
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apoptosis of drug-resistant tumor cells. The different effects between CA4-P and
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Z-GP-DAVLBH on the polarization of TAMs may be attributed to various reasons.
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tumor vessels to cause hypoxia in tumor tissues [34], which has been demonstrated to
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be an important factor promoting macrophage M2 polarization [35]. On the contrary,
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Z-GP-DAVLBH had a negligible effect on the expression of HIF-1α on both
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HepG2/ADM and MCF-7/ADR tumors (Fig. S8). Our study extends the
382
understanding of the underlying mechanisms of VDA-mediated antitumor effects.
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TAMs are a major cellular constituent of the tumor microenvironment and
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display an M1 or M2 phenotype in response to different stimuli [36], and TAMs are
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one of the most interesting targets for cancer therapy. So far, the major strategy for
386
targeting TAMs is to inhibit signaling through myeloid cell receptors such as
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colony-stimulating factor-1 receptor using genetic methods or pharmacological
388
inhibitors, which have been applied in preclinical or clinical trials to abolish the
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macrophage-mediated pro-tumor effects [37, 38]. However, as these strategies target
390
all macrophages, they have systemic toxicities that restrict their clinical applications
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[39]. Reprograming TAMs toward M1 macrophages by activating costimulatory
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CD40 with an agonist antibody [40], administering Toll-like receptor agonists [41, 42],
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inhibiting colony-stimulating factor-1 receptor [43] or using pharmaceuticals [44] has
394
the potential to inhibit tumor growth and enhance the chemotherapeutic efficacy in
395
sensitive tumors. In addition, reprogramming TAM polarization toward the antitumor
396
M1 phenotype has been shown to contribute to RRx-001-mediated MDR reversal [45].
397
In the present study, we also found that Z-GP-DAVLBH overcame MDR by skewing
398
TAMs toward M1 macrophages. Our results provide evidence to support the
399
hypothesis that reprogramming TAMs toward M1 macrophages is effective for
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circumventing tumor MDR. Furthermore, compared with the non-selective
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distribution of the ABCB-1 inhibitor, Z-GP-DAVLBH selectively accumulates in the
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tumor and has minimal effects on body weight or major organs in tumor-bearing mice
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[24], indicating that Z-GP-DAVLBH may achieve great efficacy in tumors with MDR
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in future preclinical and clinical trials. In this regard, this study sheds new light on the
405
application of VDAs in the treatment of resistant tumors. GM-CSF is generally recognized as one of the most important inflammatory
407
cytokines; it has been used to bolster antitumor immune responses due to its role in
408
re-educating TAMs toward the antitumor M1 phenotype [46, 47]. Vaccination with
409
irradiated B16 melanoma cells engineered to secrete GM-CSF stimulates more potent
410
antitumor immunity [48]. Intra-tumoral injection of GM-CSF has been demonstrated
411
to induce TAM polarization toward the antitumor M1 phenotype [49, 50]. However,
412
GM-CSF can also promote tumor progression [51-53]. The role of GM-CSF in
413
antitumor therapy is controversial and depends on the concentration and the overall
414
cytokine milieu [54]. In the present study, we found that Z-GP-DAVLBH treatment
415
resulted
416
Z-GP-DAVLBH-mediated TAM repolarization toward the antitumor M1 phenotype.
417
GM-CSF is secreted by cancer cells, ECs and stromal cells in the tumor
418
microenvironment
419
Z-GP-DAVLBH-mediated regulation of GM-CSF secretion must be studied further.
420
Besides GM-CSF, LPS and INF-γ, there are two further important activators of M1
421
macrophages [8, 32]. LPS is a pathogen-associated molecular mainly derived from
422
gram-negative bacteria, it engages the Toll-like receptor 4 on the surface of
423
macrophages to activate transcription factors to exert an inflammatory response to
424
fight against pathogenic insult [55]. INF-γ is a cytokine that produced predominantly
425
by natural killer and natural killer T cells, and is critical for innate and adaptive
426
immunity against viral, some bacterial and protozoal infections [56]. However,
up-regulation
of
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the
[54].
However,
GM-CSF,
the
which
is
mechanisms
involved
in
underlying
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Z-GP-DAVLBH had a negligible effect on these factors, which may due to the fact
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that the macrophage M1 polarization was caused by Z-GP-DAVLBH-mediated
429
internal tumor vascular disruption, but not by external infections. In conclusion, this study showed that Z-GP-DAVLBH skewed TAMs from the
431
M2 phenotype toward the M1 phenotype by up-regulating GM-CSF secretion and
432
ultimately reversed tumor MDR. Our study provides new evidence that VDAs are
433
promising agents for reversing MDR and sheds new light on the mechanisms
434
underlying M1 macrophage repolarization in the treatment of tumors with MDR.
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Acknowledgments
This study was supported by the National Natural Science Foundation of China
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(U1401225, 81630095 and 81573455), Natural Science Foundation of Guangdong
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Province (2017A030310453), National Science and Technology Major Project
440
(2017ZX09101003-008-008) and Pearl River Scholar Funded Scheme (D. M. Zhang).
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. Z-GP-DAVLBH inhibits HepG2/ADM tumor xenograft growth and induces TAM skewing toward the M1 phenotype. (A) The IC50 values of
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Z-GP-DAVLBH in HepG2 and HepG2/ADM cells. Cells were treated with various concentrations of Z-GP-DAVLBH for 24 h and the cell viabilities were detected by MTT assay. (B-D) Mice bearing HepG2/ADM tumor xenografts received an i.v.
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injection of Z-GP-DAVLBH (2.0 mg/kg) or vehicle (1% DMSO) once every two days. The (B) tumor growth curves, (C) tumor images and (D) tumor weight are shown. (E)
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Representative images and (F) quantification of H&E, ABCB1, CD31, Ki67 and TUNEL in HepG2/ADM tumors. (G) Z-GP-DAVLBH decreased M2 macrophages (Arg-1+/F4/80+) and increased M1 macrophages (iNOS+/F4/80+) in HepG2/ADM tumors. Representative images of immunofluorescence and quantification are shown.
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Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle group.
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Figure 2. Z-GP-DAVLBH inhibits MCF-7/ADR tumor xenograft growth and
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induces TAM skewing toward the M1 phenotype. (A) The IC50 values of Z-GP-DAVLBH in MCF-7 and MCF-7/ADR cells. Cells were treated with various concentrations of Z-GP-DAVLBH for 24 h and the cell viabilities were detected by MTT assay. (B-D) Mice bearing MCF-7/ADR tumor xenografts received an i.v. injection of Z-GP-DAVLBH (2.0 mg/kg) or vehicle (1% DMSO) once every other day. The (B) tumor growth curves, (C) tumor images and (D) tumor weight are shown. (E-F) Z-GP-DAVLBH decreased microvessel density and induced cancer cell apoptosis. (E) Representative images and (F) quantification of H&E, ABCB1, CD31,
ACCEPTED MANUSCRIPT Ki67 and TUNEL in MCF-7/ADR tumors. (G) Z-GP-DAVLBH decreased M2 macrophages (Arg-1+/F4/80+) and increased M1 macrophages (iNOS+/F4/80+) in MCF-7/ADR tumors. Representative images of immunofluorescence and the
< 0.01 and ***P < 0.001 compared with the vehicle group.
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quantification are shown. Data are presented as mean ± SEM (n = 5). *P < 0.05, **P
Figure 3. M1 macrophage depletion attenuates the antitumor effect of
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Z-GP-DAVLBH. (A-C) Mice bearing HepG2/ADM tumor xenografts were pretreated with GdCl3 (10 mg/kg) twice per week for two weeks to selectively deplete
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M1 macrophages, and the mice then received an i.v. injection of Z-GP-DAVLBH (2.0 mg/kg). The (A) tumor volume, (B) tumor images and (C) tumor weight are shown. (D-E) Immunofluorescence analysis of the effects of GdCl3 and Z-GP-DAVLBH on M1 macrophages. (D) Representative images of the immunofluorescence assay and
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(E) the quantification of M1 macrophages (iNOS+/F4/80+). (F) Representative images and (G) the quantification of necrosis, Ki67 index and apoptotic cells (TUNEL) in tumors from (B). Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01
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and ***P < 0.001 compared with the vehicle group. #P < 0.05 and
###
P < 0.001
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compared with the Z-GP-DAVLBH group.
Figure 4. ELISA analysis of cytokine levels in serum and tumors. Mice bearing HepG2/ADM tumors received an i.v injection of 2 mg/kg Z-GP-DAVLBH. Blood and tumors were collected at the indicated times. Serum and tumor homogenates were analyzed by ELISA. Z-GP-DAVLBH increased the secretion of GM-CSF and IL-6 in (A) serum and (B) tumors and inhibited the secretion of M-CSF and IL-10 in (C) serum and (D) tumors. Data are presented as mean ± SEM (n = 3). *P < 0.05, **P <
ACCEPTED MANUSCRIPT 0.01 and ***P < 0.001 compared with the 0 h group.
Figure 5. GM-CSF neutralizing antibody abates the antitumor effects of Z-GP-DAVLBH on HepG2/ADM tumor xenografts. (A-C) Mice bearing tumors
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were treated with vehicle (1% DMSO), Z-GP-DAVLBH (2 mg/kg), anti-GM-CSF (2 mg/kg) or anti-CM-CSF combined with Z-GP-DAVLBH. (A) Tumor growth curves, (B) tumor images and (C) tumor weight are shown. (D-E) Immunofluorescence
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analysis of the effects of anti-GM-CSF and Z-GP-DAVLBH on M1 macrophages. (D) Representative images of the immunofluorescence assays and (E) the quantification
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of M1 macrophages (iNOS+/F4/80+). (F) Representative images and (G) the quantification of necrosis, Ki67 index and apoptotic cells (TUNEL) in tumors from (B). Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle group. #P < 0.05 and ###P < 0.001 compared with the
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Figure 6. GM-CSF-educated M1 macrophage conditioned medium enhances the
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in vitro antitumor effects of DAVLBH. (A-B) THP-1 cells were polarized to M1
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macrophages by sequential treatment with PMA (100 nM) and GM-CSF (50 ng/mL). (A) ELISA assays of IL-6 and TNF-α. (B) Western blotting for F4/80, iNOS, CD68 and CD86. (C-E) Conditioned medium (CM) was derived from GM-CSF-educated M1 macrophages. HepG2/ADM and MCF-7/ADR cells were treated with DAVLBH alone or in combination with CM for 24 h, and the cells were then collected for western blotting analysis or Annexin V-PI assays. Representative images of (C) Annexin V-PI analysis of HepG2/ADM cells, (D) Annexin V-PI analysis of MCF-7/ADR cells and (E) western blotting. Data are presented as mean ± SEM (n =
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ACCEPTED MANUSCRIPT Highlights • A vascular disrupting agent, Z-GP-DAVLBH, inhibits the growth of multidrug-resistant HepG2/ADM and MCF-7/ADR tumor xenografts but has no significant cytotoxicity toward these
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two cancer cell lines in vitro. • Z-GP-DAVLBH induces the repolarization of tumor-associated macrophages to the M1
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• M1 macrophages contribute to the anti-cancer effect of Z-GP-DAVLBH both in vitro and in vivo.
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M1 macrophage repolarization.
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• Granulocyte-macrophage colony-stimulating factor plays a key role in Z-GP-DAVLBH-induced
S0304-3835(18)30038-7
DOI:
10.1016/j.canlet.2018.01.016
Reference:
CAN 13684
To appear in:
Cancer Letters
Received Date: 29 November 2017 Revised Date:
1 January 2018
Accepted Date: 8 January 2018
Please cite this article as: X. Lei, M. Chen, X. Li, M. Huang, Q. Nie, N. Ma, H. Chen, N. Xu, W. Ye, D. Zhang, A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.01.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A vascular disrupting agent overcomes tumor multidrug resistance
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by skewing macrophage polarity toward the M1 phenotype
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Xueping Lei1, 2,#, Minfeng Chen1, 2,#, Xiaobo Li1, 2, Maohua Huang1, 2, Qiulin Nie1, 2,
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Nan Ma1, 2, Heru Chen1, 2, Nanhui Xu1, 2, Wencai Ye1, 2,*, Dongmei Zhang1, 2,*
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Affiliations:
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1
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2
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College of Pharmacy, Jinan University, Guangzhou 510632, China
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University,
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Guangzhou 510632, China
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#: Lei Xueping and Chen Minfeng are co-first authors.
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Corresponding Authors: Dongmei Zhang, College of Pharmacy, Jinan University,
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601 Huangpu Avenue. Wes, Guangzhou 510632, China; Phone: +86-20-85222653;
14
E-mail: [email protected]. or Wencai Ye, College of Pharmacy, Jinan
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University, 601 Huangpu Avenue. Wes, Guangzhou 510632, China; Phone:
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+86-20-85220004; E-mail: [email protected].
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Abstract Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC)
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transporters is the major obstacle for chemotherapeutic success. Although attempts
30
have been made to circumvent ABC transporter-mediated MDR in past decades, there
31
is still no effective agent in clinic. Here, we identified a vascular disrupting agent,
32
Z-GP-DAVLBH, that significantly inhibited the growth of multidrug-resistant human
33
hepatoma HepG2/ADM and human breast cancer MCF-7/ADR tumor xenografts,
34
although these cells were insensitive to Z-GP-DAVLBH in vitro. Z-GP-DAVLBH
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increased the secretion of granulocyte-macrophage colony-stimulating factor in tumor
36
tissues and serum of tumor-bearing mice to skew tumor-associated macrophages from
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the pro-tumor M2 phenotype to the antitumor M1 phenotype, thereby contributing to
38
the induction of HepG2/ADM and MCF-7/ADR cell apoptosis. Our findings shed
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new light on the underlying mechanisms of VDAs in the treatment of drug-resistant
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tumors and provide strong evidence that Z-GP-DAVLBH should be a promising agent
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for overcoming MDR.
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Keywords: multidrug resistance; vascular disrupting agent; tumor-associated
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macrophages; granulocyte-macrophage colony-stimulating factor
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Abbreviations
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MDR: multidrug resistance; ABCB-1: ATP-binding cassette transporter B1; TAM:
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tumor-associated macrophage; VDA: vascular disrupting agent; iNOS: inducible
49
nitric oxide synthase; Arg-1: arginase 1; Ly6G: lymphocyte antigen 6 complex locus
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G6D; IFN-β: interferon-β; CA4-P: combretastatin A4 phosphate. GM-CSF:
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granulocyte-macrophage
colony-stimulating
factor;
M-CSF:
macrophage 2
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colony-stimulating
factor;
IL-6:
interleukin-6;
IL-10:
interleukin-10;
LPS:
53
lipopolysaccharide; INF-γ: interferon gamma; TNF-α: tumor necrosis factor alpha;
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HIF-1α: hypoxia-inducible factor.
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1. Introduction
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Multidrug resistance (MDR) is the phenomenon in which tumor cells are
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resistance to a wide range of chemotherapeutic drugs, and MDR has been considered
59
one of the major reasons for chemotherapeutic failure [1]. The overexpression of
60
ATP-binding cassette (ABC) transporters is a major cause of MDR and has a broad
61
spectrum effect on the efflux of chemotherapy drugs that are frequently used in clinic,
62
such as vinca alkaloids, doxorubicin (DOX) and taxanes [2]. Recently, the
63
identification of effective ABC transporter inhibitors to reverse MDR is the major
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therapeutic approach. However, although four generations of ABC transporter
65
inhibitors have been emerged and tested in preclinical or clinical trials, non-specific
66
toxicity limits their successful use in circumventing MDR in clinic [3-5]. In addition,
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nano-sized delivery systems, such as liposomal ABC transporter inhibitors, are
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proposed to reduce the non-selective inhibition of ABC transporters; however, these
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strategies also fail to reverse tumor MDR in clinic [6]. Therefore, it is necessary to
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explore new strategies or drugs for overcoming tumor MDR.
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Tumor-associated macrophages (TAMs) are derived from circulating monocytes
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or resident tissue macrophages [7]. TAMs display two main phenotypes, M1 and M2,
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in response to diverse microenvironmental stimuli; M1 macrophages have antitumor
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activity, and M2 macrophages exhibit tumor-promoting effects [8]. During
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chemotherapy, TAMs often polarize to M2 macrophages and then promote tumor
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angiogenesis, metastasis and suppress antitumor immunity, leading to tumor 3
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resistance to chemotherapy [9-13]. Strategies to deplete TAMs, block M2 macrophage
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programming and reprogram macrophages to the M1 phenotype have been proposed
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to augment the efficacy of chemotherapy [14-16]. The inhibition of tumor angiogenesis has been considered a potential strategy for
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treating tumors with MDR [17, 18], as its targets are endothelial cells (ECs), not
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cancer cells [19], and EC genome is relatively stable compared with that of cancer
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cells [20]. Tumor vessel disruption is the other important strategy for targeted
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treatment. Vascular disrupting agents (VDAs) selectively target tumor ECs to disrupt
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established tumor vasculature, resulting in widespread necrosis in the tumor core [21].
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VDAs have been shown to possess potential preclinical and clinical activity in
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multiple types of tumors that are sensitive to chemotherapy [22, 23]. However,
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whether VDAs can overcome tumor MDR and the underlying mechanisms remain to
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be investigated.
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In our previous study, we found that Z-GP-DAVLBH, a VDA that disrupts tumor
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vessels by targeting pericytes, has broad spectrum antitumor activity against
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chemotherapy-sensitive tumor xenografts [24], but its effect on tumors with MDR is
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still unclear. Here, we show that Z-GP-DAVLBH overcomes MDR by skewing TAMs
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toward
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colony-stimulating factor (GM-CSF) plays a key role in Z-GP-DAVLBH-induced M1
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macrophage repolarization.
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the
cytotoxic
M1
phenotype
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that
granulocyte-macrophage
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2. Materials and methods
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2.1 Reagents
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DAVLBH and Z-GP-DAVLBH (purity > 98%) were synthesized according to
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previously described methods [24]. They were dissolved into DMSO to prepare a 4
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stock solution at 20 mM and were stored at -20°C protected from light. The anti-
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ATP-binding cassette transporter B1 (ABCB1) antibody was from Santa Cruz
104
Biotechnology (Santa Cruz, CA). The antibodies against Ki67, poly ADP-ribose
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polymerase
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(HRP)-conjugated secondary antibodies were obtained from Cell Signaling
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Technology (Danvers, MA). The antibody against CD31, the neutralizing antibody
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against GM-CSF and human recombinant GM-CSF (rhGM-CSF) were obtained from
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R&D Systems (Minneapolis, MN). Antibodies against F4/80, inducible nitric oxide
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synthase (iNOS), CD68, CD86, Arginase 1 (Arg-1), lymphocyte antigen 6 complex
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locus G6D (Ly6G), CD11b, interferon-β (IFN-β) and hypoxia-inducible factor
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(HIF-1α) were purchased from Abcam (Cambridge, UK). The DeadEnd Colorimetric
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TUNEL System was obtained from Promega (Madison, WI). The Annexin
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V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit
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was from Thermo Fisher Scientific (Waltham, MA). Enzyme-linked immunosorbent
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assay (ELISA) kits for GM-CSF, macrophage colony-stimulating factor (M-CSF),
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interleukin-6 (IL-6), interleukin-10 (IL-10), lipopolysaccharide (LPS), interferon
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gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) were purchased from Multi
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Sciences (Hangzhou, China). The 17β-estradiol slow release pellet (Cat. SE-121,
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60-day release, 0.36 mg/pellet) was obtained from Innovative Research of America
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(Sarasota, FL). DOX, vincristine, gadolinium chloride (GdCl3), phorbol 12-myristate
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13-acetate (PMA) and other agents were from Sigma-Aldrich (St. Louis, MO).
cleaved-PARP,
β-actin
and
horseradish
peroxidase
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(PARP),
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2.2 Cells and cell culture
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The human hepatocellular carcinoma HepG2 cell line, the human breast
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adenocarcinoma MCF-7 cell line and the human monocytic THP-1 cell line were from 5
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cell line (DOX-selected ABCB1-overexpressing cells) was a kind gift from Prof.
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Kwok-Pui Fung (Chinese University of Hong Kong, Hong Kong) [25]. The
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multidrug-resistant MCF-7/ADR cell line (DOX-selected ABCB1-overexpressing
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cells) was generously provided by Prof. Li-Wu Fu (Sun Yat-Sen University, China)
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[26]. All the cells were cultured in RPMI-1640 with 10% FBS (Life Technologies)
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and 1% penicillin-streptomycin at 37°C with 5% CO2. Macrophage-conditioned
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medium was generated by incubating M1 macrophages in serum-free RPMI 1640
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medium for 48 h. In addition, HepG2/ADM and MCF-7/ADR cells were cultured in
136
medium containing 1.2 µM DOX to maintain MDR. The cell lines used in this study
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were authenticated no cross-contamination of other human cell lines using the STR
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Multi-amplification Kit (Microreader TM21 ID System), and all cell lines were tested
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negative for mycoplasma using the Mycoplasma Detection Set (M&C Gene
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Technology).
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2.3 Cell proliferation assay
1×104 cells per well were seeded in 96-well plates and cultured for 24 h.
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Adherent cells were treated with various concentrations of DAVLBH, DOX or
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vincristine,
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5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) were added to
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each well and incubated at 37°C for 4 h before reading the absorbance at 570 nm with
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a plate reader.
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After
another
24
h
incubation,
30
µL
5
mg/mL
3-(4,
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2.4 In vivo assay Four- to six-week-old female and male BALB/c nu/nu mice were purchased from 6
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the Experimental Animal Ethics Committee of Jinan University (Guangzhou, China).
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HepG2/ADM cells and MCF-7/ADR cells (2 × 106) suspended in 200 µL of a 50%
155
mixture of matrigel were inoculated subcutaneously into the backs of female
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(MCF-7/ADR cells) or male (HepG2/ADM cells) BALB/c nu/nu mice. Before
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MCF-7/ADR cells transplantation, the mice were anesthetized and subcutaneously
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implanted with 17β-estradiol supplementation. When the tumor volume reached
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approximately 200 mm3, tumor-bearing mice were randomized to the appropriate
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groups (5 mice per group). The mice received an intravenous (i.v.) injection of test
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compounds once every other day. Mice in the vehicle group received only saline
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(containing 1% DMSO). GdCl3 (10 mg/kg diluted in saline) was injected into mice
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twice a week to selectively deplete M1 macrophages [27]. To neutralize GM-CSF, a
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neutralizing antibody against GM-CSF was administered via intraperitoneal (i.p.)
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injection at a dose of 2 mg/kg in parallel with Z-GP-DAVLBH treatment. Tumor
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volume was measured with an electronic caliper (Mitutoyo, Tokyo, Japan) every two
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days and calculated using the following formula: a × b2 × 0.5, where a is the longest
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diameter, and b is the diameter perpendicular to a. At the end of the experiment, the
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mice were anesthetized by an i.p. injection of 5 ml/kg of 1% pentobarbital sodium salt,
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and the tumors were removed, weighed, and photographed. Then, one section of each
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tumor was fixed in 4% paraformaldehyde for pathological examination; the other
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sections were stored at -80°C for ELISA analysis.
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2.5 Histology and immunohistochemical analysis
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Fixed tumors were embedded in paraffin and sectioned at a thickness of 5 µm.
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Hematoxylin-eosin (H&E) staining was performed according to standard procedures. 7
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178
antibodies overnight at 4°C. Next, the slides were incubated with HRP-conjugated
179
secondary antibodies, stained with a DAB kit, and counterstained with hematoxylin.
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TUNEL staining was detected by the DeadEnd Colorimetric TUNEL System kit
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according to the manufacturer’s instructions. Three images from each slide were taken
182
with an Olympus BX 53 microscope, and integrated optical density (IOD) values and
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the Ki67- or TUNEL-staining cells were analyzed with Image-Pro Plus 6.0 software
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(Media Cybernetics, Inc., Rockville, MD).
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2.6 Immunofluorescence analysis
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After successive soaks in 10%, 20% and 30% sucrose solution for 24 h, the fixed
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tumors were embedded in OCT and subsequently cut into 5-µm-thick slides. The
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slides were incubated with an anti-F4/80 antibody and antibodies against iNOS or
190
Arg-1 overnight at 4°C and then incubated with appropriate Alexa Fluor
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dye-conjugated secondary antibodies for 1 h at room temperature. DAPI was used for
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nuclear staining, and slices were observed with a Zeiss LSM 800 confocal
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microscope.
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2.7 Enzyme-linked immunosorbent assay The concentrations of GM-CSF, M-CSF, IL-6, IL-10, TNF-α, LPS and INF-γ
were measured using ELISA kits according to the manufacturers’ instructions.
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2.8 Generation and characterization of M1 macrophages
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M1 macrophages were generated from THP-1 cells according to a previous
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protocol with some modifications [28]. Briefly, THP-1 cells were treated with 100 nM 8
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PMA for 24 h to induce differentiation into macrophage-like cells. Then, the cells
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were stimulated with 50 ng/mL rhGM-CSF for 6 days. On the seventh day, the cells
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were harvested for further experiments.
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2.9 Western blotting
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Western blotting was performed as previously described [29]. Briefly, cells were
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collected and lysed with RIPA buffer, and total protein was analyzed by western
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blotting.
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2.10 Annexin V/PI assay
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HepG2/ADM and MCF-7/ADR cells seeded in 6-well plates were treated with
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DAVLBH (5 µM or 6 µM) alone or in combination with conditioned medium from
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GM-CSF-educated M1 macrophages for 24 h. The cells were then harvested and
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analyzed by flow cytometry (Guava Technologies, Millipore, Billerica, MA) using an
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Annexin V-FITC/PI apoptosis detection kit according to the manufacturer’s protocol.
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2.11 Statistical analysis
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The data are presented as mean ± SEM after analysis using GraphPad Prism 5.0
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(GraphPad Software, Inc., San Diego, CA). The two-tailed unpaired t-test was used to
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compare differences between two groups, and differences among more than two
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groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. P <
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0.05 indicated a significant difference.
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3. Results
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3.1 Z-GP-DAVLBH-mediated inhibition of HepG2/ADM tumor growth may be 9
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associated with the skewing of TAMs from the M2 to the M1 phenotype First, we evaluated the antitumor effect of Z-GP-DAVLBH on HepG2/ADM cells,
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which overexpress ABCB-1 and are resistant to DOX and vincristine compared with
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parental cells (Fig. S1A-C). We found that the anti-proliferative effect of
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Z-GP-DAVLBH on HepG2/ADM cells was much lower than that on HepG2 cells,
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with IC50 values of 16 µM and 0.55 µM, respectively (Fig. 1A). In addition,
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Z-GP-DAVLBH stimulates ABCB1 ATPase activity (Fig. S2). These results indicated
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that Z-GP-DAVLBH was the substrate of ABCB-1 thus exerting poor antitumor
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activity in the ABCB-1-overexpressing cancer cells in vitro. Consistent with previous
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reports
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Z-GP-DAVLBH effectively destroyed tumor vessels in HepG2/ADM xenografts
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within 4 h by selectively targeting tumor pericytes (Fig. S3A). As a result, 2.0 mg/kg
239
Z-GP-DAVLBH dramatically inhibited tumor growth, with an inhibitory rate of
240
approximately 75%. Tumor volume in the vehicle group increased from 159.06 ±
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33.35 to 635.91 ± 292.86 mm3, whereas that in the Z-GP-DAVLBH group merely
242
increased from 145.29 ± 48.92 to 159.60 ± 44.48 mm3. In addition, the average tumor
243
weight was 0.88 ± 0.26 g in the vehicle group and 0.38 ± 0.20 g in the
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Z-GP-DAVLBH-treated group (Fig. 1B-D). Further pathological examination
245
revealed that Z-GP-DAVLBH caused widespread necrosis, decreased the Ki67
246
proliferative index and CD31-positive microvessel density. However, Z-GP-DAVLBH
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had a negligible effect on tumor ABCB-1 expression, indicating that ABCB-1 is not
248
involved in the antitumor effect of Z-GP-DAVLBH. In addition, we found that
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Z-GP-DAVLBH treatment increased the population of TUNEL-positive cells (Fig. 1E,
250
F). These results indicate that Z-GP-DAVLBH inhibits HepG2/ADM tumor growth,
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and this effect may be associated with tumor vessel disruption and cancer cell
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tumor
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apoptosis. Given that M1 macrophages promote tumor cell apoptosis [8], we next explored
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whether Z-GP-DAVLBH-mediated HepG2/ADM apoptosis is associated with the
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induction of macrophages toward the M1 phenotype. Immunofluorescence assays of
256
macrophages in HepG2/ADM tumor xenografts with polarization state-specific
257
markers revealed that Z-GP-DAVLBH reprogramed TAMs from an M2- to an
258
M1-dominant phenotype, as evidenced by the increase in iNOS+/F4/80+ M1
259
macrophages and the reduction in Arg-1+/F4/80+ M2 macrophages (Fig. 1G).
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However, Z-GP-DAVLBH had a negligible effect on the Ly6G, CD11b and IFN-β
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staining tumor-associated N1 neutrophils [30, 31] (Fig. S4).
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3.2 Z-GP-DAVLBH-mediated inhibition of MCF-7/ADR tumor growth may be
264
associated with the skewing of TAMs from the M2 to the M1 phenotype We next tested the effect of Z-GP-DAVLBH on another multidrug-resistant cell
266
line, MCF-7/ADR. These cells overexpressed ABCB-1 and were resistant to DOX
267
and vincristine (Fig. S5). The anti-proliferative effect of Z-GP-DAVLBH on
268
MCF-7/ADR cells was much lower than that on MCF-7 cells (Fig. 2A), with IC50
269
values of 19.92 µM and 0.91 µM, respectively. These results were consistence with
270
the effect on HepG2/ADM cells (Fig. 1A). Moreover, Z-GP-DAVLBH (2 mg/kg)
271
significantly suppressed MCF-7/ADR tumor xenograft growth, with an inhibitory rate
272
of approximately 70% (Fig. 2B-D). We also found that Z-GP-DAVLBH effectively
273
destroyed the vessels in MCF-7/ADR tumor xenografts within 4 h by selectively
274
targeting tumor pericytes (Fig. S3B). H&E staining showed that Z-GP-DAVLBH (2
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mg/kg) treatment led to extensive necrosis in both the core and periphery of
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MCF-7/ADR tumors. The CD31-positive microvessel density and the Ki67
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proliferative index, rather than ABCB-1 expression, were dramatically decreased in
278
tumors
279
immunofluorescence analyses showed that Z-GP-DAVLBH skewed TAMs from the
280
M2 phenotype to the M1 phenotype, as evidenced by the increased iNOS+/F4/80+ M1
281
macrophages and the decreased Arg-1+/F4/80+ M2 macrophages (Fig. 2G). Taken
282
together, these results indicate that Z-GP-DAVLBH has potent antitumor activity
283
against MCF-7/ADR tumor xenografts, similar to that against HepG2/ADM tumor
284
xenografts, and that this effect may be associated with the skewing of TAMs from the
285
M2 to the M1 phenotype.
the
Z-GP-DAVLBH-treated
group
(Fig.
2E-F).
Furthermore,
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3.3 M1 macrophage depletion attenuates the antitumor effect of Z-GP-DAVLBH
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on HepG2/ADM tumor xenografts
To further validate whether M1 macrophages are primarily responsible for the
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Z-GP-DAVLBH-induced suppression of tumor growth, GdCl3, a selective M1
291
macrophage scavenger that induces the apoptosis of inflammatory macrophages via
292
competitive inhibition of Ca2+ mobilization and damage to plasma membranes [28],
293
was used to selectively deplete the M1 population. Mice bearing HepG2/ADM tumors
294
were pretreated with GdCl3 (10 mg/kg) for two weeks to deplete M1 macrophages.
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We found that GdCl3-induced M1 depletion significantly attenuated the antitumor
296
effect
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GdCl3+Z-GP-DAVLBH group was 24%, which was much lower than that in the
298
Z-GP-DAVLBH-alone group, in which the inhibitory rate was 47% (Fig. 3A-C).
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Tumors in the GdCl3-treated group were confirmed to lack iNOS+/F4/80+ M1
300
macrophages, and the Z-GP-DAVLBH-mediated increase in M1 macrophages was
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blocked by GdCl3 treatment (Fig. 3D, E), whereas the Arg-1+/F4/80+ M2
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of
Z-GP-DAVLBH;
the
tumor
growth
inhibitory
rate
in
the
12
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GdCl3 alleviated the Z-GP-DAVLBH-induced HepG2/ADM tumor cell apoptosis
304
(based on the percentage of TUNEL-positive cells) and the decrease in the Ki67
305
proliferative index (Fig. 3F, G). These data indicate that M1 macrophages are critical
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for the antitumor effect of Z-GP-DAVLBH on HepG2/ADM tumor xenografts.
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3.4 GM-CSF contributes to Z-GP-DAVLBH-induced TAM reprograming toward
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the M1 phenotype
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We next investigated the underlying mechanism of Z-GP-DAVLBH-induced M1
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macrophage skewing. Given that LPS, INF-γ and GM-CSF are the three major
312
stimulators of M1 macrophages [8, 32], we evaluated the changes in these growth
313
factors in serum and tumor tissue from mice bearing HepG2/ADM xenografts. The
314
ELISA assay results showed that Z-GP-DAVLBH treatment for 24 h increased
315
GM-CSF levels in both mouse serum and tumor tissue (Fig. 4A, B) but had a
316
negligible effect on the levels of LPS and INF-γ (Fig. S7). We also found that the
317
level of M-CSF, a stimulator of M2 macrophages [8], was decreased in mouse serum
318
and tumor tissue after Z-GP-DAVLBH treatment (Fig. 4C, D). In addition,
319
Z-GP-DAVLBH increased IL-6 levels (M1 macrophage marker) and decreased IL-10
320
levels (M2 macrophage marker) in mouse serum and tumor tissue (Fig. 4A-D) [8]. To
321
determine the role of GM-CSF in the antitumor effect of Z-GP-DAVLBH, a GM-CSF
322
neutralizing antibody was used to capture the secreted GM-CSF in HepG2/ADM
323
tumor xenografts. Our results showed that GM-CSF neutralizing antibody (2 mg/kg)
324
retarded the antitumor effect of Z-GP-DAVLBH (2 mg/kg); the tumor growth
325
inhibitory rate in the group treated with the GM-CSF neutralizing antibody and
326
Z-GP-DAVLBH
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was
11%,
which
was
much
lower
than
that
in
the 13
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Z-GP-DAVLBH-alone group (the inhibitory rate was 41%) (Fig. 5A-C). The
328
GM-CSF neutralizing antibody decreased the population of iNOS+/F4/80+ M1
329
macrophages and attenuated the Z-GP-DAVLBH-mediated increase in M1
330
macrophages (Fig. 5D, E). In addition, our results showed that the GM-CSF antibody
331
abated
332
(TUNEL-positive cells) and the decrease in the Ki67 proliferative index (Fig. 5F, G).
333
These data indicate that GM-CSF is a key regulator of Z-GP-DAVLBH-induced M1
334
macrophage skewing, which contributes to the antitumor effects of Z-GP-DAVLBH.
HepG2/ADM
tumor
cell
apoptosis
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Z-GP-DAVLBH-induced
SC
the
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3.5 M1 macrophage-conditioned medium promotes Z-GP-DAVLBH-induced
337
apoptosis of cancer cells with MDR in vitro
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Next, we further investigated whether GM-CSF-stimulated M1 macrophages
339
contribute to Z-GP-DAVLBH-induced apoptosis of multidrug-resistant cancer cells.
340
THP-1 cells in the monocyte state were stimulated with PMA (100 nM) and GM-CSF
341
(50 ng/mL) to differentiate into an M1 macrophage-like phenotype, as characterized
342
by the increased secretion of IL-6 and TNF-α (Fig. 6A) and the overexpression of
343
F4/80, iNOS, CD68 and CD86 compared with unstimulated THP-1 cells (Fig. 6B). As
344
Z-GP-DAVLBH can be hydrolyzed into DAVLBH in tumor tissue [24], we combined
345
M1 macrophage-conditioned medium with DAVLBH to test the cytotoxicity in
346
HepG2/ADM and MCF-7/ADR cells. Annexin V-FITC/PI assays showed that
347
treatment with M1 macrophage-conditioned medium or DAVLBH (5 µM and 6 µM)
348
alone had a negligible effect on HepG2/ADM and MCF-7/ADR cells, whereas
349
DAVLBH (5 µM and 6 µM) combined with M1 macrophage-conditioned medium
350
induced the apoptosis of approximately 40% and 60% of HepG2/ADM cells (Fig. 5C)
351
and of approximately 30% and 40% of MCF-7/ADR cells (Fig. 5D). Western blotting
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353
treatment with the combination of M1 macrophage-conditioned medium and
354
DAVLBH, while almost no cleaved-PARP was observed in the groups treated with
355
M1 macrophage-conditioned medium or DAVLBH alone (Fig. 5E). Taken together,
356
these data indicate that DAVLBH is not sufficient to induce the apoptosis of
357
HepG2/ADM and MCF-7/ADR cells and that M1 macrophages promote such cell
358
apoptosis.
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4. Discussion
For decades, MDR has hampered the success of cancer chemotherapy. Attempts
362
have been made to identify selective ABCB-1 inhibitors that overcome MDR without
363
non-specific toxicity, but there is still no such effective and safe agent in clinic. Here,
364
we show that Z-GP-DAVLBH, a pericyte-targeting VDA, selectively disrupted the
365
tumor vasculature, increased the secretion of GM-CSF, skewed TAMs toward the
366
antitumor M1 phenotype, induced tumor cell apoptosis, and ultimately inhibited the
367
growth of multidrug-resistant HepG2/ADM and MCF-7/ADR tumor xenografts.
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VDAs are considered promising agents for cancer treatment [21]. However,
369
TAMs have been shown to hamper the efficacy of VDAs [33]. For example, a
370
frequently used VDA, CA4-P, triggers TIE-2+ M2 macrophage recruitment [34],
371
which promotes tumor angiogenesis, metastasis and tumor regrowth and these are
372
some of the significant reasons for the failure of this treatment. In contrast to CA4-P,
373
Z-GP-DAVLBH did not increase M2 macrophages but rather skewed TAMs toward
374
the antitumor M1 phenotype by increasing GM-CSF secretion and then induced
375
apoptosis of drug-resistant tumor cells. The different effects between CA4-P and
376
Z-GP-DAVLBH on the polarization of TAMs may be attributed to various reasons.
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tumor vessels to cause hypoxia in tumor tissues [34], which has been demonstrated to
379
be an important factor promoting macrophage M2 polarization [35]. On the contrary,
380
Z-GP-DAVLBH had a negligible effect on the expression of HIF-1α on both
381
HepG2/ADM and MCF-7/ADR tumors (Fig. S8). Our study extends the
382
understanding of the underlying mechanisms of VDA-mediated antitumor effects.
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TAMs are a major cellular constituent of the tumor microenvironment and
384
display an M1 or M2 phenotype in response to different stimuli [36], and TAMs are
385
one of the most interesting targets for cancer therapy. So far, the major strategy for
386
targeting TAMs is to inhibit signaling through myeloid cell receptors such as
387
colony-stimulating factor-1 receptor using genetic methods or pharmacological
388
inhibitors, which have been applied in preclinical or clinical trials to abolish the
389
macrophage-mediated pro-tumor effects [37, 38]. However, as these strategies target
390
all macrophages, they have systemic toxicities that restrict their clinical applications
391
[39]. Reprograming TAMs toward M1 macrophages by activating costimulatory
392
CD40 with an agonist antibody [40], administering Toll-like receptor agonists [41, 42],
393
inhibiting colony-stimulating factor-1 receptor [43] or using pharmaceuticals [44] has
394
the potential to inhibit tumor growth and enhance the chemotherapeutic efficacy in
395
sensitive tumors. In addition, reprogramming TAM polarization toward the antitumor
396
M1 phenotype has been shown to contribute to RRx-001-mediated MDR reversal [45].
397
In the present study, we also found that Z-GP-DAVLBH overcame MDR by skewing
398
TAMs toward M1 macrophages. Our results provide evidence to support the
399
hypothesis that reprogramming TAMs toward M1 macrophages is effective for
400
circumventing tumor MDR. Furthermore, compared with the non-selective
401
distribution of the ABCB-1 inhibitor, Z-GP-DAVLBH selectively accumulates in the
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tumor and has minimal effects on body weight or major organs in tumor-bearing mice
403
[24], indicating that Z-GP-DAVLBH may achieve great efficacy in tumors with MDR
404
in future preclinical and clinical trials. In this regard, this study sheds new light on the
405
application of VDAs in the treatment of resistant tumors. GM-CSF is generally recognized as one of the most important inflammatory
407
cytokines; it has been used to bolster antitumor immune responses due to its role in
408
re-educating TAMs toward the antitumor M1 phenotype [46, 47]. Vaccination with
409
irradiated B16 melanoma cells engineered to secrete GM-CSF stimulates more potent
410
antitumor immunity [48]. Intra-tumoral injection of GM-CSF has been demonstrated
411
to induce TAM polarization toward the antitumor M1 phenotype [49, 50]. However,
412
GM-CSF can also promote tumor progression [51-53]. The role of GM-CSF in
413
antitumor therapy is controversial and depends on the concentration and the overall
414
cytokine milieu [54]. In the present study, we found that Z-GP-DAVLBH treatment
415
resulted
416
Z-GP-DAVLBH-mediated TAM repolarization toward the antitumor M1 phenotype.
417
GM-CSF is secreted by cancer cells, ECs and stromal cells in the tumor
418
microenvironment
419
Z-GP-DAVLBH-mediated regulation of GM-CSF secretion must be studied further.
420
Besides GM-CSF, LPS and INF-γ, there are two further important activators of M1
421
macrophages [8, 32]. LPS is a pathogen-associated molecular mainly derived from
422
gram-negative bacteria, it engages the Toll-like receptor 4 on the surface of
423
macrophages to activate transcription factors to exert an inflammatory response to
424
fight against pathogenic insult [55]. INF-γ is a cytokine that produced predominantly
425
by natural killer and natural killer T cells, and is critical for innate and adaptive
426
immunity against viral, some bacterial and protozoal infections [56]. However,
up-regulation
of
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the
[54].
However,
GM-CSF,
the
which
is
mechanisms
involved
in
underlying
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Z-GP-DAVLBH had a negligible effect on these factors, which may due to the fact
428
that the macrophage M1 polarization was caused by Z-GP-DAVLBH-mediated
429
internal tumor vascular disruption, but not by external infections. In conclusion, this study showed that Z-GP-DAVLBH skewed TAMs from the
431
M2 phenotype toward the M1 phenotype by up-regulating GM-CSF secretion and
432
ultimately reversed tumor MDR. Our study provides new evidence that VDAs are
433
promising agents for reversing MDR and sheds new light on the mechanisms
434
underlying M1 macrophage repolarization in the treatment of tumors with MDR.
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Acknowledgments
This study was supported by the National Natural Science Foundation of China
438
(U1401225, 81630095 and 81573455), Natural Science Foundation of Guangdong
439
Province (2017A030310453), National Science and Technology Major Project
440
(2017ZX09101003-008-008) and Pearl River Scholar Funded Scheme (D. M. Zhang).
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. Z-GP-DAVLBH inhibits HepG2/ADM tumor xenograft growth and induces TAM skewing toward the M1 phenotype. (A) The IC50 values of
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Z-GP-DAVLBH in HepG2 and HepG2/ADM cells. Cells were treated with various concentrations of Z-GP-DAVLBH for 24 h and the cell viabilities were detected by MTT assay. (B-D) Mice bearing HepG2/ADM tumor xenografts received an i.v.
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injection of Z-GP-DAVLBH (2.0 mg/kg) or vehicle (1% DMSO) once every two days. The (B) tumor growth curves, (C) tumor images and (D) tumor weight are shown. (E)
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Representative images and (F) quantification of H&E, ABCB1, CD31, Ki67 and TUNEL in HepG2/ADM tumors. (G) Z-GP-DAVLBH decreased M2 macrophages (Arg-1+/F4/80+) and increased M1 macrophages (iNOS+/F4/80+) in HepG2/ADM tumors. Representative images of immunofluorescence and quantification are shown.
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Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle group.
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Figure 2. Z-GP-DAVLBH inhibits MCF-7/ADR tumor xenograft growth and
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induces TAM skewing toward the M1 phenotype. (A) The IC50 values of Z-GP-DAVLBH in MCF-7 and MCF-7/ADR cells. Cells were treated with various concentrations of Z-GP-DAVLBH for 24 h and the cell viabilities were detected by MTT assay. (B-D) Mice bearing MCF-7/ADR tumor xenografts received an i.v. injection of Z-GP-DAVLBH (2.0 mg/kg) or vehicle (1% DMSO) once every other day. The (B) tumor growth curves, (C) tumor images and (D) tumor weight are shown. (E-F) Z-GP-DAVLBH decreased microvessel density and induced cancer cell apoptosis. (E) Representative images and (F) quantification of H&E, ABCB1, CD31,
ACCEPTED MANUSCRIPT Ki67 and TUNEL in MCF-7/ADR tumors. (G) Z-GP-DAVLBH decreased M2 macrophages (Arg-1+/F4/80+) and increased M1 macrophages (iNOS+/F4/80+) in MCF-7/ADR tumors. Representative images of immunofluorescence and the
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quantification are shown. Data are presented as mean ± SEM (n = 5). *P < 0.05, **P
Figure 3. M1 macrophage depletion attenuates the antitumor effect of
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Z-GP-DAVLBH. (A-C) Mice bearing HepG2/ADM tumor xenografts were pretreated with GdCl3 (10 mg/kg) twice per week for two weeks to selectively deplete
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M1 macrophages, and the mice then received an i.v. injection of Z-GP-DAVLBH (2.0 mg/kg). The (A) tumor volume, (B) tumor images and (C) tumor weight are shown. (D-E) Immunofluorescence analysis of the effects of GdCl3 and Z-GP-DAVLBH on M1 macrophages. (D) Representative images of the immunofluorescence assay and
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(E) the quantification of M1 macrophages (iNOS+/F4/80+). (F) Representative images and (G) the quantification of necrosis, Ki67 index and apoptotic cells (TUNEL) in tumors from (B). Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01
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compared with the Z-GP-DAVLBH group.
Figure 4. ELISA analysis of cytokine levels in serum and tumors. Mice bearing HepG2/ADM tumors received an i.v injection of 2 mg/kg Z-GP-DAVLBH. Blood and tumors were collected at the indicated times. Serum and tumor homogenates were analyzed by ELISA. Z-GP-DAVLBH increased the secretion of GM-CSF and IL-6 in (A) serum and (B) tumors and inhibited the secretion of M-CSF and IL-10 in (C) serum and (D) tumors. Data are presented as mean ± SEM (n = 3). *P < 0.05, **P <
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Figure 5. GM-CSF neutralizing antibody abates the antitumor effects of Z-GP-DAVLBH on HepG2/ADM tumor xenografts. (A-C) Mice bearing tumors
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were treated with vehicle (1% DMSO), Z-GP-DAVLBH (2 mg/kg), anti-GM-CSF (2 mg/kg) or anti-CM-CSF combined with Z-GP-DAVLBH. (A) Tumor growth curves, (B) tumor images and (C) tumor weight are shown. (D-E) Immunofluorescence
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analysis of the effects of anti-GM-CSF and Z-GP-DAVLBH on M1 macrophages. (D) Representative images of the immunofluorescence assays and (E) the quantification
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of M1 macrophages (iNOS+/F4/80+). (F) Representative images and (G) the quantification of necrosis, Ki67 index and apoptotic cells (TUNEL) in tumors from (B). Data are presented as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the vehicle group. #P < 0.05 and ###P < 0.001 compared with the
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Figure 6. GM-CSF-educated M1 macrophage conditioned medium enhances the
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in vitro antitumor effects of DAVLBH. (A-B) THP-1 cells were polarized to M1
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macrophages by sequential treatment with PMA (100 nM) and GM-CSF (50 ng/mL). (A) ELISA assays of IL-6 and TNF-α. (B) Western blotting for F4/80, iNOS, CD68 and CD86. (C-E) Conditioned medium (CM) was derived from GM-CSF-educated M1 macrophages. HepG2/ADM and MCF-7/ADR cells were treated with DAVLBH alone or in combination with CM for 24 h, and the cells were then collected for western blotting analysis or Annexin V-PI assays. Representative images of (C) Annexin V-PI analysis of HepG2/ADM cells, (D) Annexin V-PI analysis of MCF-7/ADR cells and (E) western blotting. Data are presented as mean ± SEM (n =
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ACCEPTED MANUSCRIPT Highlights • A vascular disrupting agent, Z-GP-DAVLBH, inhibits the growth of multidrug-resistant HepG2/ADM and MCF-7/ADR tumor xenografts but has no significant cytotoxicity toward these
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two cancer cell lines in vitro. • Z-GP-DAVLBH induces the repolarization of tumor-associated macrophages to the M1
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• M1 macrophages contribute to the anti-cancer effect of Z-GP-DAVLBH both in vitro and in vivo.
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M1 macrophage repolarization.
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• Granulocyte-macrophage colony-stimulating factor plays a key role in Z-GP-DAVLBH-induced