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Nuclear and fluorescent labeled PD-1-Liposome-DOX-64Cu/ IRDye800CW allows improved breast tumor targeted imaging and therapy Yang Du, Xiaolong Liang, Yuan Li, Ting Sun, Zhengyu Jin, Huadan Xue, and Jie Tian Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00649 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017
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Molecular Pharmaceutics
Nuclear and fluorescent labeled PD-1-Liposome-DOX-64Cu/IRDye800CW allows improved breast tumor targeted imaging and therapy Yang Du1,+, Xiaolong Liang2,+, Yuan Li3,1, +, Ting Sun3,1, Zhengyu Jin3, Huadan Xue3,*, Jie Tian1,* 1
Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, The State Key Laboratory of
Management and Control for Complex Systems, Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China 2
Department of Ultrasonography, Peking University Third Hospital, Beijing 100191, China
3
Department of Radiology, Peking Union Medical College Hospital, Beijing, China
+
These authors contributed equally to this paper.
*Corresponding author: Jie Tian, E-mails: [email protected], Key Laboratory of Molecular Imaging of Chinese
Academy of Sciences, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China. Tel.: +86
10 62527995, Fax.: +86 10 62527995; Huadan Xue, [email protected], Department of Radiology, Peking
Union Medical College Hospital, Beijing, China
Ethical approval: The care and use of animals were followed by all applicable international, national and/or institutional guidelines. Conflicts of interest： ： No potential conflicts of interest were declared by the authors.
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ABSTRACT The over-expression of programmed cell death-1 (PD-1) in tumors as breast cancer makes it a possible target for cancer imaging and therapy. Advances in molecular imaging, including radionuclide imaging and near infrared fluorescence (NIRF) imaging, enable the detection of tumors with high sensitivity. In this study, we aim to develop a novel PD-1 antibody targeted positron emission tomography (PET) and NIRF labeled liposome loaded with doxorubicin (DOX) and evaluate its application for in vivo cancer imaging and therapy. IRDye800CW and 64Cu were conjugated to liposomes with PD-1 antibody labeling, and DOX was inside the liposomes to form theranostic
nanoparticles.
The
4T1
tumors
were
successfully
visualized
with
PD-1-Liposome-DOX-64Cu/IRDye800CW using NIRF/PET imaging. The bioluminescent imaging (BLI)
results
showed
that
tumor
growth
was
significantly
inhibited
in
the
PD-1-Liposome-DOX-treated group than the IgG control. Our results highlight the potential of using dual-labeled theranostic PD-1 mAb-targeted Liposome-DOX-64Cu/IRDye800CW for the management of breast tumor.
KEYWORDS: Programmed cell death-1 (PD-1), near infrared fluorescence (NIRF) imaging, PET imaging, doxorubicin (DOX), breast cancer, therapy
ABBREVIATIONS PD-1
Programmed cell death-1
DOX
Doxorubicin
FMI
Fluorescence imaging 2
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Molecular Pharmaceutics
PET
Positron emission tomography
NIRF
Near infrared fluorescence
BLI
Bioluminescent imaging
TILs
Tumor infiltrating lymphocytes
mAb
Monoclonal antibody
INTRODUCTION Breast cancer, especially triple-negative breast cancer, which is deficient of human epidermal growth factor receptor 2, estrogen receptor, progesterone receptor expression, generally involves high-grade and aggressive tumors and is difficult to eradicate using conventional chemotherapy leading to the poor survival
1, 2
. Therefore, it is urgently needed to develop novel imaging and
therapeutic methods to improve the breast cancer management. As the development of preclinical molecular imaging, new opportunities have been generated to noninvasively observe the biodistribution and tumor targeting of nanoparticles. Both radionuclide imaging and NIRF imaging allow for the sensitive detection of tumors in vivo, and each has its own complementary advantages that outweigh the limitations of other modalities. The specific expression of the programmed cell death-1 (PD-1) checkpoint on tumor-infiltrating lymphocytes (TILs) was demonstrated using a radiotracer
64
Cu-labeled anti-mouse PD-1 for
immunoPET imaging 3. As an inhibitory receptor, PD-1 is expressed on activated T cells and B-cells that inhibit antitumor immunity 4. The PD-1 blockade was recently demonstrated to effectively treat bladder cancer 5. The positive responses to PD-1 blockade are shown in several advanced tumors, such as non-small cell lung cancer, melanoma, renal cell cancer, and ovarian 3
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cancer 6. It has been demonstrated that PD-1+/FOXP3+ Tregs were expressed in the breast tumor microenvironment, suggesting PD-L1 therapy is possible in breast cancer
7, 8
. Because PD-1 is
selectively expressed in the tumors, it represents a potential target for the antibody-guided targeting of cancer with radionuclides and fluorophores. Chemotherapy is a standard cancer treatment regimen using cytotoxic antitumor drugs
9, 10
.
Nowadays, cytotoxic chemotherapy, particularly anthracycline-based regimens as doxorubicin (DOX), remains the major treatment for triple negative breast cancer 11, 12. Despite the wide spread use of traditional chemotherapeutics, the poor specificity and limited accumulation of anticancer agents in tumors, caused by their nonspecific distribution throughout the body, can result in various adverse side effects. Thus, the development of more effective and safe drugs is urgently needed. Nanotechnology has been becoming an indispensable tool in clinical diagnostics and therapeutics in recent years. In particular, liposomes have received great attentions due to their bilayer structure, easy functionalization, and ability to load chemotherapeutic drugs or other theranostic agents 13. Meanwhile, liposomes have a good therapeutic effect on cancer due to their proper size, good biocompatibility, low toxicity, and immunogenicity characteristics 14, 15. Among the different clinically approved nanomedicines for cancer therapy, liposomes exhibit superior application prospects, several formulations as PEGylated liposomal DOX (DOXil/Caelyx) and liposomal daunorubicin (DaunoXome, Galen) have been approved by Food and Drug Administration 16. Although the tumor-targeting efficacy in cancer was improved by liposomes, in vivo drug distribution and early feedback of therapeutic effect should be precisely monitored as well. 4
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In this study, we synthesized dual-labeled PD-1 mAb-targeted liposomes and evaluated their tumor-targeting and imaging abilities for 4T1 mammary tumors using NIRF and nuclear dual-imaging modalities. PET is a highly sensitive and quantitative tomographic imaging modality. We reasoned that the fabrication of a probe with both NIRF dye and PET isotope allowing sensitive and accurate pharmacokinetic monitoring and tumor targeting evaluation 17. In addition, DOX was enveloped in the PD-1 mAb-targeted liposomes to assess its possible use for antitumor therapy. The treatment efficacy was further assessed using bioluminescent imaging (BLI). Moreover, the possible antitumor mechanisms of PD-1-Liposome-DOX were analyzed. The results showed here suggested that the use of PD-1-Liposome-DOX as a theranostic agent for breast cancer has great potential for clinical application.
EXPERIMENTAL SECTION Materials The purified anti-mouse PD-1 monoclonal antibody (mAb) (RMP1-14), which is a rat IgG2a antibody, and isotype control rat IgG2a (2A3) were bought from BioXCell (West Lebanon, NH, USA). IRDye800CW was provided by LiCor Biosciences (Lincoln, Nebraska, USA). 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
(DSPE),
mono
(N-hydroxysuccinimidyl
ester)
of
1,4,7,10-Tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetic acid derivatives (DOTA-NHS ester) and cholesterol were the products of Macrocyclics and Avanti Polar Lipids, respectively. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinimide (DSPE-PEG-NHS) was purchased from NOF Co. in Japan. DOX was obtained from Beijing 5
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Huafeng United Technology Co.
Preparation of DSPE-PEG-PD-1 and DSPE-PEG-IgG2a DSPE-PEG-PD-1 and DSPE-PEG-IgG2a were first synthesized. PD-1 mAb with an amino group was reacted with DSPE-PEG-NHS with an NHS group to obtain stable amide bonding. Briefly, PD-1 mAb was mixed with DSPE-PEG-NHS were mixed with a molar ratio of 1:10 in aqueous 80 % DMSO (pH 8.5 − 9.0). The reaction was moderately stirred for 2 h at 37 °C. The final product was purified by SEC3000 HPLC (mobile phase: 0.1 M ammonium acetate buffer at pH 5.5, eluted rate: 1 mL/min). The final solution was concentrated using an Amicon centrifugal filter and lyophilized.
Preparation of PD-1-Liposome-DOX and IgG-Liposome- DOX PD-1-Liposome-DOX was prepared by the ethanol injection method. Briefly, a mixture of DSPC, Cholesterol, DSPE-IRDye800CW, DSPE-DOTA, and DSPE-PEG-PD-1 (molar ratio: 49:43.9:0.1:2:2.5) was dissolved in Dimethyl Sulphoxide. Then, the solvent was injected into 350 mM ammonium sulfate solution by sonication. The liposome suspension was extruded by a polycarbonate membrane for 10 times, followed by dialysis against a sucrose solution (10 wt %, 5 mM NaCl) to remove the free ammonium sulfate. Then, DOX was mixed into the liposome suspension at a DOX/lipid (w/w) ratio of 1:10 and incubation for 24 h at room temperature. Finally, G-50 column was used to remove non-encapsulated free DOX.
Radiolabeling of Liposomes with 64Cu 6
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A 64CuCl2 stock solution (~2.0 mCi) was first dispersed in sodium acetate buffer (0.2 mL, 100 mM, pH 6.5). Then, liposomes (0.5 mL) in phosphate buffer saline (PBS) were also exchanged with acetate buffer by PD-10 column. The liposome fraction (1.5 mL) collected was later labeled with 64Cu radiotracer (incubation for 2 h at 43 °C). Finally, PD-10 column was used to purify the product. The percentage of
64
Cu labeled on liposomes was calculated by comparing the
radioactivity ratio of liposomes before and after purification.
Characterization of PD-1-Liposome-DOX and IgG-Liposome DOX Size and zeta potential of the PD-1-Liposome-DOX and IgG-Liposome DOX were evaluated by a dynamic light scattering (DLS) analyzer (90Plus/BI-MAS, Brookhaven Instruments Co., USA). Parameters including scattering angle of 90° and wavelength of 632.8 nm were used to performed the measurements at room temperature (n = 6). Data were automatically analyzed by the instrument. The results were presented as mean diameter ± SD. Transmission electron microscopy (TEM) measurement was applied to observe the structure of PD-1-Liposome-DOX and IgG-Liposome DOX, which was performed on a H-7650 apparatus (Tokyo, Japan, accelerating voltage=100 kV). Briefly, the liposome suspension (250 mmol/L) was first immersed and incubated for 10 min in formvar-coated copper grid (300-mesh). Then, the grids were taken out and negatively stained with aqueous uranyl acetate solution (2 wt.%), which was freshly prepared and sterile filtered for 10 min at room temperature. Before imaging, the copper grids were washed by deionized water and air dried.
Cell Culture 7
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The mouse breast tumor cell line 4T1-fLuc was cultured and maintained in RPMI 1640 medium (Hyclone, Thermo Scientific, USA) supplemented with 10% fetal calf serum (Hyclone, Thermo Scientific). The cells were cultured at 37 °C in a 5 % CO2 incubator.
Cell Survival Assay Cytotoxicity was assessed in vitro using CCK-8 kit (CCK-8, Dojindo, Japan). After seeding 96-well plates and culturing overnight, the 4T1-fLuc cells were incubated with free DOX, PD-1-liposome-DOX, or IgG-liposome-DOX for 24 h before washed with PBS. Next, the cells were incubated with 0.5 mg/mL WST-1 in serum-free medium for 1 h. Absorbance values (450 nm) were measured with microplate reader (BIO TEK, USA).
Establishment of the Tumor-Bearing Animal Model Female Balb/c mice (5-wk old, 18-24 g) were bought from the Department of Experimental Animals, Peking University Health Science Center. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Peking University with Permit Number: 2011-0039, and the experimental procedures were performed according to the approved guidelines. The mice were implanted with 4T1-fLuc cells (1 × 106) subcutaneously (s.c.).
In Vivo Fluorescence Imaging (FMI) FMI was carried out to examine the biodistribution and tumor-targeting effects of PD-1-Liposome-DOX-IRDye800CW in vivo. FMI was performed by utilizing an IVIS Imaging Spectrum System (PerkinElmer Inc., USA). IVIS Living Imaging 3.0 software (PerkinElmer Inc., 8
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USA) was utilized for the data analysis. The FMI data were collected dynamically after injection of PD-1-Liposome-DOX-IRDye800CW into the 4T1 mammary tumor-bearing mice (n = 3). IgG-Liposome-DOX-IRDye800CW was utilized as control (n = 3). For quantitative comparisons, the ratios of fluorescence signal intensities in the regions of interest corresponding to the tumor and muscle regions were determined. The following formula for the tumor to background ratio was used:
Tumor to background ratio =
Fluorescence light intensity tumor Fluorescence light intensity muscle
PET Imaging PET images were obtained using small animal PET (SuperArgus, GE Healthcare, Sedecal, Spain). For PET reconstruction, a two-dimensional algorithm was used. The tumor bearing mice were
imaged
approximately
PD-1-Liposome-DOX-DOTA-64Cu
14 or
day
after
4T1
cell
IgG-Liposome-DOX-DOTA-64Cu
implantation. was
injected
intravenously. Mice were anesthetized for around 5 min by isoflurane in an air-oxygen mixture before PET image acquisition, and the images were acquired at 12 and 24 h after probe injection (n = 3). Moreover, the biodistribution was studied at 12 and 24 h (n = 4).
Drug Treatment There were four groups of tumor-bearing mice: (1) PBS, (2) free DOX, (3) IgG-Liposome-DOX, and (4) PD-1-Liposome-DOX. The mice were given 5 mg/kg DOX. The IgG-Liposome-DOX and PD-1-Liposome-DOX treatment groups were given an equivalent DOX dosage of 5 mg/kg intravenously every 2 days. The PD-1 mAb (BioXcell, West Lebanon, NH) was given 200 µg 9
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through intraperitoneal injection.
In Vivo BLI and Tumor Volume Measurement To monitor and quantify tumor growth, the tumor-bearing mice were given with D-luciferin (150 mg/kg) (Fremont, CA, USA) intraperitoneally and imaged using the small animal optical molecular imaging system (PerkinElmer Inc., USA) on days 0, 3, 6, 9, 12, and 15 during drug treatment. All imaged mice were anesthetized with 2 % isoflurane and warmed until they awakened. The tumor largest (a) and smallest (b) diameters were measured every 2 days, and the tumor volume was calculated as V = ab2/2.
Statistics The GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA) was used for the data analysis. Three independent experiments were repeated, and the data was presented as mean ± SEM. To compare the differences between groups, one-way analysis of variance followed by Tukey’s multiple comparisons test was performed. The means of two groups was compared using two-tailed Student’s t test. Kaplan-Meier method was used for the comparison of survival rate. *P < 0.05 was set as the significance level.
RESULTS Synthesis and Characterization of PD-1-Liposome-DOX and IgG-Liposome-DOX In this study, the ethanol injection method was used to prepare the multifunctional liposomes. 10
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Formulations were made with either PD-1 mAb or IgG2a mAb (Figure 1). By using ammonium/pH gradient method, DOX was highly efficient loaded into the liposomes. The remote encapsulation of DOX into the liposomes exhibited significant advantages at a high drug to lipid molar ratio, as it can improve the drug delivery and enhance cancer therapeutic effect. The properties of PD-1-Liposome-DOX and IgG-Liposome-DOX, including particle size, particle charge, and serum stability, were characterized, and the data were shown in Table 1 and Figure 2. High DOX encapsulation efficiency in both liposomes were achieved (95.45 ± 0.21% and 96.24 ± 0.35%) (Table 1). The sizes and surface potentials of these two kinds of drug loaded liposomes were comparable, the particle size of both liposomes showed narrow size distribution with diameters about 85 nm. Although their zeta potential was low (−3.56 ± 0.3 and −4.16 ± 0.5 mV), it was strong enough to keep good colloidal stability in the presence of the PEG chains on the particles surface. The TEM images revealed that both liposomes showed spherical and homogeneous NPs with a size range from 70 to 100 nm (Fig. 2A and Fig. 2B), well consistent with the dynamic light scattering (DLS) measurement shown in Fig. 2C and Fig. 2D. The stability of the PD-1-Liposome-DOX and IgG-Liposome-DOX were further assessed by mixing these liposomes in serum containing DMEM. Hydrodynamic particle size showed no obvious change in the DMEM solutions after 48h (Fig. 2E), showing great potential for further application in biological system.
Effects of the Different Treatments on Cell Survival in Vitro After characterizing the nanoparticles in vitro, we further studied the antitumor effects of free DOX, PD-1-Liposome-DOX, and IgG-Liposome-DOX on 4T1-fLuc cell viability. The results 11
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showed that both PD-1-Liposome-DOX and IgG-Liposome-DOX decreased the cell survival rate and increased the inhibition rate compared with free DOX at both 1 and 10 µg/mL (Fig. 3A). To further confirm the results, we performed ex vivo BLI imaging of 4T1-fLuc cells to determine the number of live cells following the different treatments. BLI intensity was further quantified and analyzed in triplicate. The data revealed that both PD-1-Liposome-DOX and IgG-Liposome-DOX were effective against tumor cells, similar to free DOX, and showed decreased BLI intensities compared with PBS-treated cells ex vivo (Fig. 3B). The above results indicated that PD-1-Liposome-DOX and IgG-Liposome-DOX retained the cell toxicity effects of DOX and can be used for in vivo antitumor chemotherapy.
Biodistribution of PD-1-Liposome-DOX with IRDye800CW/64Cu Dual-Modality NIRF/PET Imaging on 4T1 Mammary Tumors To explore whether PD-1-Liposome-DOX displayed enhanced tumor-targeting effects in vivo, nanoparticles were dual labeled with IRDye800CW and 64Cu for in vivo NIRF and PET imaging. The UV−Vis absorption and fluorescence properties and the Radio Thin-Layer Chromatography of nanoparticles were characterized, and the data are shown in Supplemental Fig. 1 and Supplemental Fig. 2. FMI was dynamically examined from 0, 5, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after intravenous administration of nanoparticles. A higher tumor to background ratio of PD-1-Liposome-DOX-IRDye800CW
was
from
2
to
48
h
compared
to
IgG-Liposome-DOX-IRDye800CW, and the most significant tumor to background ratio difference was around 2.4-fold higher compared to the IgG control found at 24 h (Fig. 4A). Typical FMI 12
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images at 24 h are shown in Fig. 4B, and BLI was performed to determine the exact location of tumors in situ (Fig. 4B). The FMI signal was stronger in the PD-1-Liposome-DOX-IRDye800CW group compared to the IgG-Liposome-DOX-IRDye800CW group. To further confirm our observations, we dissected out the tumor and major organs after the in vivo observations at 24 h. The NIRF signal was higher in tumors in the PD-1-Liposome-DOX-IRDye800CW group compared to IgG control (Fig. 4C). PET imaging of 4T1 mammary tumors was performed at 12 and 24 h post-injection of PD-1-Liposome-DOX-DOTA-64Cu. Representative PET images of mice are shown in Fig. 5A. PD-1-Liposome-DOX-DOTA-64Cu displayed a higher PET signal in tumors compared to IgG-Liposome-DOX-DOTA-64Cu at both 12 and 24 h. The tumor-to-muscle ratio of PD-1-Liposome-DOX-DOTA-64Cu was approximately 1.40-fold and 1.8-fold higher compared to IgG-Liposome-DOX at 12 h and 24 h, respectively (Fig. 5B). To validate this observation, the biodistribution of the nanoparticles was examined in tumors and different organs. In general, higher
tumor
uptake
was
found
at both 12
and
24
h after
administration of
PD-1-Liposome-DOX-DOTA-64Cu compared to the IgG control (Fig. 5C and 5D). For example, 64
Cu uptake was 2.34 ± 1.25 %ID/g for PD-1-Liposome-DOX-DOTA-64Cu and 1.27 ± 0.65 %ID/g
for the IgG control at 24 h post-injection. Moreover, it was observed that the highest uptake of 64
Cu was in the spleen, which functions in priming T cells for antigen surveillance and attack
before deploying them throughout the body, and the signals in tumors are lymphocytes that have infiltrated the tumor. Taken together, the PET and NIRF imaging results demonstrated PD-1 mAb-targeted liposomes improved in vivo targeted tumor imaging.
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Comparison of the Different Treatment Effects Using 4T1 Mammary Tumor Xenografts The different treatment effects were further studied. Tumor growth was dynamically monitored from days 1 to 15 using BLI imaging, which is a sensitive indicator of tumor growth (Fig. 6A and 6B). The results showed that BLI light intensity greatly increased in the PBS group, whereas relatively a mild increase in BLI light intensity was observed in the free DOX and IgG-Liposome-DOX groups during the 15-day observation. The PD-1-Liposome-DOX group exhibited lower light intensities compared to the free DOX and IgG-Liposome-DOX groups during the 15-day observation. We also measured the tumor volumes, and they were consistent with the BLI data showing that PD-1-Liposome-DOX exhibited better antitumor efficacy than other treatments. Furthermore, the survival rate was also monitored, and the PD-1-liposome-DOX treatment prolonged the survival of mice compared to other groups.
DISCUSSION Although a single imaging modality can provide some insights into a disease process, in vivo multimodality imaging can offer a new prospective. In this study, we provide proof-of-principle for targeting PD-1-expressing breast tumors in vivo with dual-labeled IRDye800CW and 64Cu in 4T1 a mammary tumor mouse model. In addition, PD-1-Liposome-DOX exhibited significantly enhanced therapeutic efficacy compared to IgG-Liposome-DOX and free DOX. This is the first report, to the best of our knowledge, on the use of PD-1 mAb-targeted liposomes for targeted imaging and therapy of breast tumors. The active targeting of cancers is one of the most important considerations for cancer imaging and therapy. PD-1 over-expression is involved in a diverse array of tumor types because of its 14
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participation in signaling pathways regulating attenuated CD8+ T cell function and enhanced Treg activity, which creates an inhibitory environment in the tumors
18-20
. Thus, the selective PD-1
expression in the tumor represents a potential target for the antibody-guided targeting of tumors with fluorophores and radionuclides. Moreover, the expression of PD-L1 on tumor cells is a potent biomarker to predict the responses of immunotherapies to PD-1 or PD-L1 21. However, at present, using immunohistochemistry to assess tumor PD-1/PD-L1 expression has been proven to be a partial and imperfect predictor of anti-PD-1/anti-PD-L1 immune therapeutic effects
22
. FMI and
immunoPET imaging are non-invasive means by which PD-1 expression can be measured throughout an entire tumor simultaneously. In this study, we examined PD-1 as a biomarker for targeted breast tumor imaging and therapy using NIRF/PET imaging. Indeed, our in vivo FMI data revealed a relatively higher signal for PD-1-Liposome-DOX in the tumor regions compared to IgG-Liposome-DOX, which was confirmed by the ex vivo imaging of tumors. A shortcoming of FMI is the limited penetration depth of the emitted light into tissue. A new approach to deal with the limited tissue penetration of FMI is to combine it with radionuclide detection. Therefore, we performed PET imaging by labeling the nanoparticles with
64
Cu. Consistent with the NIRF
observations, our PET imaging and biodistribution data revealed that PD-1-Liposome-DOX exhibited better tumor-targeting effects and longer retention in tumors compared to the IgG. Targeted cancer therapy depends on the specific-expression of tumor biomarkers for the selective delivery of therapeutic agents to tumors while reducing the side effects to normal organs 16
. As for chemotherapy, achieving increased tumor-targeting treatment efficacy and decreased
organ toxicities currently remain a challenge. In this study, we encapsulated DOX in the liposomal nanoparticles with PD-1 mAb labeling. The results showed that compared to the 15
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IgG-Liposome-DOX and free DOX, PD-1-Liposome-DOX exhibited better antitumor activity, as demonstrated by BLI imaging and tumor volume measurement, and also longer survival. The two possible underlying antitumor mechanisms were delineated. On the one hand, PD-1 mAb as a potent biomarker increased the tumor-targeting effects of Liposome-DOX specifically in the tumor regions as shown by the in vivo NIRF and PET imaging and biodistribution results. On the other hand, it was reported that anti-PD-1 mAb combined with chemotherapy exhibited an encouraging antitumor effect in advanced non-small cell lung cancer
23
. Previous works provide
evidence that PD-L1 expression may mediate chemotherapy resistance and also may provide the rationale for a combined chemotherapeutic agents and anti-PD-1/PD-L1 mAbs for cancer treatment
24
. In this study, our flow cytometry data on TILs in supplemental Fig. 3 revealed that
there were relatively more CD3+ immune T cells and CD8+ T cells, which mainly have an antitumor growth function. This finding indicated that PD-1 mAb may simultaneously works as an adjuvant immunotherapy for DOX chemotherapy. As efficient drug nanocarriers, liposomes carry hydrophobic or hydrophilic drugs, showing good biocompatibility. Many liposome nanoparticles have been fabricated and approved by FDA for clinical applications. However, for achieving more personalized cancer therapy, theranostic agents with both therapeutic and imaging functions are urgently needed for image guidance and therapeutic
response
monitoring.
Compared
to
the
conventional
liposome,
PD-1-Liposome-DOX-64Cu/IRDye800CW described in this study can improve the in vivo tumor targeting and therapy ability of liposomes. By taking advantages of each imaging modality, pharmacokinetics and therapeutic response of drugs, and the prognosis-associated disease status can be better monitored and undertaken. Nevertheless, the problem we have to consider is more 16
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experiments are needed to verify the biological safety of multifunctional liposomes before its clinical application. Taken together, our study indicates that PD-1-expressing breast tumors can be visualized sensitively and specifically using dual-modality NIRF/PET imaging with the newly developed PD-1-Liposome-DOX-64Cu/IRDye800CW conjugate. Moreover, the PD-1-Liposome-DOX exhibited enhanced antitumor therapeutic effects. This is the first report, to our knowledge, on the use of PD-1 mAb as a target, IRDye800CW/64Cu as imaging agents, and DOX as a chemotherapeutic drug for the noninvasive NIRF/PET imaging and chemo- and immunotherapy of 4T1 mammary tumors. Our results demonstrate the efficacy of the application of our targeted nanoparticles to treat breast cancer, and highlight the promising clinical translational ability of PD-1-liposome-DOX for breast tumor management and suggest a potential strategy for theranostics for other PD-1-expressing tumors.
ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (81227901, 81470083, 81527805, 81571810, 61231004, 81771846), the Research and Development Program of China (973) under Grant (2014CB748600, 2015CB755500), the Strategic Priority Research Program from Chinese Academy of Sciences (XDB02060010), the Beijing Natural Science Foundation
(Z16110200010000)
and
grants
from
Peking
(BYSY2015023).
FINANCIAL DISCLOSURE 17
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No potential conflicts of interest were declared by authors.
SUPPORTING INFORMATION The supplemental method of analysis of the tumor infiltrating lymphocytes (TILs) composition, and supplemental results for UV−Vis absorption and fluorescence properties, the radio-labeling yield of PD-1-Liposome-64Cu, and the effects of PD-1-Liposome-DOX on TILs were supplied as supporting Information.
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23. Kanda, S.; Goto, K.; Shiraishi, H.; Kubo, E.; Tanaka, A.; Utsumi, H.; Sunami, K.; Kitazono, S.; Mizugaki, H.; Horinouchi, H.; Fujiwara, Y.; Nokihara, H.; Yamamoto, N.; Hozumi, H.; Tamura, T., Safety and efficacy of nivolumab and standard chemotherapy drug combination in patients with advanced non-small-cell lung cancer: a four arms phase Ib study. Ann Oncol 2016, 27 (12), 2242-2250. 24. Rizvi, N. A.; Hellmann, M. D.; Brahmer, J. R.; Juergens, R. A.; Borghaei, H.; Gettinger, S.; Chow, L. Q.; Gerber, D. E.; Laurie, S. A.; Goldman, J. W.; Shepherd, F. A.; Chen, A. C.; Shen, Y.; Nathan, F. E.; Harbison, C. T.; Antonia, S., Nivolumab in Combination With Platinum-Based Doublet Chemotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer. J Clin Oncol 2016, 34 (25), 2969-79.
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Table 1. Characterization of PD-1-Liposome-DOX and IgG-Liposome-DOX. Size Formulation (nm)
IgG-Liposome-DOX
Zeta potential
Entrapment efficiency
(mV)
(%)
PDI
83.52±3.55
0.201
−3.56 ± 0.3
95.45 ± 0.21
PD-1-Liposome-DOX 84.56±2.58
0.189
−4.16 ± 0.5
96.24 ± 0.35
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Figure 1 Schematic illustration of PD-1 mAb-targeted multifunctional liposome with DOX loading. 140x105mm (300 x 300 DPI)
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Figure 2. TEM images and size distribution of nanoparticles. (A) and (C) IgG-Liposome-DOX; (B) and (D) PD-1-Liposome-DOX. Diameter of both liposomes in serum containing DMEM vs time measured by DLS (E). Both liposomes showed excellent serum stability. 140x77mm (300 x 300 DPI)
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Figure 3 Evaluation of PD-1-Liposome-DOX treatment on cell survival compared to the IgG control and free DOX in vitro. (A) The 4T1-fLuc tumor cell survival and inhibition rates after different treatments were evaluated using CCK8 kit. (B) BLI imaging of 4T1-fLuc tumor cells following different treatments in triplicate, and statistics for the BLI light intensities in 4T1-fLuc cells following different treatments. ns indicates no significant difference, P > 0.05. 140x105mm (300 x 300 DPI)
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Figure 4 FMI of PD-1-Liposome-DOX-IRDye800CW in 4T1 mammary tumor-bearing mice in vivo and ex vivo. (A) Comparison of the tumor to background ratio between the PD-1-Liposome-DOX-IRDye800CW and IgGLiposome-DOX-IRDye800CW groups. (B) BLI and FMI imaging of tumors in situ in the PD-1-Liposome-DOXIRDye800CW and IgG-Liposome-DOX-IRDye800CW groups. (C) The ex vivo FMI imaging of tumors (T) and major organs, including the liver (L), kidney (K), spleen (S), and heart (H), 24 h after the in vivo observations. **P < 0.001. 140x88mm (300 x 300 DPI)
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Figure 5 PET imaging and biodistribution of PD-1-Liposome-DOX-DOTA-64Cu in the 4T1 mammary tumorbearing mice. (A) PET images of 4T1 mammary tumor-bearing mice at 12 and 24 h post-injection of PD-1Liposome-DOX-DOTA-64Cu. (B) The tumor to muscle ratio of PD-1-Liposome-DOX and IgG-Liposome-DOX at both 12 h and 24 h time points. Biodistribution of PD-1-Liposome-DOX-DOTA-64Cu in 4T1 tumors 12 h (C) and 24 h (D) post-injection. 140x105mm (300 x 300 DPI)
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Figure 6 Comparison of PD-1-Liposome-DOX treatment efficacy with other treatments in vivo. (A) Dynamic in vivo BLI monitoring of tumor growth after different treatments from days 1 to 15. (B) BLI light intensities and tumor volume of different treatment groups at different time points, and the Kaplan-Meier survival plots of survival rate (n = 10). *P < 0.05. 140x113mm (300 x 300 DPI)
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PD-1-expressing breast tumors can be visualized sensitively and specifically using dual-modality NIRF/PET imaging with newly developed PD-1-Liposome-DOX-64Cu/IRDye800CW. The PD-1-Liposome-DOX64Cu/IRDye800CW significantly enhanced the antitumor effects of doxorubicin through active tumor targeting effects and also the adjuvant immune therapeutic effects of PD-1 mAb. Our study highlights the potential of using dual-labeled PD-1 mAb-targeted Liposome-DOX-64Cu/IRDye800CW as theranostic agent for the management of breast cancer 88x55mm (300 x 300 DPI)
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Article
Nuclear and fluorescent labeled PD-1-Liposome-DOX-64Cu/ IRDye800CW allows improved breast tumor targeted imaging and therapy Yang Du, Xiaolong Liang, Yuan Li, Ting Sun, Zhengyu Jin, Huadan Xue, and Jie Tian Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00649 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017
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Nuclear and fluorescent labeled PD-1-Liposome-DOX-64Cu/IRDye800CW allows improved breast tumor targeted imaging and therapy Yang Du1,+, Xiaolong Liang2,+, Yuan Li3,1, +, Ting Sun3,1, Zhengyu Jin3, Huadan Xue3,*, Jie Tian1,* 1
Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, The State Key Laboratory of
Management and Control for Complex Systems, Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China 2
Department of Ultrasonography, Peking University Third Hospital, Beijing 100191, China
3
Department of Radiology, Peking Union Medical College Hospital, Beijing, China
+
These authors contributed equally to this paper.
*Corresponding author: Jie Tian, E-mails: [email protected], Key Laboratory of Molecular Imaging of Chinese
Academy of Sciences, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China. Tel.: +86
10 62527995, Fax.: +86 10 62527995; Huadan Xue, [email protected], Department of Radiology, Peking
Union Medical College Hospital, Beijing, China
Ethical approval: The care and use of animals were followed by all applicable international, national and/or institutional guidelines. Conflicts of interest： ： No potential conflicts of interest were declared by the authors.
1
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ABSTRACT The over-expression of programmed cell death-1 (PD-1) in tumors as breast cancer makes it a possible target for cancer imaging and therapy. Advances in molecular imaging, including radionuclide imaging and near infrared fluorescence (NIRF) imaging, enable the detection of tumors with high sensitivity. In this study, we aim to develop a novel PD-1 antibody targeted positron emission tomography (PET) and NIRF labeled liposome loaded with doxorubicin (DOX) and evaluate its application for in vivo cancer imaging and therapy. IRDye800CW and 64Cu were conjugated to liposomes with PD-1 antibody labeling, and DOX was inside the liposomes to form theranostic
nanoparticles.
The
4T1
tumors
were
successfully
visualized
with
PD-1-Liposome-DOX-64Cu/IRDye800CW using NIRF/PET imaging. The bioluminescent imaging (BLI)
results
showed
that
tumor
growth
was
significantly
inhibited
in
the
PD-1-Liposome-DOX-treated group than the IgG control. Our results highlight the potential of using dual-labeled theranostic PD-1 mAb-targeted Liposome-DOX-64Cu/IRDye800CW for the management of breast tumor.
KEYWORDS: Programmed cell death-1 (PD-1), near infrared fluorescence (NIRF) imaging, PET imaging, doxorubicin (DOX), breast cancer, therapy
ABBREVIATIONS PD-1
Programmed cell death-1
DOX
Doxorubicin
FMI
Fluorescence imaging 2
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PET
Positron emission tomography
NIRF
Near infrared fluorescence
BLI
Bioluminescent imaging
TILs
Tumor infiltrating lymphocytes
mAb
Monoclonal antibody
INTRODUCTION Breast cancer, especially triple-negative breast cancer, which is deficient of human epidermal growth factor receptor 2, estrogen receptor, progesterone receptor expression, generally involves high-grade and aggressive tumors and is difficult to eradicate using conventional chemotherapy leading to the poor survival
1, 2
. Therefore, it is urgently needed to develop novel imaging and
therapeutic methods to improve the breast cancer management. As the development of preclinical molecular imaging, new opportunities have been generated to noninvasively observe the biodistribution and tumor targeting of nanoparticles. Both radionuclide imaging and NIRF imaging allow for the sensitive detection of tumors in vivo, and each has its own complementary advantages that outweigh the limitations of other modalities. The specific expression of the programmed cell death-1 (PD-1) checkpoint on tumor-infiltrating lymphocytes (TILs) was demonstrated using a radiotracer
64
Cu-labeled anti-mouse PD-1 for
immunoPET imaging 3. As an inhibitory receptor, PD-1 is expressed on activated T cells and B-cells that inhibit antitumor immunity 4. The PD-1 blockade was recently demonstrated to effectively treat bladder cancer 5. The positive responses to PD-1 blockade are shown in several advanced tumors, such as non-small cell lung cancer, melanoma, renal cell cancer, and ovarian 3
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cancer 6. It has been demonstrated that PD-1+/FOXP3+ Tregs were expressed in the breast tumor microenvironment, suggesting PD-L1 therapy is possible in breast cancer
7, 8
. Because PD-1 is
selectively expressed in the tumors, it represents a potential target for the antibody-guided targeting of cancer with radionuclides and fluorophores. Chemotherapy is a standard cancer treatment regimen using cytotoxic antitumor drugs
9, 10
.
Nowadays, cytotoxic chemotherapy, particularly anthracycline-based regimens as doxorubicin (DOX), remains the major treatment for triple negative breast cancer 11, 12. Despite the wide spread use of traditional chemotherapeutics, the poor specificity and limited accumulation of anticancer agents in tumors, caused by their nonspecific distribution throughout the body, can result in various adverse side effects. Thus, the development of more effective and safe drugs is urgently needed. Nanotechnology has been becoming an indispensable tool in clinical diagnostics and therapeutics in recent years. In particular, liposomes have received great attentions due to their bilayer structure, easy functionalization, and ability to load chemotherapeutic drugs or other theranostic agents 13. Meanwhile, liposomes have a good therapeutic effect on cancer due to their proper size, good biocompatibility, low toxicity, and immunogenicity characteristics 14, 15. Among the different clinically approved nanomedicines for cancer therapy, liposomes exhibit superior application prospects, several formulations as PEGylated liposomal DOX (DOXil/Caelyx) and liposomal daunorubicin (DaunoXome, Galen) have been approved by Food and Drug Administration 16. Although the tumor-targeting efficacy in cancer was improved by liposomes, in vivo drug distribution and early feedback of therapeutic effect should be precisely monitored as well. 4
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In this study, we synthesized dual-labeled PD-1 mAb-targeted liposomes and evaluated their tumor-targeting and imaging abilities for 4T1 mammary tumors using NIRF and nuclear dual-imaging modalities. PET is a highly sensitive and quantitative tomographic imaging modality. We reasoned that the fabrication of a probe with both NIRF dye and PET isotope allowing sensitive and accurate pharmacokinetic monitoring and tumor targeting evaluation 17. In addition, DOX was enveloped in the PD-1 mAb-targeted liposomes to assess its possible use for antitumor therapy. The treatment efficacy was further assessed using bioluminescent imaging (BLI). Moreover, the possible antitumor mechanisms of PD-1-Liposome-DOX were analyzed. The results showed here suggested that the use of PD-1-Liposome-DOX as a theranostic agent for breast cancer has great potential for clinical application.
EXPERIMENTAL SECTION Materials The purified anti-mouse PD-1 monoclonal antibody (mAb) (RMP1-14), which is a rat IgG2a antibody, and isotype control rat IgG2a (2A3) were bought from BioXCell (West Lebanon, NH, USA). IRDye800CW was provided by LiCor Biosciences (Lincoln, Nebraska, USA). 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
(DSPE),
mono
(N-hydroxysuccinimidyl
ester)
of
1,4,7,10-Tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetic acid derivatives (DOTA-NHS ester) and cholesterol were the products of Macrocyclics and Avanti Polar Lipids, respectively. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinimide (DSPE-PEG-NHS) was purchased from NOF Co. in Japan. DOX was obtained from Beijing 5
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Huafeng United Technology Co.
Preparation of DSPE-PEG-PD-1 and DSPE-PEG-IgG2a DSPE-PEG-PD-1 and DSPE-PEG-IgG2a were first synthesized. PD-1 mAb with an amino group was reacted with DSPE-PEG-NHS with an NHS group to obtain stable amide bonding. Briefly, PD-1 mAb was mixed with DSPE-PEG-NHS were mixed with a molar ratio of 1:10 in aqueous 80 % DMSO (pH 8.5 − 9.0). The reaction was moderately stirred for 2 h at 37 °C. The final product was purified by SEC3000 HPLC (mobile phase: 0.1 M ammonium acetate buffer at pH 5.5, eluted rate: 1 mL/min). The final solution was concentrated using an Amicon centrifugal filter and lyophilized.
Preparation of PD-1-Liposome-DOX and IgG-Liposome- DOX PD-1-Liposome-DOX was prepared by the ethanol injection method. Briefly, a mixture of DSPC, Cholesterol, DSPE-IRDye800CW, DSPE-DOTA, and DSPE-PEG-PD-1 (molar ratio: 49:43.9:0.1:2:2.5) was dissolved in Dimethyl Sulphoxide. Then, the solvent was injected into 350 mM ammonium sulfate solution by sonication. The liposome suspension was extruded by a polycarbonate membrane for 10 times, followed by dialysis against a sucrose solution (10 wt %, 5 mM NaCl) to remove the free ammonium sulfate. Then, DOX was mixed into the liposome suspension at a DOX/lipid (w/w) ratio of 1:10 and incubation for 24 h at room temperature. Finally, G-50 column was used to remove non-encapsulated free DOX.
Radiolabeling of Liposomes with 64Cu 6
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A 64CuCl2 stock solution (~2.0 mCi) was first dispersed in sodium acetate buffer (0.2 mL, 100 mM, pH 6.5). Then, liposomes (0.5 mL) in phosphate buffer saline (PBS) were also exchanged with acetate buffer by PD-10 column. The liposome fraction (1.5 mL) collected was later labeled with 64Cu radiotracer (incubation for 2 h at 43 °C). Finally, PD-10 column was used to purify the product. The percentage of
64
Cu labeled on liposomes was calculated by comparing the
radioactivity ratio of liposomes before and after purification.
Characterization of PD-1-Liposome-DOX and IgG-Liposome DOX Size and zeta potential of the PD-1-Liposome-DOX and IgG-Liposome DOX were evaluated by a dynamic light scattering (DLS) analyzer (90Plus/BI-MAS, Brookhaven Instruments Co., USA). Parameters including scattering angle of 90° and wavelength of 632.8 nm were used to performed the measurements at room temperature (n = 6). Data were automatically analyzed by the instrument. The results were presented as mean diameter ± SD. Transmission electron microscopy (TEM) measurement was applied to observe the structure of PD-1-Liposome-DOX and IgG-Liposome DOX, which was performed on a H-7650 apparatus (Tokyo, Japan, accelerating voltage=100 kV). Briefly, the liposome suspension (250 mmol/L) was first immersed and incubated for 10 min in formvar-coated copper grid (300-mesh). Then, the grids were taken out and negatively stained with aqueous uranyl acetate solution (2 wt.%), which was freshly prepared and sterile filtered for 10 min at room temperature. Before imaging, the copper grids were washed by deionized water and air dried.
Cell Culture 7
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The mouse breast tumor cell line 4T1-fLuc was cultured and maintained in RPMI 1640 medium (Hyclone, Thermo Scientific, USA) supplemented with 10% fetal calf serum (Hyclone, Thermo Scientific). The cells were cultured at 37 °C in a 5 % CO2 incubator.
Cell Survival Assay Cytotoxicity was assessed in vitro using CCK-8 kit (CCK-8, Dojindo, Japan). After seeding 96-well plates and culturing overnight, the 4T1-fLuc cells were incubated with free DOX, PD-1-liposome-DOX, or IgG-liposome-DOX for 24 h before washed with PBS. Next, the cells were incubated with 0.5 mg/mL WST-1 in serum-free medium for 1 h. Absorbance values (450 nm) were measured with microplate reader (BIO TEK, USA).
Establishment of the Tumor-Bearing Animal Model Female Balb/c mice (5-wk old, 18-24 g) were bought from the Department of Experimental Animals, Peking University Health Science Center. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Peking University with Permit Number: 2011-0039, and the experimental procedures were performed according to the approved guidelines. The mice were implanted with 4T1-fLuc cells (1 × 106) subcutaneously (s.c.).
In Vivo Fluorescence Imaging (FMI) FMI was carried out to examine the biodistribution and tumor-targeting effects of PD-1-Liposome-DOX-IRDye800CW in vivo. FMI was performed by utilizing an IVIS Imaging Spectrum System (PerkinElmer Inc., USA). IVIS Living Imaging 3.0 software (PerkinElmer Inc., 8
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USA) was utilized for the data analysis. The FMI data were collected dynamically after injection of PD-1-Liposome-DOX-IRDye800CW into the 4T1 mammary tumor-bearing mice (n = 3). IgG-Liposome-DOX-IRDye800CW was utilized as control (n = 3). For quantitative comparisons, the ratios of fluorescence signal intensities in the regions of interest corresponding to the tumor and muscle regions were determined. The following formula for the tumor to background ratio was used:
Tumor to background ratio =
Fluorescence light intensity tumor Fluorescence light intensity muscle
PET Imaging PET images were obtained using small animal PET (SuperArgus, GE Healthcare, Sedecal, Spain). For PET reconstruction, a two-dimensional algorithm was used. The tumor bearing mice were
imaged
approximately
PD-1-Liposome-DOX-DOTA-64Cu
14 or
day
after
4T1
cell
IgG-Liposome-DOX-DOTA-64Cu
implantation. was
injected
intravenously. Mice were anesthetized for around 5 min by isoflurane in an air-oxygen mixture before PET image acquisition, and the images were acquired at 12 and 24 h after probe injection (n = 3). Moreover, the biodistribution was studied at 12 and 24 h (n = 4).
Drug Treatment There were four groups of tumor-bearing mice: (1) PBS, (2) free DOX, (3) IgG-Liposome-DOX, and (4) PD-1-Liposome-DOX. The mice were given 5 mg/kg DOX. The IgG-Liposome-DOX and PD-1-Liposome-DOX treatment groups were given an equivalent DOX dosage of 5 mg/kg intravenously every 2 days. The PD-1 mAb (BioXcell, West Lebanon, NH) was given 200 µg 9
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through intraperitoneal injection.
In Vivo BLI and Tumor Volume Measurement To monitor and quantify tumor growth, the tumor-bearing mice were given with D-luciferin (150 mg/kg) (Fremont, CA, USA) intraperitoneally and imaged using the small animal optical molecular imaging system (PerkinElmer Inc., USA) on days 0, 3, 6, 9, 12, and 15 during drug treatment. All imaged mice were anesthetized with 2 % isoflurane and warmed until they awakened. The tumor largest (a) and smallest (b) diameters were measured every 2 days, and the tumor volume was calculated as V = ab2/2.
Statistics The GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA) was used for the data analysis. Three independent experiments were repeated, and the data was presented as mean ± SEM. To compare the differences between groups, one-way analysis of variance followed by Tukey’s multiple comparisons test was performed. The means of two groups was compared using two-tailed Student’s t test. Kaplan-Meier method was used for the comparison of survival rate. *P < 0.05 was set as the significance level.
RESULTS Synthesis and Characterization of PD-1-Liposome-DOX and IgG-Liposome-DOX In this study, the ethanol injection method was used to prepare the multifunctional liposomes. 10
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Formulations were made with either PD-1 mAb or IgG2a mAb (Figure 1). By using ammonium/pH gradient method, DOX was highly efficient loaded into the liposomes. The remote encapsulation of DOX into the liposomes exhibited significant advantages at a high drug to lipid molar ratio, as it can improve the drug delivery and enhance cancer therapeutic effect. The properties of PD-1-Liposome-DOX and IgG-Liposome-DOX, including particle size, particle charge, and serum stability, were characterized, and the data were shown in Table 1 and Figure 2. High DOX encapsulation efficiency in both liposomes were achieved (95.45 ± 0.21% and 96.24 ± 0.35%) (Table 1). The sizes and surface potentials of these two kinds of drug loaded liposomes were comparable, the particle size of both liposomes showed narrow size distribution with diameters about 85 nm. Although their zeta potential was low (−3.56 ± 0.3 and −4.16 ± 0.5 mV), it was strong enough to keep good colloidal stability in the presence of the PEG chains on the particles surface. The TEM images revealed that both liposomes showed spherical and homogeneous NPs with a size range from 70 to 100 nm (Fig. 2A and Fig. 2B), well consistent with the dynamic light scattering (DLS) measurement shown in Fig. 2C and Fig. 2D. The stability of the PD-1-Liposome-DOX and IgG-Liposome-DOX were further assessed by mixing these liposomes in serum containing DMEM. Hydrodynamic particle size showed no obvious change in the DMEM solutions after 48h (Fig. 2E), showing great potential for further application in biological system.
Effects of the Different Treatments on Cell Survival in Vitro After characterizing the nanoparticles in vitro, we further studied the antitumor effects of free DOX, PD-1-Liposome-DOX, and IgG-Liposome-DOX on 4T1-fLuc cell viability. The results 11
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showed that both PD-1-Liposome-DOX and IgG-Liposome-DOX decreased the cell survival rate and increased the inhibition rate compared with free DOX at both 1 and 10 µg/mL (Fig. 3A). To further confirm the results, we performed ex vivo BLI imaging of 4T1-fLuc cells to determine the number of live cells following the different treatments. BLI intensity was further quantified and analyzed in triplicate. The data revealed that both PD-1-Liposome-DOX and IgG-Liposome-DOX were effective against tumor cells, similar to free DOX, and showed decreased BLI intensities compared with PBS-treated cells ex vivo (Fig. 3B). The above results indicated that PD-1-Liposome-DOX and IgG-Liposome-DOX retained the cell toxicity effects of DOX and can be used for in vivo antitumor chemotherapy.
Biodistribution of PD-1-Liposome-DOX with IRDye800CW/64Cu Dual-Modality NIRF/PET Imaging on 4T1 Mammary Tumors To explore whether PD-1-Liposome-DOX displayed enhanced tumor-targeting effects in vivo, nanoparticles were dual labeled with IRDye800CW and 64Cu for in vivo NIRF and PET imaging. The UV−Vis absorption and fluorescence properties and the Radio Thin-Layer Chromatography of nanoparticles were characterized, and the data are shown in Supplemental Fig. 1 and Supplemental Fig. 2. FMI was dynamically examined from 0, 5, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after intravenous administration of nanoparticles. A higher tumor to background ratio of PD-1-Liposome-DOX-IRDye800CW
was
from
2
to
48
h
compared
to
IgG-Liposome-DOX-IRDye800CW, and the most significant tumor to background ratio difference was around 2.4-fold higher compared to the IgG control found at 24 h (Fig. 4A). Typical FMI 12
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images at 24 h are shown in Fig. 4B, and BLI was performed to determine the exact location of tumors in situ (Fig. 4B). The FMI signal was stronger in the PD-1-Liposome-DOX-IRDye800CW group compared to the IgG-Liposome-DOX-IRDye800CW group. To further confirm our observations, we dissected out the tumor and major organs after the in vivo observations at 24 h. The NIRF signal was higher in tumors in the PD-1-Liposome-DOX-IRDye800CW group compared to IgG control (Fig. 4C). PET imaging of 4T1 mammary tumors was performed at 12 and 24 h post-injection of PD-1-Liposome-DOX-DOTA-64Cu. Representative PET images of mice are shown in Fig. 5A. PD-1-Liposome-DOX-DOTA-64Cu displayed a higher PET signal in tumors compared to IgG-Liposome-DOX-DOTA-64Cu at both 12 and 24 h. The tumor-to-muscle ratio of PD-1-Liposome-DOX-DOTA-64Cu was approximately 1.40-fold and 1.8-fold higher compared to IgG-Liposome-DOX at 12 h and 24 h, respectively (Fig. 5B). To validate this observation, the biodistribution of the nanoparticles was examined in tumors and different organs. In general, higher
tumor
uptake
was
found
at both 12
and
24
h after
administration of
PD-1-Liposome-DOX-DOTA-64Cu compared to the IgG control (Fig. 5C and 5D). For example, 64
Cu uptake was 2.34 ± 1.25 %ID/g for PD-1-Liposome-DOX-DOTA-64Cu and 1.27 ± 0.65 %ID/g
for the IgG control at 24 h post-injection. Moreover, it was observed that the highest uptake of 64
Cu was in the spleen, which functions in priming T cells for antigen surveillance and attack
before deploying them throughout the body, and the signals in tumors are lymphocytes that have infiltrated the tumor. Taken together, the PET and NIRF imaging results demonstrated PD-1 mAb-targeted liposomes improved in vivo targeted tumor imaging.
13
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Comparison of the Different Treatment Effects Using 4T1 Mammary Tumor Xenografts The different treatment effects were further studied. Tumor growth was dynamically monitored from days 1 to 15 using BLI imaging, which is a sensitive indicator of tumor growth (Fig. 6A and 6B). The results showed that BLI light intensity greatly increased in the PBS group, whereas relatively a mild increase in BLI light intensity was observed in the free DOX and IgG-Liposome-DOX groups during the 15-day observation. The PD-1-Liposome-DOX group exhibited lower light intensities compared to the free DOX and IgG-Liposome-DOX groups during the 15-day observation. We also measured the tumor volumes, and they were consistent with the BLI data showing that PD-1-Liposome-DOX exhibited better antitumor efficacy than other treatments. Furthermore, the survival rate was also monitored, and the PD-1-liposome-DOX treatment prolonged the survival of mice compared to other groups.
DISCUSSION Although a single imaging modality can provide some insights into a disease process, in vivo multimodality imaging can offer a new prospective. In this study, we provide proof-of-principle for targeting PD-1-expressing breast tumors in vivo with dual-labeled IRDye800CW and 64Cu in 4T1 a mammary tumor mouse model. In addition, PD-1-Liposome-DOX exhibited significantly enhanced therapeutic efficacy compared to IgG-Liposome-DOX and free DOX. This is the first report, to the best of our knowledge, on the use of PD-1 mAb-targeted liposomes for targeted imaging and therapy of breast tumors. The active targeting of cancers is one of the most important considerations for cancer imaging and therapy. PD-1 over-expression is involved in a diverse array of tumor types because of its 14
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participation in signaling pathways regulating attenuated CD8+ T cell function and enhanced Treg activity, which creates an inhibitory environment in the tumors
18-20
. Thus, the selective PD-1
expression in the tumor represents a potential target for the antibody-guided targeting of tumors with fluorophores and radionuclides. Moreover, the expression of PD-L1 on tumor cells is a potent biomarker to predict the responses of immunotherapies to PD-1 or PD-L1 21. However, at present, using immunohistochemistry to assess tumor PD-1/PD-L1 expression has been proven to be a partial and imperfect predictor of anti-PD-1/anti-PD-L1 immune therapeutic effects
22
. FMI and
immunoPET imaging are non-invasive means by which PD-1 expression can be measured throughout an entire tumor simultaneously. In this study, we examined PD-1 as a biomarker for targeted breast tumor imaging and therapy using NIRF/PET imaging. Indeed, our in vivo FMI data revealed a relatively higher signal for PD-1-Liposome-DOX in the tumor regions compared to IgG-Liposome-DOX, which was confirmed by the ex vivo imaging of tumors. A shortcoming of FMI is the limited penetration depth of the emitted light into tissue. A new approach to deal with the limited tissue penetration of FMI is to combine it with radionuclide detection. Therefore, we performed PET imaging by labeling the nanoparticles with
64
Cu. Consistent with the NIRF
observations, our PET imaging and biodistribution data revealed that PD-1-Liposome-DOX exhibited better tumor-targeting effects and longer retention in tumors compared to the IgG. Targeted cancer therapy depends on the specific-expression of tumor biomarkers for the selective delivery of therapeutic agents to tumors while reducing the side effects to normal organs 16
. As for chemotherapy, achieving increased tumor-targeting treatment efficacy and decreased
organ toxicities currently remain a challenge. In this study, we encapsulated DOX in the liposomal nanoparticles with PD-1 mAb labeling. The results showed that compared to the 15
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IgG-Liposome-DOX and free DOX, PD-1-Liposome-DOX exhibited better antitumor activity, as demonstrated by BLI imaging and tumor volume measurement, and also longer survival. The two possible underlying antitumor mechanisms were delineated. On the one hand, PD-1 mAb as a potent biomarker increased the tumor-targeting effects of Liposome-DOX specifically in the tumor regions as shown by the in vivo NIRF and PET imaging and biodistribution results. On the other hand, it was reported that anti-PD-1 mAb combined with chemotherapy exhibited an encouraging antitumor effect in advanced non-small cell lung cancer
23
. Previous works provide
evidence that PD-L1 expression may mediate chemotherapy resistance and also may provide the rationale for a combined chemotherapeutic agents and anti-PD-1/PD-L1 mAbs for cancer treatment
24
. In this study, our flow cytometry data on TILs in supplemental Fig. 3 revealed that
there were relatively more CD3+ immune T cells and CD8+ T cells, which mainly have an antitumor growth function. This finding indicated that PD-1 mAb may simultaneously works as an adjuvant immunotherapy for DOX chemotherapy. As efficient drug nanocarriers, liposomes carry hydrophobic or hydrophilic drugs, showing good biocompatibility. Many liposome nanoparticles have been fabricated and approved by FDA for clinical applications. However, for achieving more personalized cancer therapy, theranostic agents with both therapeutic and imaging functions are urgently needed for image guidance and therapeutic
response
monitoring.
Compared
to
the
conventional
liposome,
PD-1-Liposome-DOX-64Cu/IRDye800CW described in this study can improve the in vivo tumor targeting and therapy ability of liposomes. By taking advantages of each imaging modality, pharmacokinetics and therapeutic response of drugs, and the prognosis-associated disease status can be better monitored and undertaken. Nevertheless, the problem we have to consider is more 16
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experiments are needed to verify the biological safety of multifunctional liposomes before its clinical application. Taken together, our study indicates that PD-1-expressing breast tumors can be visualized sensitively and specifically using dual-modality NIRF/PET imaging with the newly developed PD-1-Liposome-DOX-64Cu/IRDye800CW conjugate. Moreover, the PD-1-Liposome-DOX exhibited enhanced antitumor therapeutic effects. This is the first report, to our knowledge, on the use of PD-1 mAb as a target, IRDye800CW/64Cu as imaging agents, and DOX as a chemotherapeutic drug for the noninvasive NIRF/PET imaging and chemo- and immunotherapy of 4T1 mammary tumors. Our results demonstrate the efficacy of the application of our targeted nanoparticles to treat breast cancer, and highlight the promising clinical translational ability of PD-1-liposome-DOX for breast tumor management and suggest a potential strategy for theranostics for other PD-1-expressing tumors.
ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (81227901, 81470083, 81527805, 81571810, 61231004, 81771846), the Research and Development Program of China (973) under Grant (2014CB748600, 2015CB755500), the Strategic Priority Research Program from Chinese Academy of Sciences (XDB02060010), the Beijing Natural Science Foundation
(Z16110200010000)
and
grants
from
Peking
(BYSY2015023).
FINANCIAL DISCLOSURE 17
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No potential conflicts of interest were declared by authors.
SUPPORTING INFORMATION The supplemental method of analysis of the tumor infiltrating lymphocytes (TILs) composition, and supplemental results for UV−Vis absorption and fluorescence properties, the radio-labeling yield of PD-1-Liposome-64Cu, and the effects of PD-1-Liposome-DOX on TILs were supplied as supporting Information.
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23. Kanda, S.; Goto, K.; Shiraishi, H.; Kubo, E.; Tanaka, A.; Utsumi, H.; Sunami, K.; Kitazono, S.; Mizugaki, H.; Horinouchi, H.; Fujiwara, Y.; Nokihara, H.; Yamamoto, N.; Hozumi, H.; Tamura, T., Safety and efficacy of nivolumab and standard chemotherapy drug combination in patients with advanced non-small-cell lung cancer: a four arms phase Ib study. Ann Oncol 2016, 27 (12), 2242-2250. 24. Rizvi, N. A.; Hellmann, M. D.; Brahmer, J. R.; Juergens, R. A.; Borghaei, H.; Gettinger, S.; Chow, L. Q.; Gerber, D. E.; Laurie, S. A.; Goldman, J. W.; Shepherd, F. A.; Chen, A. C.; Shen, Y.; Nathan, F. E.; Harbison, C. T.; Antonia, S., Nivolumab in Combination With Platinum-Based Doublet Chemotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer. J Clin Oncol 2016, 34 (25), 2969-79.
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Molecular Pharmaceutics
Table 1. Characterization of PD-1-Liposome-DOX and IgG-Liposome-DOX. Size Formulation (nm)
IgG-Liposome-DOX
Zeta potential
Entrapment efficiency
(mV)
(%)
PDI
83.52±3.55
0.201
−3.56 ± 0.3
95.45 ± 0.21
PD-1-Liposome-DOX 84.56±2.58
0.189
−4.16 ± 0.5
96.24 ± 0.35
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Molecular Pharmaceutics
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Figure 1 Schematic illustration of PD-1 mAb-targeted multifunctional liposome with DOX loading. 140x105mm (300 x 300 DPI)
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Molecular Pharmaceutics
Figure 2. TEM images and size distribution of nanoparticles. (A) and (C) IgG-Liposome-DOX; (B) and (D) PD-1-Liposome-DOX. Diameter of both liposomes in serum containing DMEM vs time measured by DLS (E). Both liposomes showed excellent serum stability. 140x77mm (300 x 300 DPI)
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Figure 3 Evaluation of PD-1-Liposome-DOX treatment on cell survival compared to the IgG control and free DOX in vitro. (A) The 4T1-fLuc tumor cell survival and inhibition rates after different treatments were evaluated using CCK8 kit. (B) BLI imaging of 4T1-fLuc tumor cells following different treatments in triplicate, and statistics for the BLI light intensities in 4T1-fLuc cells following different treatments. ns indicates no significant difference, P > 0.05. 140x105mm (300 x 300 DPI)
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Molecular Pharmaceutics
Figure 4 FMI of PD-1-Liposome-DOX-IRDye800CW in 4T1 mammary tumor-bearing mice in vivo and ex vivo. (A) Comparison of the tumor to background ratio between the PD-1-Liposome-DOX-IRDye800CW and IgGLiposome-DOX-IRDye800CW groups. (B) BLI and FMI imaging of tumors in situ in the PD-1-Liposome-DOXIRDye800CW and IgG-Liposome-DOX-IRDye800CW groups. (C) The ex vivo FMI imaging of tumors (T) and major organs, including the liver (L), kidney (K), spleen (S), and heart (H), 24 h after the in vivo observations. **P < 0.001. 140x88mm (300 x 300 DPI)
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Figure 5 PET imaging and biodistribution of PD-1-Liposome-DOX-DOTA-64Cu in the 4T1 mammary tumorbearing mice. (A) PET images of 4T1 mammary tumor-bearing mice at 12 and 24 h post-injection of PD-1Liposome-DOX-DOTA-64Cu. (B) The tumor to muscle ratio of PD-1-Liposome-DOX and IgG-Liposome-DOX at both 12 h and 24 h time points. Biodistribution of PD-1-Liposome-DOX-DOTA-64Cu in 4T1 tumors 12 h (C) and 24 h (D) post-injection. 140x105mm (300 x 300 DPI)
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Molecular Pharmaceutics
Figure 6 Comparison of PD-1-Liposome-DOX treatment efficacy with other treatments in vivo. (A) Dynamic in vivo BLI monitoring of tumor growth after different treatments from days 1 to 15. (B) BLI light intensities and tumor volume of different treatment groups at different time points, and the Kaplan-Meier survival plots of survival rate (n = 10). *P < 0.05. 140x113mm (300 x 300 DPI)
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PD-1-expressing breast tumors can be visualized sensitively and specifically using dual-modality NIRF/PET imaging with newly developed PD-1-Liposome-DOX-64Cu/IRDye800CW. The PD-1-Liposome-DOX64Cu/IRDye800CW significantly enhanced the antitumor effects of doxorubicin through active tumor targeting effects and also the adjuvant immune therapeutic effects of PD-1 mAb. Our study highlights the potential of using dual-labeled PD-1 mAb-targeted Liposome-DOX-64Cu/IRDye800CW as theranostic agent for the management of breast cancer 88x55mm (300 x 300 DPI)
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