Proteasome Inhibitor–Loaded Micelles Enhance Antitumor Activity Through Macrophage Reprogramming by NF-κB Inhibition

Accepted Manuscript Proteasome inhibitor loaded micelles enhance antitumor activity through macrophage reprograming by NF-κB inhibition Hailiang Wu, Anqi Tao, John D. Martin, Sabina Quader, Xueying Liu, Kei Takahashi, Louise Hespel, Yutaka Miura, Yoshihiro Hayakawa, Tatsuro Irimura, Horacio Cabral, Kazunori Kataoka PII:

S0022-3549(17)30217-4

DOI:

10.1016/j.xphs.2017.03.031

Reference:

XPHS 709

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 17 January 2017 Revised Date:

25 March 2017

Accepted Date: 27 March 2017

Please cite this article as: Wu H, Tao A, Martin JD, Quader S, Liu X, Takahashi K, Hespel L, Miura Y, Hayakawa Y, Irimura T, Cabral H, Kataoka K, Proteasome inhibitor loaded micelles enhance antitumor activity through macrophage reprograming by NF-κB inhibition, Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.xphs.2017.03.031. 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 Proteasome inhibitor loaded micelles enhance antitumor activity through macrophage reprograming by NF-κB inhibition Hailiang Wu1, Anqi Tao2, John D. Martin2, Sabina Quader1, Xueying Liu1, Kei Takahashi3, Louise

RI PT

Hespel2, Yutaka Miura2, Yoshihiro Hayakawa3,4, Tatsuro Irimura3,5, Horacio Cabral2*, Kazunori

SC

Kataoka1,6*

1. Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14,

M AN U

Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.

2. Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

3. Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical

TE D

Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 4. Division of Pathogenic Biochemistry, Department of Bioscience, Institute of Natural Medicine,

EP

University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. 5. Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

AC C

6. Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

*Corresponding authors E-mail: [email protected], [email protected]

ACCEPTED MANUSCRIPT Abstract Macrophage reprogramming towards a tumor attacking phenotype is a promising treatment strategy, yet such strategies are scarce and it is not clear how to combine them with cytotoxic therapies that

RI PT

are often used to treat solid tumors. Here, we evaluate whether a micelle-encapsulated proteasome inhibitor, i.e. the peptide aldehyde drug MG132, which is cytotoxic to cancer cells, can reprogram macrophages to attack the tumor. Through in vitro studies, we demonstrated that the proteasome

SC

inhibition reduces nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling – a known promoter of tumor-supporting macrophages and chemoresistance – in both cancer cells

M AN U

and macrophages. In in vivo studies, we showed that, while free MG132 did not affect the macrophage phenotype in tumors even at its maximum tolerated dose, the micellar formulation of MG132 safely achieved simultaneous cancer cell killing and macrophage reprogramming, thereby,

TE D

enhancing the antitumor efficacy in a syngeneic, orthotopic breast cancer model.

Keywords: Macrophage reprogramming, NF-κB, Proteasome inhibitor, polymeric micelles,

AC C

EP

MG132, breast cancer

ACCEPTED MANUSCRIPT Introduction The presence of tumor-associated macrophages (TAMs) correlates with poor prognosis in several cancers.1-3 TAM programming promotes tumor progression through immunosuppression of

RI PT

CD8+ T-cells, matrix remodeling, angiogenesis, tumor invasion, metastasis, and drug resistance.4-6 Accordingly, preclinical studies have demonstrated the treatment benefit of switching the phenotype of TAMs from the pro-tumor M2 phenotype into the antitumor M1 phenotype in combination with

SC

chemotherapy and/or radiation.7-11 These treatment strategies are promising in patients as well.12-14 TAM reprogramming strategies are combinable with cytotoxic therapies, as the latter induce

M AN U

immunogenic cell death that renders apoptotic tumor cells as endogenous vaccines.15 On the other hand, some drugs promote lymphodepletion or favor the generation of M2 macrophages and therefore oppose TAM-targeting strategies.16 Thus, translatable therapeutic strategies capable of killing cancer cells while promoting macrophage polarization to M1 are of great importance.

TE D

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) plays an essential role in inflammatory signals of TAMs and cancer cells. Thus, NF-κB is central to strategies that aim

EP

to modulate the immune system and kill cancer cells simultaneously.17 Indeed, NF-κB induces macrophages to increase cancer cell invasiveness,18 while NF-κB inhibition polarizes TAMs to

AC C

attack cancer cells.19 Independent of immune effects, NF-κB is an anti-apoptotic signal to cancer cells thereby promoting chemoresistance.20 Commonly used chemotherapeutics can upregulate NF-κB, which makes them less suitable for combining with macrophage reprogramming strategies.21 Contrarily, as NF-κB is activated through phosphorylation of the inhibitor protein IκB, which leads to degradation of IκB through the ubiquitin-proteasome pathway, proteasome inhibitors can reduce levels of NF-κB22-25 and ameliorate NF-κB mediated resistance to chemotherapy,26 while exerting cytotoxic effects in cancer cells. Thus, proteasome inhibitors appear to be well-suited for targeting both cancer cells and TAMs in solid tumors, but their effects have not been investigated.

ACCEPTED MANUSCRIPT Among proteasome inhibitors, the peptide aldehyde Cbz-leu-leu-leucinal (MG132) is highly potent and rapidly blocks proteasomes, and presents significant inhibition of NF-κB activation,23,24,27 which could be exploited for treating cancer cells and reprogramming TAMs.

RI PT

However, MG132 is readily oxidized in vivo and its bioavailability is poor.28 Polymeric micelles incorporating the peptide aldehyde proteasome inhibitor MG132 protect the aldehyde group of the drug in the harsh in vivo environment, and become activated after accumulating at the tumor

SC

site.29,30 Moreover, the drug release of MG132-loaded micelles (MG132/m) is triggered at endosomal pH, which increases the therapeutic efficiency and reduces toxicity through selective

M AN U

intracellular drug activation.29 Here, we sought to determine whether MG132/m could achieve simultaneous targeted cytotoxic effects against cancer cells, NF-κB inhibition and macrophage

Materials and methods Materials

TE D

reprogramming.

EP

α-Methoxy-ω-amino-poly(ethylene glycol) (MeO-PEG-NH2; Mw = 12,000) was purchased from NOF Co, Inc. (Tokyo, Japan). β-Benzyl-L-aspartate N-carboxy anhydride

AC C

(BLA-NCA) was bought from Chuo Kaseihin Co., Inc. (Tokyo, Japan). Dimethyl sulfoxide (DMSO), N,N-dimethylacetamide (DMAc), dichloromethane (DCM), acetic anhydride and hydrazine monohydrate were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Diethyl ether was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). MG132 (Z-Leu-Leu-Leu-H) was purchased from Peptide institute, Inc. (Osaka, Japan). Spectra/Por dialysis tube (MWCO = 6-8000 Da) was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Bortezomib, Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Dainippon Sumitomo Pharma Co.

ACCEPTED MANUSCRIPT Ltd. (Osaka, Japan). Cell Counting Kit-8 and DAPI were purchased from Dojindo Laboratories (Kumamoto, Japan). Anti-CD68 monoclonal antibody, anti-NOS2 monoclonal antibody, and anti-mouse IgG AlexaFluor488, anti-mouse IgG AlexaFluor647 were bought from Abcam

RI PT

(Cambridge, UK), while anti-CD163 antibody was purchased from CosmoBio Co., Ltd (Tokyo, Japan). Anti-NF-κB p100/p52 antibody and goat anti-mouse AlexaFluor488 IgG were also purchased from Abcam. Goat anti-rabbit AlexaFluor488 IgG and goat anti-rabbit AlexaFluor647

SC

IgG were purchased from ThermoFisher Scientific (Waltham, MA USA).

M AN U

Cell line

Murine breast carcinoma cells 4T1 and J774 A-1 macrophages (JCRB cell bank, Japan) were cultured in DMEM supplemented 10% FBS, 1% penicillin and streptomycin at 37 °C under 5% CO2. 4T1 cells with luminescent nuclear reporter of NF-κB (4T1-NF-κB-luc) were prepared by as

TE D

previously reported.31 Briefly, 4T1 cells (5 x 105/well) were transfected with pGL4.32 vector using Lipofectamine 2000. 4T1-NF-κB-luc cells were then selected by using Hygromycin B (100 ng/mL)

EP

and cloned by limiting dilution. The luminescent signal from 4T1-NF-κB-luc cells was confirmed

Animals

AC C

and recorded by In Vivo Imaging System (IVIS, PerkinElmer Japan Co., Ltd.).

Immunocompetent female BALB/c mice at 6 weeks of age were used as hosts for the

breast tumor model. All animals were obtained from Charles River Laboratories (Tokyo, Japan), and treated in accordance with the policies of the Animal Ethics Committee of The University of Tokyo and the Innovation Center of NanoMedicine.

Synthesis of block copolymer and preparation of proteasome inhibitor (MG132)-loaded micelles

ACCEPTED MANUSCRIPT (MG132/m) Poly(ethylene

glycol)-b-poly(β-benzyl-L-aspartate)

(PEG-PBLA)

copolymer

was

prepared according to the previously reported method29, i.e. ring-opening polymerization of

RI PT

BLA-NCA initiated by MeO-PEG-NH2. The degree of polymerization of the PBLA block of PEG-PBLA was determined to be 40 by 1H-NMR spectra. Then, the amine at the ω-end of PEG-PBLA was acetylated by adding excess acetic anyhydride in DMSO. The resulting product

introduce

hydrazide

units

on

the

polyamino

SC

(PEG-PBLA-Ac) was then aminolyzed with excess of hydrazine monohydrate in DMSO to acid

side

moieties.29

The

obtained

M AN U

PEG-poly(aspartate-hydrazide) was washed using cold ether 3 times. MG132 was mixed with PEG-poly(aspartate-hydrazide) in DMAc to conjugate the drug to the polymer via a Schiff base. MG132/m

self-assembled

after

drop-wise

mixing

of

the

DMAc

solution

of

PEG-poly(aspartate-hydrazide-MG132) in water.29 The size of the micelles was determined to be 40

TE D

nm with a narrow PDI (0.10) by using Zetasizer (Malvern, UK). Empty micelles were prepared by drop-wise addition of a 1 mg/ml DMAc solution of PEG-PBLA in water. The size of the empty

AC C

EP

micelles was determined to be 80 nm with narrow PDI (0.08).

In vitro cytotoxicity study

4T1-NF-κB-luc cells were seeded in 96-well plates at 2.5 x 104 cells/well. Twenty-four

hours later, free MG132 or MG132/m were added to make final concentrations of 32-, 16-, 8-, 4-, 2-, 1- and 0.5-µg/ml. The cell viability was determined after 6-, 24-, 48- and 72-h incubation by adding 10 µl of CCK8 into each well and culturing for 2 h at 37 °C. Cell viability was measured by the absorbance at 450 nm using Infinite M1000 PRO microplate reader (Tecan Trading AG, Switzerland). Luminescent NF-κB level was quantified by the photon flux (photon/s) by IVIS after

ACCEPTED MANUSCRIPT addition of 300 µg/well of D-luciferin (Promega; Madison, WI). The bioluminescent signal was quantified by IVIS software.

RI PT

In vitro evaluation of NF-κB levels in macrophages J774 A-1 macrophages (5000 per well) were cultured in DMEM containing 10% (v/v) FBS for 24 h before drug administration. MG132/m of different concentrations were added and

SC

incubated with the macrophages for 24 h before immunostaining. Free MG132 was used as control. The cells were fixed by 4% PFA, followed by permeabilization with 0.5% Tween 20 in PBS in

M AN U

order to detect intracellular antigen. Cells were immersed in Blocking One solution before incubating with primary antibody (anti-NF-κB p100/p52 antibody with 1:200 dilution) and followed by secondary antibody (anti-Rabbit Alexa 488 with 1:300 dilution). Hoechst was used to stain cell nucleus and washing buffer was made of 0.2% Tween 20, 0.2% BSA in PBS. The NF-κB

TE D

expression in macrophages was evaluated by IN Cell Analyzer 2200 (GE Healthcare Life Sciences).

EP

In vitro macrophage polarization

To determine the amount of in vitro macrophage polarization induced by MG132/m, J774

AC C

A-1 macrophages (5000 per well) were plated to 8-well glass-based chamber slides together with 4T1-NF-κB-Luc cells (5000 per well). After incubation for 24 h, cells were treated with different concentrations of MG132/m or free MG132. Then, the cells were incubated for 24 h in 5% CO2 at 37 °C. After that, the cells were fixed by 4% PFA, and treated with Blocking One solution prior to incubating with primary antibodies (anti-NOS2 antibody with 1:200 dilution for M1-type macrophage, anti-CD163 antibody with 1:200 dilution for M2-type macrophage) and followed by secondary antibodies (goat anti-rabbit Alexa Fluor 647 IgG for NOS2-Ab, goat anti-mouse AlexaFluor488 IgG for CD163-Ab). Hoechst was used to stain cell nucleus. The polarization of

ACCEPTED MANUSCRIPT macrophages treated with each concentration of MG132 micelle was observed by confocal laser scanning microscopy (LSM780, Carl Zeiss) after staining.

RI PT

In vivo NF-κB activity in tumors Mice bearing orthotopic 4T1-NF-κB-luc tumors were intravenously injected with PBS, free MG132 drug (8 mg/kg), or MG132/m (16- or 64-mg/kg). Luminescent signal from the

SC

4T1-NF-κB-luc tumors was recorded as photon flux by IVIS after intraperitoneal injection of 100 mg/kg of D-luciferin at 6-, 24-, 48- and 72-h post-treatment of free MG132 or MG132/m. The level

M AN U

of NF-κB was monitored as the photon flux per tumor volume by using the following equation: NF-κB level in tumor = Photon flux/(a×b2/2) where a and b were the major and minor diameters of the tumor measured by a caliper, respectively. In addition, the effect of the polymer forming the micelles on the NF-κB signal of tumors was

EP

block copolymer basis.

TE D

evaluated by intravenously injecting empty micelles prepared from PEG-PBLA at 10 mg/kg in a

Assessment of macrophage population by immunofluorescence

AC C

To study the distribution of macrophages and their phenotype in non-treated tumors,

4T1-NF-κB-luc tumors (n = 4) were harvested after 8 days of inoculating the cancer cells, fixed in 4% formaldehyde, incubated in 30% sucrose and embedded in Tissue-Tek OCT Compound (Sakura-finetek Japan). Sections from each tumor were cut by a Cryostat (Leica CM1950) and stained for macropage distribution by using anti-CD68 mouse antibody, M1-macropage phenotype (NOS2+) by using anti-NOS2 antibody, and for M2-macrophage phenotype (CD163+) with anti-CD163 antibody, followed by staining with fluorescent secondary antibodies. For evaluation of the effect of the drugs on the macrophage populations within tumors, mice were treated with free

ACCEPTED MANUSCRIPT MG132 (8 mg/kg) or MG132/m (64 mg/kg), and 48 h after treatment, tumors were collected and processed for histological analysis as mentioned above. The tissues were imaged by using a confocal laser-scanning microscope (LSM780 Meta, Zeiss; Germany) with a 20× objective. The

RI PT

amount of macrophages (CD68+) was evaluated by ImageJ, and their phenotype was assessed by the colocalization coefficients of M1 (NOS2) or M2 (CD163) with macrophage (CD68) markers by

Anticancer efficacy against orthotopic breast tumor model

SC

using built-in Zeiss analytic software ZEN lite.

M AN U

To establish the orthotropic breast tumor model, 1 × 106 4T1-NF-κB-luc cells were injected into the right mammary pad of female BALB/c mice. Eight-days later, mice bearing tumors (n = 5) were intravenously injected with PBS, free MG132 drug (8 mg/kg), or MG132/m (16- or 64-mg/kg) every 3 days for a total of 3 times, i.e. on days 0, 3 and 6. Anticancer efficacy was

TE D

evaluated in terms of tumor volume, which was calculated by the following equation: V= a×b2/2

EP

where a and b were the major and minor diameters of the tumor measured by a caliper, respectively.

AC C

The body weight of mice was measured as a parameter of the toxicity during the treatment.

Statistical analysis

Statistical analysis was performed by one-way analysis of the variance (ANOVA). P

values lower than 0.05 were considered statistically significant.

Results Effect of MG132/m on the NF-κB levels in breast cancer cells in vitro We first hypothesized that MG132 and MG132/m would reduce NF-κB activity of breast

ACCEPTED MANUSCRIPT carcinoma cells in vitro. We transfected 4T1 murine mammary carcinoma cells with a luminescent nuclear reporter of NF-κB (4T1-NF-κB-luc cells). Introduction of the reporter did not affect the activity of MG132 and MG132/m (Table 1). We exposed 4T1-NF-κB-luc cells to MG132 or

RI PT

MG132/m at different concentrations and for various times, and assessed luminescent photon flux units by IVIS. Both MG132 (Figure 1A) and MG132/m (Figure 1B) decreased the NF-κB levels. However, micelles showed consistent inhibitory effects with dose, while the inhibitory effect of free

SC

MG132 varied widely with dosage. Specifically, MG132 inhibited NF-κB at concentrations higher than 8 µg/ml, while it increased the NF-κB levels at lower doses after 72 h incubation. In contrast,

M AN U

MG132/m inhibited NF-κB at 72 h incubation at all concentrations assayed. As a matter of fact, the dose needed for 50% inhibition of NF-κB activity at 72 h is approximately 7 µg/ml MG132 (Figure 1C) compared to approximately 1µg/ml MG132/m (Figure 1D)

To investigate whether the inhibition is from proteasome inhibition or an effect of the

TE D

micelle formulation, 4T1-NF-κB-luc cells were exposed to bortezomib, a clinically approved proteasome inhibitor, and polymeric micelles incorporating a platinum anticancer drug,

EP

DACHPt/m.32,33 DACHPt/m did not inhibit or enhance NF-κB activity, while bortezomib decreased the NF-κB levels at 48 h incubation (Supplementary Figure S1), which is in agreement with

AC C

previous studies, supporting the effect of proteasome inhibition on the NF-κB levels.

Effect of MG132-loaded micelles on the NF-κB levels in macrophages in vitro The effect of the drugs on the NF-κB levels in J774 A-1 macrophages was evaluated by fluorescence immunohistochemistry. The macrophages were exposed for 24 h to various concentrations of free MG132 and MG132/m ranging from 50 to 3 µg/ml based on our results from 4T1-NF-κB-Luc cells (Figure 1). Non-treated macrophages were used as control. After drug

ACCEPTED MANUSCRIPT exposure, the macrophages were fixed, permeabilized and marked with anti-NF-κB p100/p52 antibody and an Alexa 488 secondary antibody. The fluorescence intensity corresponding to the NF-κB levels inside the macrophages was normalized to the intensity of non-treated macrophages.

RI PT

MG132/m achieved strong inhibition of NF-κB in the range of 25 to 3 ng/ml (Figure 2), while free MG132 increased the NF-κB levels in the macrophages except at its lowest concentration, i.e. 3 ng/ml, which confirmed the effective suppression of NF-κB in macrophages by delivering MG132

SC

through pH-sensitive polymeric micelles.

M AN U

Effect of MG132-loaded micelles on macrophage polarization in vitro

After demonstrating the inhibitory effect of MG132/m on NF-κB levels, we next sought to examine the effect of MG132/m on macrophage reprogramming because NF-κB inhibition reprograms TAMs to the antitumor M1 phenotype.19 Thus, we first co-cultured the macrophages

TE D

with 4T1-NF-κB-Luc cells for 24 h, and then treated them with different concentrations of MG132/m or free MG132. Several markers have been used for studying the spectrum of M1 and

EP

M2 phenotypes. In this study, we focused on M1 macrophages expressing NOS2, as it is correlated with the production of high levels of reactive nitrogen species, which are cytotoxic to cancer cells.

AC C

M2 macrophages were marked by using anti-CD163 antibody, which is a scavenger receptor associated with the regulation of cytokine release. These markers have prognostic value in the clinic.3 Thus, after 24 h incubation, the phenotype of the macrophages was evaluated by immunofluorescence microscopy after marking M1 phenotype by using anti-NOS2 antibody and M2 phenotype with anti-CD163 antibody. Thus, macrophages incubated with 4T1-NF-κB-Luc cells showed increased M2 phenotype (Figure 3; green). Addition of 100 ng/ml of MG132/m on a MG132 basis promoted the M1 phenotype, while free MG132 was able to promote M1 phenotype even at lower doses, as marked by anti-NOS2 antibody (Figure 3; red).

ACCEPTED MANUSCRIPT

Effect of MG132/m on the NF-κB levels in cancer cells in vivo We next aimed to determine whether our tumor model reflected the results of the in vitro

RI PT

study. In this study, we used the maximum tolerated dose (MTD) of MG132, i.e. 8 mg/kg,29 and safe doses of MG132/m, i.e. 16 and 64 mg/kg. Bioluminescent NF-κB signal from 4T1-NF-κB-luc tumors was compared between different groups at 6, 24, 48 and 72 h after a single dose (Figure 4A).

SC

NF-κB expression per tumor volume (photon flux/mm3) was inhibited by approximately 40% after 48 h by 64mg/kg MG132/m, while no change was detected in PBS (Figure 4B). The NF-κB

M AN U

inhibitory effect of 64mg/kg MG132/m was maintained until 72 h. MG132/m at a lower dose of 16 mg/kg did not exert any significant effect on the levels of NF-κB. As for free MG132, the relative NF-κB luminescence showed over 60% enhancement at 48 h (Figure 4B) and more than 100%

TE D

increase after 72 h.

To confirm that the differential effect on NF-κB from MG132/m compared with MG132

EP

was not caused by the polymer of the micelles, we reproduced the experiment with empty micelles without any drug cargo. Compared with MG132/m, empty micelles did not affect NF-κB

AC C

luminescence (Figure 4C).

Effect of MG132/m on TAMs polarization in vivo After demonstrating the inhibitory effect of MG132/m on NF-κB levels, we next sought to examine the effect of MG132/m on macrophage reprogramming. Specifically, we hypothesized that NF-κB expression decreased by 64 mg/kg MG132/m might coincide with a M2 to M1 reprogramming of TAMs. Thus, the distribution of NOS2+ and CD163+ macrophages was analyzed by double immunofluorescent staining in 4T1 tumor tissues. As expected, anti-NOS2 and

ACCEPTED MANUSCRIPT anti-CD163 antibodies were found to mark different populations of macrophages (Figure 5A,D). Furthermore, NOS2 and CD163 were expressed in cells that also were marked by the pan-macrophage marker CD68 (Figures 5B,C). The CD163/CD68 colocalization ratio was much

RI PT

higher than the NOS2/CD68 colocalization ratio, indicating the M2 phenotype of the TAMs in 4T1 tumors.

Breast tumors treated with PBS, 8mg/kg MG132 and 64mg/kg MG132/m (n = 4) were

SC

then harvested 48 h after treatment, when NF-κB level varied most distinctly between different groups (Figure 2 and Figure 4). After fixation, 2 pieces of 5-µm tissue sections were cut and

M AN U

stained with CD68/NOS2 for M2 macrophages and CD68/CD163 for M1 macrophages. Therefore, the colocalization of CD68+/NOS2+ macrophages, or CD68+/CD163+ macrophages was considered as the proportion of M2 or M1 in the whole macrophage population. Here, we used colocalization coefficient calculated by ZEN lite software to assess macrophage polarization.34 We found that the

TE D

proportion of M2 tumor-promoting macrophages was greatly reduced by MG132/m (Figure 6). The colocalization coefficient of CD163+ and CD68+ macrophages in the micelle group is significantly

EP

lower than PBS or free drug groups (Figure 5D)

AC C

As the next step, the distribution of M1 phenotype macrophage (NOS2+) was assessed by the same method (Figure 6). While the proportion of M1 macrophages was low in the control and free MG132-treated tissues (Figures 6E,F), increased NOS2+ population was observed in micelle-treated breast tumors (Figure 6G). The colocalization coefficient of NOS2 (red) with CD68 (green) did not differed between tumors treated by PBS and MG132 (Figure 6H). However, the colocalization coefficient with MG312/m treatment was significantly higher than with free MG132 treatment (Figure 6H).

ACCEPTED MANUSCRIPT Antitumor activity of MG132/m against orthotopic breast tumor After confirming the in vitro and in vivo NF-κB inhibitory effect of the MG132/m, and their ability to reprogram TAMs in breast tumors, we proceeded to study the antitumor efficacy

RI PT

against a syngeneic, orthotopic breast tumor model. We injected 1 × 106 4T1-NF-κB-luc cells into the mammary gland on the left side of BALB/c mice. Breast tumors were allowed to grow for 8 days, and then treated with free MG132 at its MTD, i.e. 8mg/kg,29 MG132/m at 16 or 64mg/kg, and

SC

PBS (n = 5) by intravenous injections at days 0, 3 and 6. The low dose of MG132 was necessary because of the high toxicity of the free drug, whereas micelles did not cause body weight loss even

M AN U

at 64 mg/kg29. The anticancer efficacy was evaluated by tumor size measurements. At day 9, 1 tumor out of 5 in 64 mg/kg MG132/m group was eliminated (Figure 7A, +). Moreover, MG132/m at 64 mg/kg significantly inhibited the growth rate of the orthotopic tumors as compared with PBS and free MG132 (Figure 7A). The safety of the treatments was further confirmed by following the

TE D

body weight changes throughout the experiment (Figure 7B). Thus, the ability of MG132/m at 64 mg/kg to kill cancer cells while promoting macrophage polarization to M1 promoted enhanced

AC C

Discussion

EP

antitumor effects, supporting our proposed strategy.

This study highlights the interplay of the immune system with the cytotoxic treatment of

cancer. Here, we demonstrate that the nanomedicine based proteasome inhibitor MG132/m, previously known to have cytotoxic effects on cancer cells29,30, can also reprogram intratumoral macrophages to the tumor-suppressive M1 phenotype in a syngeneic, orthotopic breast cancer model. Other therapies that reprogram macrophages have proven combinable with chemotherapy, radiation therapy, and immune checkpoint blockade therapy in preclinical and clinical studies.10,12 Proteasome inhibitors are a particularly attractive therapy to combine with other cytotoxic

ACCEPTED MANUSCRIPT treatments, as their mechanism of action is distinct from standard chemotherapies and they reverse multi-drug resistance.35 Our study also indicates how responsive the pro-tumor immune microenvironment is to

RI PT

the dose of proteasome inhibitors. Here, we demonstrated that MG132/m reprograms macrophages as it blocks NF-κB activity even at low doses, while the effect of free MG132 on NF-κB is highly dose dependent and in some cases serves to increase NF-κB expression. In fact, previous studies

SC

have shown that high doses of MG132 increase the concentration reactive oxygen species.36 Increased ROS could lead to increased NF-κB levels.37 Our data along with that of prior studies

M AN U

suggests that the stable, pH-sensitive linkage in MG132/m is critical to the macrophage reprogramming ability of this nanomedicine.

In contrast, a previous study has shown that free MG132 is capable of blocking NF-κB activity leading to reduced angiogenesis in a preclinical model of pancreatic cancer.27 Blocking

TE D

angiogenesis leading to vessel normalization is another strategy to reprogram macrophages and likely is combinable with MG132/m.38 Normalization of the vasculature could also increase the

EP

transvascular transport of MG132/m, with its hydrodynamic diameter of approximately 40 nanometers39, although the compact size of MG132/m dictates that it will likely penetrate

AC C

efficiently without an adjunct therapy.32,40 The interplay of the immune system and blood vessels through NF-κB highlights the interaction of inflammation, angiogenesis, and tumor progression41 while underscoring the urgent need to develop cytotoxic agents that simultaneously can tame – not exacerbate – the insidious tumor microenvironment. MG132/m is one such nanomedicine with anti-cytotoxic and stromal-normalizing effects that is likely combinable with a wide range of treatments for solid tumors.

ACCEPTED MANUSCRIPT Acknowledgements This work was supported by “the Center of Innovation, Science and Technology based

RI PT

Radical Innovation and Entrepreneurship Program (COI STREAM)” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (K.K.) and Grants-in-Aid for

SC

Scientific Research B (JP16H03179; H.C.) and Young Scientists B (JP25750172; H.C.) from the Japan Society for the Promotion of Science (JSPS). J.D.M. is an International Research Fellow of

M AN U

the JSPS.

1.

TE D

References

Komohara Y, Jinushi M, Takeya M: Clinical significance of macrophage

heterogeneity in human malignant tumors. Cancer science 105:1-8, 2014 2.

Aharinejad S, Salama M, Paulus P, et al: Elevated CSF1 serum concentration

EP

predicts poor overall survival in women with early breast cancer. Endocrine-related cancer 20:777-783, 2013 3.

Edin S, Wikberg ML, Dahlin AM, et al: The distribution of macrophages with a

AC C

M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PloS one 7:e47045, 2012 4.

Coussens LM, Raymond WW, Bergers G, et al: Inflammatory mast cells

up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes & development 13:1382-1397, 1999 5.

Lin EY, Nguyen AV, Russell RG, et al: Colony-stimulating factor 1 promotes

progression of mammary tumors to malignancy. The Journal of experimental medicine 193:727-740, 2001 6.

Hanahan D, Coussens LM: Accessories to the crime: functions of cells recruited

to the tumor microenvironment. Cancer Cell 21:309-22, 2012 7.

DeNardo DG, Brennan DJ, Rexhepaj E, et al: Leukocyte complexity predicts

ACCEPTED MANUSCRIPT breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov 1:54-67, 2011 8.

Ruffell B, Chang-Strachan D, Chan V, et al: Macrophage IL-10 blocks CD8+ T

cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells. Cancer Cell 26:623-637, 2014 Shiao SL, Ruffell B, DeNardo DG, et al: TH2-polarized CD4+ T cells and

RI PT

9.

macrophages limit efficacy of radiotherapy. Cancer immunology research 3:518-525, 2015 10.

Ruffell B, Coussens LM: Macrophages and therapeutic resistance in cancer.

Cancer Cell 27:462-472, 2015 11.

Gunderson AJ, Kaneda MM, Tsujikawa T, et al: Bruton tyrosine kinase–

12.

SC

dependent immune cell cross-talk drives pancreas cancer. Cancer discovery 6:270-285, 2016 Ries CH, Cannarile MA, Hoves S, et al: Targeting tumor-associated

M AN U

macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer cell 25:846-859, 2014 13.

Tap WD, Wainberg ZA, Anthony SP, et al: Structure-guided blockade of CSF1R

kinase in tenosynovial giant-cell tumor. New England Journal of Medicine 373:428-437, 2015 14.

Coussens LM, Zitvogel L, Palucka AK: Neutralizing tumor-promoting chronic

inflammation: a magic bullet? Science 339:286-91, 2013

Galluzzi L, Buque A, Kepp O, et al: Immunological effects of conventional

TE D

15.

chemotherapy and targeted anticancer agents. Cancer cell 28:690-714, 2015 16.

Rébé C, Ghiringhelli F: Cytotoxic effects of chemotherapy on cancer and

11:2645-2654, 2015 17.

EP

immune cells: how can it be modulated to generate novel therapeutic strategies? Future Oncology Saccani A, Schioppa T, Porta C, et al: p50 nuclear factor-κB overexpression in

AC C

tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance. Cancer research 66:11432-11440, 2006 18.

Hagemann T, Wilson J, Kulbe H, et al: Macrophages induce invasiveness of

epithelial cancer cells via NF-κB and JNK. The Journal of Immunology 175:1197-1205, 2005 19.

Hagemann T, Lawrence T, McNeish I, et al: “Re-educating” tumor-associated

macrophages by targeting NF-κB. The Journal of experimental medicine 205:1261-1268, 2008 20.

Wang C-Y, Guttridge DC, Mayo MW, et al: NF-κB induces expression of the

Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Molecular and cellular biology 19:5923-5929, 1999 21.

Cheng Q, Lee HH, Li Y, et al: Upregulation of Bcl-x and Bfl-1 as a potential

mechanism of chemoresistance, which can be overcome by NF-[kappa] B inhibition. Oncogene

ACCEPTED MANUSCRIPT 19:4936, 2000 22.

Lee DH, Goldberg AL: Proteasome inhibitors: valuable new tools for cell

biologists. Trends in cell biology 8:397-403, 1998 23.

Ortiz Lazareno PC, Hernandez Flores G, Dominguez Rodriguez JR, et al:

MG132 proteasome inhibitor modulates proinflammatory cytokines production and expression of

RI PT

their receptors in U937 cells: involvement of nuclear factor κB and activator protein 1. Immunology 124:534-541, 2008 24.

Nakajima S, Kato H, Takahashi S, et al: Inhibition of NF-κB by MG132

through ER stress-mediated induction of LAP and LIP. FEBS letters 585:2249-2254, 2011 25.

Shembade N, Ma A, Harhaj EW: Inhibition of NF-κB signaling by A20 through

26.

SC

disruption of ubiquitin enzyme complexes. Science 327:1135-1139, 2010

Cusack JC, Liu R, Houston M, et al: Enhanced chemosensitivity to CPT-11 with

M AN U

proteasome inhibitor PS-341: implications for systemic nuclear factor-κB inhibition. Cancer research 61:3535-3540, 2001 27.

Matsuo Y, Sawai H, Ochi N, et al: Proteasome inhibitor MG132 inhibits

angiogenesis in pancreatic cancer by blocking NF-κB activity. Digestive diseases and sciences 55:1167-1176, 2010 28.

Lindsten K, Menéndez-Benito V, Masucci MG, et al: A transgenic mouse model

29.

TE D

of the ubiquitin/proteasome system. Nature biotechnology 21:897-902, 2003 Quader S, Cabral H, Mochida Y, et al: Selective intracellular delivery of

proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy. Journal of Controlled Release 188:67-77, 2014 Matsumoto Y, Miyamoto Y, Cabral H, et al: Enhanced efficacy against cervical

EP

30.

carcinomas through polymeric micelles physically incorporating the proteasome inhibitor MG132.

AC C

Cancer science 107:773-781, 2016 31.

Ryu BJ, Lee H, Kim S-H, et al: PF-3758309, p21-activated kinase 4 inhibitor,

suppresses migration and invasion of A549 human lung cancer cells via regulation of CREB, NF-κB, and β-catenin signalings. Molecular and cellular biochemistry 389:69-77, 2014 32.

Cabral H, Matsumoto Y, Mizuno K, et al: Accumulation of sub-100 nm

polymeric micelles in poorly permeable tumours depends on size. Nat Nanotechnol 6:815-23, 2011 33.

Cabral H, Nishiyama N, Okazaki S, et al: Preparation and biological properties

of dichloro (1, 2-diaminocyclohexane) platinum (II)(DACHPt)-loaded polymeric micelles. Journal of Controlled Release 101:223-232, 2005 34.

Dunn KW, Kamocka MM, McDonald JH: A practical guide to evaluating

colocalization in biological microscopy. American Journal of Physiology-Cell Physiology

ACCEPTED MANUSCRIPT 300:C723-C742, 2011 35.

Zhang Y, Shi Y, Li X, et al: Proteasome inhibitor MG132 reverses multidrug

resistance of gastric cancer through enhancing apoptosis and inhibiting P-gp. Cancer biology & therapy 7:540-546, 2008 36.

Han YH, Park WH: MG132 as a proteasome inhibitor induces cell growth

level. Human & experimental toxicology 29:607-614, 2010 37.

Morgan MJ, Liu Z-g: Crosstalk of reactive oxygen species and NF-κB signaling.

Cell research 21:103-115, 2011 38.

RI PT

inhibition and cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH

Huang Y, Yuan J, Righi E, et al: Vascular normalizing doses of antiangiogenic

Proc Natl Acad Sci U S A 109:17561-6, 2012

Chauhan VP, Stylianopoulos T, Martin JD, et al: Normalization of tumour blood

M AN U

39.

SC

treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy.

vessels improves the delivery of nanomedicines in a size-dependent manner. Nat Nanotechnol 7:383-8, 2012 40.

Matsumoto Y, Nichols JW, Toh K, et al: Vascular bursts enhance permeability

of tumour blood vessels and improve nanoparticle delivery. Nature nanotechnology, 2016 41.

Martin JD, Fukumura D, Duda DG, et al: Reengineering the Tumor

TE D

Microenvironment to Alleviate Hypoxia and Overcome Cancer Heterogeneity. Cold Spring Harbor Perspectives in Medicine 6:a027094, 2016

EP

Figure Captions

AC C

Figure 1. MG132 and MG132/m affect NF-κB activity of breast tumor cells differently in vitro. The response of NF-κB levels of 4T1-NF-κB-luc cells to various concentrations of MG132 (A) and MG132/m (B) at 6-, 24-, 48- and 72-h incubation. The response of NF-κB levels was plotted as a function of the dose of free MG132 (C) and MG132/m (D), which decreases NF-κB levels to 50% at a lower dose (red dotted line) at 72 h incubation time. Figure 2. MG132 and MG132/m affect NF-κB activity of macrophages differently in vitro. The response of NF-κB levels in macrophages to various concentrations of MG132 (white bars) and MG132/m (black bars) after 24 h incubation. Data are expressed as mean ± S.D. (n = 3).

ACCEPTED MANUSCRIPT Figure 3. M1-phenotype macrophages are induced with MG132 and MG132/m treatment in vitro. Representative confocal images of immunofluorescent stainings of NOS2 (M1 phenotype; red) and CD163 (M2 phenotype; green) in macrophages co-cultured with 4T1-NF-κB-luc cells

RI PT

treated with MG132/m or free MG132 for 24 h. Nuclei was stained with Hoechst (blue). Scale bar: 50 µm.

Figure 4. Inhibitory effect of MG132 on the NF-κB levels of 4T1-NF-κB-luc tumors is

SC

restricted to micelles. (A) Representative bioluminescent images of NF-κB-luminescence after PBS, MG132, and MG132/m administration. (B) Quantification of NF-κB luminescence intensity

M AN U

relative to the initial intensity at the start of the experiment (time = 0) by tumor volume after administration of PBS, MG132, and MG132/m (n = 5). (C) Quantification of NF-κB luminescence intensity relative to the initial intensity at the start of the experiment (time = 0) by tumor volume after administration of PBS or empty micelles (n = 5). Data are expressed as mean ± SE (n = 5). *P

TE D

< 0.05. **P < 0.001 by one-way ANOVA.

Figure 5. Presence of different macrophage phenotypes marked by CD68 and either

EP

expressing NOS2 or CD163. Images of immunofluorescent stainings in 4T1 breast tumors of (A) NOS2 (red) and CD163 (green), (B) CD163 (red) and CD68 (green) and (C) NOS2 (red) and CD68

AC C

(green). Nuclei are shown by DAPI staining (blue). (D), Colocalization coefficient of NOS2/CD163, CD163/CD68 (M2 phenotype) and NOS2/CD68 (M1 phenotype). Scale bar: 50 µm. Figure 6. Effect of MG132/m treatment on macrophage phenotype. Representative confocal images of immunofluorescent stainings of CD68 and CD163 in consecutive sections of the breast tumors treated with PBS (A), free MG132 (B) or MG132/m (C) at 48 h post treatment. (D), Colocalization coefficient of CD163 (red) with CD68 (green) showed that M2 phenotype (CD163+) macrophage was significantly limited by MG132/m compared with PBS and free drug. Representative confocal images of immunofluorescent stainings of CD68 and NOS2 in consecutive

ACCEPTED MANUSCRIPT sections of the breast tumors treated with PBS (E), free MG132 (F) or MG132/m (G) at 48 h post treatment. (H), Colocalization coefficient of NOS2 (red) with CD68 (green) showed that the fraction of M1 phenotype macrophages was significantly increased by MG132/m compared with

RI PT

PBS and free drug. *P < 0.05 by one-way ANOVA. Scale bar: 50 µm. Figure 7. MG132-loaded micelles (MG132/m) inhibit the growth of a syngeneic, orthotopic breast tumor model in a dose-dependent manner. Volume of 4T1-NF-κB-luc tumors treated with

SC

free MG132 (8 mg/kg, squares), MG132/m (16 mg/kg, triangles; 64 mg/kg, cirles), or control (PBS, diamonds) intravenously injected on days 0, 3 and 6 (arrowheads) plotted against time (A). +

M AN U

denotes a 1 tumor regression in MG132/m treated group. Body weight of mice from PBS-, MG132-, and MG132/m-treated groups plotted against time (B). Data are expressed as mean ± SE (n = 5).

AC C

EP

TE D

**P < 0.01. ***P < 0.001 by one-way ANOVA.

ACCEPTED MANUSCRIPT Table 1. Fifty-percent inhibitory concentration of free MG132 and MG132-loaded micelles against 4T1 and 4T1-NF-κB-luc cells. Data are expressed as mean ± S.D. (n = 2).

4T1

Drug MG132

48 h

72 h

2.5 ± 0.2

0.5 ± 0.3

0.5 ± 0.6

N.D.

15.2 ± 0.2

5.4 ± 0.5

3.2 ± 0.3 N.D.

1.3 ± 0.2 22.6 ± 0.4

0.3 ± 0.4 10.4 ± 1.3

AC C

EP

TE D

M AN U

SC

MG132/m MG132 4T1-NF-κB-luc MG132/m a Determined by CCK8.

24 h

RI PT

Cell line

IC50 (µM)a

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT