- Email: [email protected]
Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer
Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer Xiaoxiao Liu, Xiang Gao, Songping Zheng, Bilan Wang, Yanyan Li, Chanjuan Zhao, Yagmur Muftuoglu, Song Chen, Ying Li, Haiyan Yao, Hui Sun, Qing Mao, Chao You, Gang Guo, Yuquan Wei PII: DOI: Reference:
S1549-9634(17)30067-9 doi: 10.1016/j.nano.2017.04.006 NANO 1565
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
8 July 2016 4 April 2017 10 April 2017
Please cite this article as: Liu Xiaoxiao, Gao Xiang, Zheng Songping, Wang Bilan, Li Yanyan, Zhao Chanjuan, Muftuoglu Yagmur, Chen Song, Li Ying, Yao Haiyan, Sun Hui, Mao Qing, You Chao, Guo Gang, Wei Yuquan, Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.04.006
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ACCEPTED MANUSCRIPT Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer Xiaoxiao Liu1,7,*, Xiang Gao1,*,#, Songping Zheng1,*, Bilan Wang2, Yanyan Li6, Chanjuan
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Zhao2, Yagmur Muftuoglu3, Song Chen3, Ying Li3,8, Haiyan Yao4, Hui Sun5, Qing Mao1, Chao You1, Gang Guo1 and Yuquan Wei1 1
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Department of Neurosurgery and Institute of Neurosurgery, State Key Laboratory of
Biotherapy, West China Hospital, West China Medical School, Sichuan University and
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Collaborative Innovation Center for Biotherapy, Chengdu, 610041, PR China. 2
Department of Pharmacy, West China Second University Hospital of Sichuan University,
Chengdu, 610041, PR China. 3
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Department of Pharmacology, Yale School of Medicine, Yale University, New Haven, CT
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06520, USA. 4
Southern Connecticut State University, New Haven, Connecticut, United States of America.
5
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Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven,
6
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Connecticut, United States of America. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Fudan
University, Shanghai, China. 7
Department
of
Medical
Oncology,
Cancer
Center,
State
Key
Laboratory
of
Biotherapy/Collaborative Innovation Center for Biotherapy, and West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, PR China. 8
Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University,
Dalian 116023, China *These authors are considered equal first authors.
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Correspondence: Xiang Gao
Tel: +86 28 8542 2136; Fax: +86 28 8550 2796
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Email: [email protected]
This work was supported by the National Natural Science Foundation of China
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(NSFC81502165) and the Sichuan University Outstanding Young Scholars Research Fund (2016SCU04A04).
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Conflict of interest: The authors declare no confltics of interest.
ACCEPTED MANUSCRIPT Abstract For the past few years, immunotherapy has recently shown considerable clinical benefit in
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CRC therapy, and the application of immunologic therapies in cancer treatments continues to increase perennially. Interleukin-12, an ideal candidate for tumor immunotherapy, could
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activate both innate and adaptive immunities. In this study, we developed a novel gene delivery system with a self-assembly method by MPEG-PLA and DOTAP(DMP) with
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zeta-potential value of 38.5 mV and size of 37.5 nm. The supernatant of lymphocytes treated with supernatant from Ct26 transfected pIL12 with DMP could inhibit Ct26 cells growth ex vivo. Treatment of tumor-bearing mice with DMP-pIL12 complex has significantly inhibited
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tumor growth at both the subcutaneous and peritoneal model in vivo by inhibiting
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angiogenesis, promoting apoptosis and reducing proliferation. The IL-12 plasmid and DMP
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complex may be used to treat the colorectal cancer in clinical as a new drug.
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Key word: Colorectal cancer, Immunotherapy, Interleukin-12, Nanoparticles.
ACCEPTED MANUSCRIPT Background Colorectal cancer (CRC), one of the most prevalent cancers, is a leading cause of cancer
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mortality worldwide1. An estimated 1.4 million cases are diagnosed every year, and more than 690,000 patients died from the disease in 2012 according to global cancer statistics 2. Due to
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significant strides in traditional treatments, such as surgical management, chemotherapy and radiation therapy, the average survival for advanced CRC now approaches 30 months3,4.
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However, almost half of all patients develop liver or peritoneal metastases over the course of this disease. Even with treatment, the median survival for metastatic CRC patient is still relatively low, and current treatment paradigms appear to have reached their maximum
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benefit. Therefore, for decades, considerable efforts have been devoted to developing more
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effective anti-tumor agents and better strategies for targeting tumors5-7. Over the past few years, immunotherapy has shown considerable clinical benefit in
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treating CRC, and the application of immunologic therapies in cancer treatments continues to
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increase every year. Cytokine-based immune therapy, which could induce or augment the anti-tumor immune response, has proven effective in a variety of models 3,9. Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine, was proven to be an ideal candidate for tumor immunotherapy, due to its ability to activate both innate and adaptive immune responses9-11. Specifically, IL-12 has been demonstrated to be one of the most potent anti-tumor cytokines in experimental animal models, and it proves effective in eradicating experimental tumors, eliciting long-term anti-tumor immunity, and inhibiting tumor formation and metastases12-14. However, clinical trials testing the therapeutic potential of administering IL-12 have been halted because of serious potential systemic toxicity and lower-than-anticipated efficacy15,16.
ACCEPTED MANUSCRIPT Conversely, administration of IL-12 via gene therapy is an ideal method for harnessing the power of this cytokine because expression of IL-12 can be maintained at low levels and will
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eventually subside to basal levels. To optimize the delivery of IL-12 gene therapy, more efficient gene carriers with lower levels of toxicity are strongly needed.
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While replication-deficient adenovirus (Ad) offers several important advantages as a vector for gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal
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transduction efficiency, and adverse systemic side effects17-19. Its application for gene therapy introduces serious concerns about endogenous virus recombinations, oncogenic effects, and immunological reactions. However, non-viral gene delivery systems such as the nanoparticles
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described in this work show great potential for overcoming physical and biological barriers to
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achieving therapeutic levels of transgene expression at target sites20-24. In this study, we developed a novel gene delivery system with a self-assembly method by poly(ethylene
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methoxy
glycol)-poly(lactide)
(MPEG-PLA)
and
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1,2-dioleoyl-3-trimethylammonium-propan (DOTAP). Further, we show that transfected cancer cells express and secrete high levels of IL-12, which activates the immune system. In addition, potential toxicity is dramatically reduced, and the resulting steady expression of IL-12 creates a stronger anti-tumor immune response. Also presented in this work are the pharmaceutical properties, in vitro biological activity, in vivo anti-tumor effects, anti-tumor mechanisms, and a preliminary toxicity evaluation of DOTAP/MPEG-PLA-pIL12 (DMP-pIL12) .
ACCEPTED MANUSCRIPT Methods Materials
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1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, U.S.); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
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tetrazolium bromide (MTT) from Sigma (USA); Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) from Gibco BRL (USA); methanol and acetic acid
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(HPLC grade) from Fisher Scientific (UK); Mice lymphocyte separation medium from Dakewe (China) and dimethyl sulfoxide (DMSO) and acetone from KeLong Chemicals (China). Antibodies purchased include: rat anti-mouse CD31 polyclonal antibody (BD USA),
rabbit
anti-mouse
Ki67
antibody
(Abcam,
USA),
and
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PharmingenTM,
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rhodamine-conjugated secondary antibody (Abcam, USA). A plasmid encoding p35 and p40 subunits of murine IL12 (pIL12) was constructed at the BGH site of the pVAX vector
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(Invitrogen Corp., Carlsbad, CA, U.S.), an expression vector encoding kanamycin resistance
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under the control of a separate T7 promoter/enhancer. pVax served as a negative control. All plasmid DNA were extracted using the EndoFree Plasmid Giga kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Preparation of DOTAP/MPEG-PLA (DMP), characterization of DMP and agarose gel electrophoresis of naked plasmid DNA and complexes are described in the supplementary materials.
Cell culture and transfection experiments Murine Ct26 colon carcinoma cells was purchased from American Type Culture
ACCEPTED MANUSCRIPT Collection (ATCC® Number: CRL-2638™, Manassas, USA) and were routinely cultured in RPMI 1640 medium (Gibco-BRL, Rockville, IN, USA) containing 10 % FBS (Sigma
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Chemical Co., St. Louis, MO), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in a humidified atmosphere containing 5 % CO2 at 37 °C.
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DMP and pEGFP plasmid were incubated respectively in RPMI 1640 medium without FBS for five min. After that, they were mixed and incubated for twenty min. The Ct26 cells in
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6 well cell culture plates were washed twice used RPMI 1640 medium without FBS. Pre-mixed solution was put into 6 well cell culture plates for 4 h. Then pre-mixed solution replaced by RPMI 1640 medium containing 10 % FBS. Subsequent operations were took in
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corresponding time points. DMP, containing 4 μg pEGFP, was used to transfect Ct26 cells for
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48 h. Transfected cells were photographed using an Array Scan VTI HCS Reader (Thermo Fisher Scientific Inc., Waltham, MA, U.S.). Transfection efficiencies at both ratios (pEGFP
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versus DMP at 1 : 100 and 1 : 150) were determined using FACS Calibur flow cytometry (BD
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Biosciences, San Jose, CA, U.S.).
Measurement of IL-12 expression Ct26 cells were transfected with DMP-pIL12 liposomes and cultured for 72 h before IL-12 expression was measured by RT-PCR, flow cytometry, and ELISA. IL-12 mRNA was measured by Reverse Transcription polymerase chain reaction (RT-PCR) using the PrimeScript TM RT reagent Kit (Takara Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning, China) and 2×Taq MasterMix kit (Beijing CoWin Biotech Co., Ltd., Beijing, China). The RT-PCR primers for IL-12 gene were 5’-TCC TGC TTC ACG CCT TCA-3’ (forward) and 5’-
ACCEPTED MANUSCRIPT AGT CCA GTC CAC CTC TAC AAC ATA-3’ (reverse). Transfection efficiency was examined by flow cytometry. Cells were permeabilized using a BD Cytofix/Cytoperm™
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Fixation/permeabilization Kit and stained with PE Rat Anti-Mouse IL-12 (p40/p70) overnight. The level of IL-12 expression in the cell culture supernatant at 24 h, 48 h, and 72 h were
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measured by ELISA kits (eBioscience, Inc., San Diego, CA, U.S.) According to manufacturer’s instructions, the plate was incubated with capture antibody overnight at 4 °C.
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The plates were washed in accordance with the requirements. Then, standards and samples were put into the plates at room temperature for 2 h. The detection antibody was added after washing the plates. The work of color and termination were done after 1 h. Finally, read
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absorbance at 450 nm with a reference filter of 570 nm.
Measurement of cell viability and secretion of IFN-γ
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Ct26 cells were culture in a 6 well cell culture plates, with a concentration of 2 * 105 cells
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per well. After Ct26 cells were transfected with NS (NS: normal salt), DMP, DMP-pVax (pVax: the vector plasmid), or DMP-pIL12 for 72 h, supernatant was collected from each group.
These samples were further applied to culture lymphoctytes derived from murine spleen. After 24 h, the viability of lymphocytes from each group (NS, DMP, DMP-pVax, and DMP-pIL12) was tested by the MTT method. Briefly, 20 µl of 5 mg/ml MTT was added into each well and incubated at 37 °C for 3 h. The lymphocytes were fixed on the 96 well cell culture plates by centrifuging at 3000 rpm. Media was removed and 150 µl of DMSO was added. The cell plate was shaked for 15 min in dark. Lastly absorbance at 570 nm was read by
ACCEPTED MANUSCRIPT microplate reader (Thermo, USA). Another experiments were carried out at the same time and supernatant was collected from each group of lymphocytes and further used to culture CT26
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cells and with 20 µM oxaliplatin as positive control. After 24 h, the viability of CT26 cells from each group was tested, again using the MTT method what we described above except
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using centrifuge. In addition, supernatant was collected from each group of lymphocytes after
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3 h, 8 h, and 24 h of culture to measure the expression of IFN-γ by ELISA as described.
In vivo study of the anti-tumor properties of DMP-pIL12
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This section is expounded in the supplementary materials.
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Cytotoxicity Assay
A 4 h 51Cr release assay was performed as previously described by other reports25,26. In
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the peritoneal tumor model, splenocytes from each group (NS, DMP, DMP-pVax, or
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DMP-pIL12) were harvested after the mice received two treatments. Briefly, spleen lymphocytes obtained from the four groups, as the effector cells, were treated with ammonium chloride-potassium lysis buffer to deplete erythrocytes. Ct26 (1 * 106), as the target cells, were labeled with 100 μCi 51Cr for 1 h at 37 °C, washed, and resuspended at a concentration of 1 * 105 cells/ml. A total of 200 μL of effector cells (E) and 51Cr-labeled target cells (T) were assigned at different E:T ratios to each well of a 96 well plate and incubated for 4 h at 37 °C. Supernatant (100 μL) from each group was then harvested; cellular activity was calculated by this formula: % cytotoxicity = [(experimental release − spontaneous release) / (maximum release − spontaneous release) ] × 100.
ACCEPTED MANUSCRIPT TUNEL assay of Tumor Samples Apoptotic cells in tumor tissues were detected in paraffin sections using the Dead End
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Fluorometric system (Promega, Madison, WI, U.S.), a terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, according to the manufacturer's
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instructions. All sections were observed and digitally photographed under a DM 2500 fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany), and the
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quantification of TUNEL-positive cells was assessed according to previous reports21.
Measurement of tumor cell proliferation and microvessel density
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Immunohistochemistry identifying expression of the Ki67 antigen and CD31, marker for
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assessing microvessel density (MVD), were carried out with rabbit anti-mouse Ki67 and rabbit anti-mouse CD31 antibodies using the labeled streptavidin-biotin method as previously
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described21. Quantification of MVD was estimated by counting the number of microvessels in
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five random fields at ×200 magnification. A single microvessel was defined as a discrete cluster or single cell stained positive for CD31. All sections were observed or counted by two investigators or pathologists in a blinded fashion.
Assessment of systemic toxicity induced by treatment with DMP-pIL12 To evaluate toxicity caused by treatment with DMP-pIL12, vital organs (heart, liver, spleen, lungs, and kidneys) of mice were collected and fixed in 4 % paraformaldehyde. After 24 h, samples were embedded in paraffin wax and sliced into 4-μm sections. Sections were then hydrated and stained with hematoxylin and eosin (Beyotime Biotechnology (Shanghai)
ACCEPTED MANUSCRIPT Co., Ltd., Shanghai, China) (H&E) according to the manufacturer’s instructions for histomorphometric analysis. Representative images of tissues were taken with an Olympus
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light microscope.
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Statistical analysis
All data were analyzed using GRAPHPAD PRISM software (GraphPad, San Diego, CA).
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Data from multiple groups were analyzed using ANOVA, and multiple comparisons between the groups were performed using the Newman–Keuls method after ANOVA. Survival data were plotted using Kaplan–Meier curves and analyzed using the log-rank test. Tumor volumes
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[(smaller diameter)2 (larger diameter) * 0.52] and assessment of vessel density were analyzed
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using the Student’s t test. All values were presented as the mean±the standard error of
Results
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measurement. P < 0.01 was considered to be statistically significant for all experiments.
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DMP successfully incorporates DNA DMP and DMP-pIL12 were produced using the described self-assembly method (Figure 1A). As shown in Figure 1B, the zeta-potential and size of DMP were 38.5 mV and 37.5 nm, respectively (Figure 1C). TEM images show that, morphologically, DMP adopts a bilayer spheroidal structure at around 25 nm in diameter (Figure 1D). Gel retardation assays were used to characterize the ability of the DMP liposome to carry DNA. As shown in Figure 1E, free DNA not entrapped in the DMP appeared as a bright band (lanes 1 to 3), and no other band of free DNA was observed from lanes 13 to 15. This suggests that pIL-12 DNA was completely incorporated into the DMP and that the complexes were prepared successfully
ACCEPTED MANUSCRIPT without free DNA (pIL12) at the ratio of 100 times the amount of DMP to DNA. And loading efficiency in different weight ratio of 1 : 25, 1 : 50 and 1 : 100 are 70.3 ± 5.2 % , 91.3
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± 2.3 % and 100 %, respectively.
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DMP-pIL12 has high transfection efficiency in Ct26 cells
In the ex vivo cell transfection assays, DMP had high GFP transfection efficiency in Ct26
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cells after incubation for 24 h as shown in Figure 2A. The specific transfection rate demonstrated by flow cytometry analysis was 39.2 % and 54.4 % when DMP was mixed with GFP at a ratio of 1 : 100 and 1 : 150, respectively. (Figure 2B).
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Ct26 cells were transfected with NS, DMP, DMP-pVax, or DMP-pIL12, and we then
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measured the expression of IL-12 in these group cells. The level of IL-12 mRNA in the DMP-pIL12 group was significantly higher than in the control groups (Figure 2C). We also
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tested the transfection efficacy of DMP carrying IL-12 and found the 1 : 150 ratio to be
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optimal, which resulted in a transfection rate of 50.7 % and acceptable cell toxicity as shown in Figure 2D. Therefore, we used this ratio in the remaining studies. Further, the level of IL-12 expression in the cell culture supernatant was significantly higher than in the control groups after 24 h, 48 h, and 72 h of culture (Figure 2E).
DMP-pIL12 increases the proliferation and anti-tumor effects of lymphoctyes Ct26 cells were transfected with NS, DMP, DMP-pVax, or DMP-pIL12 for 72 h, and supernatant from these groups were collected for culture of lymphocytes. After 24 h of culture, viability of lymphocytes cultured with supernatant from the DMP-pIL12 group was
ACCEPTED MANUSCRIPT significantly higher than the lymphocytes cultured with supernatant from the control groups (Figure 3A). Supernatant from the four groups of lymphocytes were then collected to culture
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fresh Ct26 cells for 24 h. Supernatant from lymphocytes cultured with supernatant from DMP-pIL12-transfected Ct26 cells significantly inhibits the growth of fresh Ct26 cells
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(Figure 3B). We then analyzed the mechanisms behind this observation and found higher levels of IFN-γ expression in this supernatant than in the corresponding control samples.
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(Figure 3C-E)
DMP-pIL12 causes in vivo anti-tumor effects
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DMP and its complexes loaded with pVax and pIL-12 were used to treat Ct26 colon
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cancer in BALB/c mice model via intra-tumor injection. DMP-pIL12 treatment showed dramatic anti-tumor effects than other complexes (DMP-pVax, DMP, and NS) as shown in
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Figure 4 (A, C, D), and no significant differences were observed among mice in the
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DMP-pVax, DMP, and NS groups. In addition, DMP-pIL12-treated mice had slightly lower body weights than mice in the control groups (Figure 4B). We also observed similar anti-tumor effects in the Ct26 peritoneal model of colon cancer. Figure 5A and Figure 5B shows that the average weight of DMP-pIL12-treated mice and tumor were significantly lower than that of mice in the DMP-pVax, DMP, and NS groups. The mice administered DMP-pIL12 had significantly fewer and smaller tumor nodules than mice in the control groups (Figure 5C and 5D). In addition, administration of DMP-pIL12 also reduced the occurrence of ascites (Figure 5E).
ACCEPTED MANUSCRIPT Administration of DMP-pIL12 increases the expression of IL-12 and secretion of IFN-γ and TNF-α
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We next explored the potential mechanisms responsible for the observed anti-tumor effects of DMP-pIL12 and tested the expression of IL-12 by flow cytometry and ELISA.
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After treatments, tumors from both mouse models were harvested, and we found that mice administered DMP-pIL12 had significantly higher levels of expression of IL-12 in the tumor
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tissue than mice from control groups (Figure 6A and Supplementary Figure 1A and 1B), but no difference in serum (Supplementary Figure 1C and 1D). The anti-tumor effects of DMP-pIL12 could also be explained by the increase of IFN-γ
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and TNF-α expression in ascites and tumor tissues after treatments (Figure 6B, Figure 6C and
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Supplementary Figure 2). Specifically, the mean TNF-α concentration in DMP-pIL12- treated tumors in the subcutaneous model was significantly higher than that in DMP-pVax-treated
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tumors, in which only a very small concentration of TNF-α was expressed. We also observed
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similar results in the peritoneal model (Figure 6B). These observations suggest that increased expression of IL-12 results in expression of TNF-α at the tumor site. In addition, we observed the highest levels of IFN-γ in the subcutaneous and peritoneal models as a result of treatment with DMP-pIL12 (Figure 6C). DMP-pIL12 treated mice had no difference at level of TNF-α and IFN-γ in serum in subcutaneous model. And DMP-pIL12 treated mice had lower level of IFN-γ in serum in peritoneal model (Supplementary Figure 3).
DMP-pIL12 increases the cytotoxicity of T lymphocytes IL-12 plays an important role in the activation of T lymphocytes. Here, the effect of
ACCEPTED MANUSCRIPT DMP-pIL12 on T lymphocytes was investigated using the standard
51
Cr-release assay. T
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the indicated ratios of effector cells to target cells (Figure 6D).
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lymphocytes from mice treated with DMP-pIL12 caused the highest level of cytotoxicity at
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DMP-pIL12 induces apoptosis of cancer cells, inhibits tumor cell proliferation, and suppress tumor angiogenesis
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Anti-tumor mechanisms of DMP-pIL12 were studied by TUNEL, the Ki67 test, and CD31 staining. Treatment with DMP-pIL12 caused a significantly increase in apoptosis within tumor cell populations compared to other groups as determined by the TUNEL assay
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(Figure 7A). Furthermore, expression of IL-12 inhibits proliferation of tumor cells. Treatment
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with DMP-pIL12 significantly inhibits cancer cell proliferation compared to other groups as determined by Ki67 staining (Figure 7B). In addition, the DMP-pIL12 treatment group also
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showed anti-angiogenesis effects in tumors compared to DMP-pVax, DMP, and NS as
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determined by CD31 staining (Figure 7C). These results suggest that DMP-pIL12 induces apoptosis of cancer cells, inhibits tumor cell proliferation, and suppresses tumor angiogenesis, thus causing the anti-tumor effects observed in this study.
Treatment with DMP-pIL12 does not cause toxicity to vital organs To examine potential toxicity caused by treatment, organs (heart, liver, spleen, lungs, and kidneys) from treated mice were harvested and stained with H&E for histopathological analyses. Sections of all vital organs from mice treated with DMP-pIL12 showed normal histological morphology, and no toxicity caused by DMP-pIL12 was found (Figure 8). In
ACCEPTED MANUSCRIPT addition, no obvious systemic toxicity was observed, as determined by measurements of
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appearance, body weight, fecal output, and urinary excretion.
Discussion
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Cancer gene therapy, the treatment of the cancer using antisense, small interfering RNA or other DNA at the site of tumor development, has gained worldwide attention in recent
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years due to reports of successful therapeutic intervention using this approach27,28. Interleukin-12 is a naturally occurring cytokine that shows promise for treating cancer by inducing specific anti-tumor immune responses29. Local administration directly into the tumor
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site has proven to be much safer than systemic delivery30. The purpose of this present study
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was to overcome the low transfection efficiency of liposomal delivery of IL-12 gene therapy
cancer.
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and to explore the improved efficacy of IL-12 gene therapy for the treatment of colorectal
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In the present study, we have developed a novel delivery system applied to gene therapy in colon cancer. The DMP gene vector presented in this work shows improved properties for gene delivery both in ex vivo and vivo, which contributes to the striking anti-tumor effects of pIL-12-based complexes. Treatment of tumor-bearing mice with DMP-pIL12 has significantly inhibited tumor growth at both the subcutaneous and peritoneal model. Intensively increased expression of IL-12, IFN-γ and TNF-α was found in tumor tissue/ascites of mice treated with DMP-pIL12 while compared with other groups such as DMP-pVax group, which might be beneficial from the higher transfection efficiency of DMP and resulted in induction of tumor cell apoptosis, inhibition of tumor cell proliferation and tumor angiogenesis as detected by
ACCEPTED MANUSCRIPT immunohistochemical staining, and also the high cytotoxicity of T lymphocytes to tumor cells. The DMP-based gene delivery system developed here improves the effectiveness of gene
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expression in both in ex vivo tumor cells and in vivo tumor tissues, giving promise to the use of this experimental therapeutic in clinical treatment of CRC.
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Cancer immunotherapy using cytokines has a high potential to activate or suppress the immune response against tumors31,32. However, the short half-life of cytokines in vivo is a
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major impediment to cytokine therapy33. Nanocarrier enables sustained cytokine release to enhance the long-term memory T-cell response. Several clinical trials have suggested that nanocarrier can be safely used for repeated subcutaneous injection and they enhance strong
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antigen specific humoral and cellular immunological responses34. So nanocarrier is a
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promising delivery system for cancer immunotherapy. In our research, we proved that DMP had high transfection effect with improved IL-12 expression, and also low toxicity.
that
enhances
Th1
immune
response,
maturation
of
cytotoxic
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polypeptides
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Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked
lymphocytes9,12,35. The present study shows that DMP-pIL12 allowed for efficient cellular uptake of pIL12 at the tumor site, and lead to local production of high levels of IL-12. We also found that DMP-pIL12 stimulated lymphocytes activity, and the subsequent higher level of TNF-α and IFN-γ production than the controls. The current results are supported by previous findings: IL-12 could stimulate the production of TNF-α and IFN-γ, which serves to mediate the destruction of cancerous cells by inducing an anti-proliferative state36-39. Besides, TNF-α is also closely related to the cell apoptosis, inflammation and immunity40-42.
ACCEPTED MANUSCRIPT We then investigated the change of angiogenesis (CD31), tumor cell apoptosis (TUNEL), and Ki67 expression in tumors of each group to determine the antitumor mechanisms of
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DMP-pIL12. Histopathology analysis showed presence of severe apoptosis and reduced tumor cells proliferation, which was further confirmed by TUNEL assay, reduced expression
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of Ki67. DMP-pIL12 suppressed tumor-associated angiogenesis more than the other three complexes. Reports have also indicated that IFN-γ could be anti-angiogenic and block new
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blood vessel formation. What’s more, pathological angiogenesis plays a crucial role in tumor growth, dissemination, and the formation of ascites43. The high expression levels of IL-12 and IFN-γ after DMP-pIL12 administration contributed to the inhibition of tumor vasculature in
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the peritoneal cavity, may explain why DMP-pIL12 has a dramatic impact on ascites
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formation in the current animal model of disseminated colon cancer. IL-12 was also reported to be able to stimulate T lymphocytes, and we found
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DMP-pIL12 stimulated high cytotoxic factors released from T lymphocytes against Ct26 cells
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as measured by 51Cr-release CTL activity in our study. This was supported by previous study that T lymphocytes provide a possibilities for developing effective cancer immunotherapies by improving T lymphocytes responses and making them less susceptible to tumor microenvironment44-47. The safety and toxicity of DMP-pIL12 were also evaluated. The results suggested that the mice were in good physical health, and DMP-pIL12, DMP-pVax, DMP were all safe formulations as indicated by i.p. and intro-tumor administration. Therefore, we concluded that the novel DMP-pIL12 complexes are promising anticancer agents with good safety profiles and might be potential candidates for clinical use for CRC treatment.
ACCEPTED MANUSCRIPT To conclude, this study has proven that pIL12 can be safely delivered via DMP directly and efficiently into tumors, and that the effects of the IL-12 cytokine are safe and effective for
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treating colon cancer lesions. Hopefully, the results of the study will confirm the efficacy of DMP-pIL12 and pave the way for future studies into the safety of delivering the IL-12 into
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colon cancer tissue.
Biodegradable DOTAP/MPEG-PLA gene carriers were prepared and investigated for
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efficacy. DMP complexes with pDNA at a 100 : 1 or higher weight ratio allow for efficient delivery of pDNA and high transfection efficiency both in ex vivo and vivo. pIL-12-DMP deters the growth of colon cancer by inhibiting angiogenesis, promoting apoptosis, and
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reducing cell proliferation. The IL-12 plasmid and DMP complex can be used as a new drug
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to more effectively treat colorectal cancer in a clinical setting.
ACCEPTED MANUSCRIPT References (1)DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, et al. Cancer
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treatment and survivorship statistics, 2014. CA Cancer J Clin. 2014, 64, 252-71. (2)Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics,
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2012. CA Cancer J Clin. 2015, 65, 87-108.
Mol Aspects Med. 2014, 39, 61-81. (4)Carballal
S,
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(3)Di Franco S, Todaro M, Dieli F, Stassi G. Colorectal cancer defeating? Challenge accepted!
Rodriguez-Alcalde
D,
Moreira
L,
Hernandez
L,
Rodriguez
L,
Rodriguez-Moranta F, et al. Colorectal cancer risk factors in patients with serrated polyposis
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syndrome: a large multicentre study. Gut. 2015.
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(5)Halama N, Zoernig I, Berthel A, Kahlert C, Klupp F, Suarez-Carmona M, et al. Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by
CE
Anti-CCR5 Therapy in Cancer Patients. Cancer Cell. 2016, 29, 587-601.
AC
(6)Linnekamp JF, Wang X, Medema JP, Vermeulen L. Colorectal cancer heterogeneity and targeted therapy: a case for molecular disease subtypes. Cancer Res. 2015, 75, 245-9. (7)Salazar R, Ciardiello F. Optimizing Anti-EGFR Therapy in Colorectal Cancer. Clin Cancer Res. 2015, 21, 5415-6. (8)Moon JJ, Huang B, Irvine DJ. Engineering nano- and microparticles to tune immunity. Adv Mater. 2012, 24, 3724-46. (9)Del Vecchio M, Bajetta E, Canova S, Lotze MT, Wesa A, Parmiani G, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res. 2007, 13, 4677-85. (10)Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of
ACCEPTED MANUSCRIPT angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995, 87, 581-6. (11)Weiss JM, Subleski JJ, Wigginton JM, Wiltrout RH. Immunotherapy of cancer by
RI P
T
IL-12-based cytokine combinations. Expert Opin Biol Ther. 2007, 7, 1705-21. (12)Yoo JK, Cho JH, Lee SW, Sung YC. IL-12 provides proliferation and survival signals to
SC
murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway. J Immunol. 2002, 169, 3637-43.
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(13)Strasly M, Cavallo F, Geuna M, Mitola S, Colombo MP, Forni G, et al. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk. J Immunol. 2001, 166, 3890-9.
ED
(14)Yao L, Sgadari C, Furuke K, Bloom ET, Teruya-Feldstein J, Tosato G. Contribution of
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natural killer cells to inhibition of angiogenesis by interleukin-12. Blood. 1999, 93, 1612-21. (15)Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects single-dose
interleukin-12
CE
of
exposure
on
interleukin-12-associated
toxicity
and
AC
interferon-gamma production. Blood. 1997, 90, 2541-8. (16)Melero I, Mazzolini G, Narvaiza I, Qian C, Chen L, Prieto J. IL-12 gene therapy for cancer: in synergy with other immunotherapies. Trends Immunol. 2001, 22, 113-5. (17)Duffy MR, Parker AL, Bradshaw AC, Baker AH. Manipulation of adenovirus interactions with host factors for gene therapy applications. Nanomedicine (Lond). 2012, 7, 271-88. (18)Coughlan L, Vallath S, Gros A, Gimenez-Alejandre M, Van Rooijen N, Thomas GJ, et al. Combined
fiber
modifications
both
to
target
alpha(v)beta(6)
and
detarget
the
coxsackievirus-adenovirus receptor improve virus toxicity profiles in vivo but fail to improve antitumoral efficacy relative to adenovirus serotype 5. Hum Gene Ther. 2012, 23, 960-79.
ACCEPTED MANUSCRIPT (19)Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, et al. Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses.
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T
2010, 2, 2290-355.
(20)Cheng X, Darong Y, Lin M, Bingan Lu, Libao C, Qiuhong L, et al. Encapsulating Gold
SC
Nanoparticles or Nanorods in Graphene Oxide Shells as a Novel Gene Vector. ACS Appl. Mater. Interfaces, 2013, 5 (7), 2715–2724.
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(21)Gao X, Wang S, Wang B, Deng S, Liu X, Zhang X, et al. Improving the anti-ovarian cancer activity of docetaxel with biodegradable self-assembly micelles through various evaluations. Biomaterials. 2015, 53, 646-58. C,
Yongmao
L,
Copolymer
Z,
Bing
Grafted
X,
Zhiqiang
Luminescent
C,
Wenguang
Carbon
Dots
L.
As
a
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Polycation-b-Polyzwitterion
Xinyun
ED
(22)Lu
Multifunctional Platform for Serum-Resistant Gene Delivery and Bioimaging. ACS Appl.
CE
Mater. Interfaces, 2014, 6 (22), 20487–20497.
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(23)Chen L, She X, Wang T, He L, Shigdar S, Duan W, et al. Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles. Nanoscale. 2015, 7, 14080-92. (24)Christina
AH,
Maryam
T.
Substrate-Mediated
Gene
Delivery
from
Glycol-Chitosan/Hyaluronic Acid Polyelectrolyte Multilayer Films. ACS Appl. Mater. Interfaces, 2013, 5 (3), 524–531 (25)Lu Y, Wei YQ, Tian L, Zhao X, Yang L, Hu B, et al. Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor. J Immunol. 2003, 170, 3162-70.
ACCEPTED MANUSCRIPT (26)Liu JY, Wei YQ, Yang L, Zhao X, Tian L, Hou JM, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood.
RI P
T
2003, 102, 1815-23.
(27)Brenner MK, Gottschalk S, Leen AM, Vera JF. Is cancer gene therapy an empty suit?
SC
Lancet Oncol. 2013, 14, e447-56.
(28)Han J, Wang Q, Zhang Z, Gong T, Sun X. Cationic bovine serum albumin based
MA NU
self-assembled nanoparticles as siRNA delivery vector for treating lung metastatic cancer. Small. 2014, 10, 524-35.
(29)Qu Y, Chen L, Lowe DB, Storkus WJ, Taylor JL. Combined Tbet and IL12 gene therapy
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elicits and recruits superior antitumor immunity in vivo. Mol Ther. 2012, 20, 644-51.
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(30)Zhao X, Bose A, Komita H, Taylor JL, Kawabe M, Chi N, et al. Intratumoral IL-12 gene therapy results in the crosspriming of Tc1 cells reactive against tumor-associated stromal
CE
antigens. Mol Ther. 2011, 19, 805-14.
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(31)Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science. 1993, 260, 547-9.
(32)Mager LF, Wasmer MH, Rau TT, Krebs P. Cytokine-Induced Modulation of Colorectal Cancer. Front Oncol. 2016, 6, 96. (33)Vignali DA, Kuchroo VK. IL-12 family cytokines: immunological playmakers. Nat Immunol. 2012, 13, 722-8. (34)Tahara Y, Akiyoshi K. Current advances in self-assembled nanogel delivery systems for immunotherapy. Adv Drug Deliv Rev. 2015, 95, 65-76.
ACCEPTED MANUSCRIPT (35)Schulz EG, Mariani L, Radbruch A, Hofer T. Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12. Immunity. 2009, 30,
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T
673-83.
(36)Brunda MJ, Gately MK. Interleukin-12: potential role in cancer therapy. Important Adv
SC
Oncol. 1995, 3-18.
(37)Yuzhalin AE, Kutikhin AG. Interleukin-12: clinical usage and molecular markers of
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cancer susceptibility. Growth Factors. 2012, 30, 176-91.
(38)Diouf I, Fievet N, Doucoure S, Ngom M, Andrieu M, Mathieu JF, et al. IL-12 producing monocytes and IFN-gamma and TNF-alpha producing T-lymphocytes are increased in
ED
placentas infected by Plasmodium falciparum. J Reprod Immunol. 2007, 74, 152-62.
PT
(39)Sangro B, Melero I, Qian C, Prieto J. Gene therapy of cancer based on interleukin 12. Curr Gene Ther. 2005, 5, 573-81.
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(40)Noguchi S, Mori T, Igase M, Mizuno T. A novel apoptosis-inducing mechanism of
AC
5-aza-2'-deoxycitidine in melanoma cells: Demethylation of TNF-alpha and activation of FOXO1. Cancer Lett. 2015, 369, 344-53. (41)Griffin GK, Newton G, Tarrio ML, Bu DX, Maganto-Garcia E, Azcutia V, et al. IL-17 and TNF-alpha sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation. J Immunol. 2012, 188, 6287-99. (42)Liu Y, Jiao F, Qiu Y, Li W, Lao F, Zhou G, et al. The effect of Gd@C82(OH)22 nanoparticles on the release of Th1/Th2 cytokines and induction of TNF-alpha mediated cellular immunity. Biomaterials. 2009, 30, 3934-45. (43)Pasquet M, Golzio M, Mery E, Rafii A, Benabbou N, Mirshahi P, et al. Hospicells
ACCEPTED MANUSCRIPT (ascites-derived stromal cells) promote tumorigenicity and angiogenesis. Int J Cancer. 2010, 126, 2090-101.
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(44)Zabala M, Lasarte JJ, Perret C, Sola J, Berraondo P, Alfaro M, et al. Induction of immunosuppressive molecules and regulatory T cells counteracts the antitumor effect of
SC
interleukin-12-based gene therapy in a transgenic mouse model of liver cancer. J Hepatol. 2007, 47, 807-15.
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(45)Zhang L, Morgan RA, Beane JD, Zheng Z, Dudley ME, Kassim SH, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015, 21,
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2278-88.
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(46)Michelin MA, Montes L, Nomelini RS, Abdalla DR, Aleixo AA, Murta EF. Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.
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Oncol Lett. 2015, 10, 1523-1526.
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(47)Cavallo F, Quaglino E, Cifaldi L, Di Carlo E, Andre A, Bernabei P, et al. Interleukin 12-activated lymphocytes influence tumor genetic programs. Cancer Res. 2001, 61, 3518-23.
ACCEPTED MANUSCRIPT Figure Legends: Figure 1. Preparation and physicochemical properties of DMP.
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(A) preparation of DMP and pIL12 complex: First, a novel gene carrier was prepared with a self-assembly method. MPEG-PLA and DOTAP were assembled into a new gene carrier,
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DOTAP/MPEG-PLA Micelles (DMP). Then pIL12 plasmid was carried into cancer cells by DMP. (B) Zeta-potential of DMP; (C) Particle size of DMP; (D) Morphological
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characteristics of DMP by TEM observation; (E) Gel retardation assay of DNA and complexes. Lane 0 and 17, DNA marker; lanes 1 to 3, naked pIL12; lane 4 to 6, DMP; lane 7 to 9, weight ratios of pIL12 with DMP (1 : 25); lane 10 to 12, weight ratios of pIL12 with
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DMP (1 : 50); lane 13 to 15, weight ratios of pIL12 with DMP (1 : 100). pIL12 was
without free DNA.
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completely incorporated into DMP at a weight ratio of 1 : 100 and complexes were prepared
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Figure 2. Transfection efficiency measurement
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Transfection efficiency measurement of DMP. DMP, containing 4 μg pEGFP, were used to transfect Ct26 cells for 48 h. The transfection efficiencies at both weight ratio (pEGFP versus DMP as 1 : 100 and 1 : 150) were determined using (A) TEM and (B) Flow Cytometry. (C) The expression of IL-12 mRNA level detected by RT-PCR in NS, DMP, DMP-pVax and DMP-pIL12 transfected Ct26 cell groups; (D) IL-12 expression detected by Flow cytometry when IL-12 was mixed with DMP at a ratio of 1 : 150; (E) Elisa detection of the IL-12 in the cell supernatant of different group (NS, DMP, DMP -pVax and DMP -pIL12). (Mean ± SEM, n=3, p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, for 24 h, 48 h and 72 h, DMP-pIL12 versus NS, DMP, DMP-pVax, C)
ACCEPTED MANUSCRIPT Figure 3. MTT test of cell activity and expression of IL12 induced the secretion of IFN-γ of cultured lymphocytes test.
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When Ct26 cells were transfected with NS, DMP, DMP-pVax or DMP-pIL12 for 72 h, the supernatant from different treatments were added into lymphocytes and treated for 24 h, and
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the lymphocytes activity was tested by MTT test. (A) When the lymphocytes were treated for 24 h, the supernatant were added into Ct26 cells and treated for 24 h, and the Ct26 cell
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activity was tested by MTT test (B). (Mean±SEM, n=6) (*: p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); *: p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B). The lymphocytes were treated with cell culture supernatant collected from NS, DMP, DMP-pVax
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or DMP-pIL12 group, and the corresponding supernatants after 3 h (C), 8 h (D) and 24 h (E)
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culture in each group were then collected for measure of IFN-γ. IL-12 DMP-pIL12-mediated IFN-γ expression was substantially higher than that of other groups. (Mean ± SEM, n=3) (p <
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0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP,
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DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, E)
Figure 4. Anti-colon cancer effect of DMP-pIL12 in subcutaneous tumor model A) tumor growth curves; B) Body weight of different groups; C) Tumor weight; D) Tumor photos of of NS, DMP, DMP-pVax and DMP-pIL12 treatment groups. (Mean ± SEM, n=6) (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, C)
ACCEPTED MANUSCRIPT Figure 5. Anti-colon cancer effect of DMP-pIL12 in peritoneal colon cancer model. (A)Body weight; (B)Tumor weight of different group; (C) Images of mouse and the
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corresponding tumor; (D) The number of tumor node in different group; (E) ascites content of NS, DMP, DMP-pVax and DMP-pIL12 treated mice. (Mean ± SEM, n=6) (p < 0.01,
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DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, D); p < 0.01,
Figure 6.
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DMP-pIL12 versus NS, DMP, DMP-pVax, E)
IL-12 expression, IFN-γ, TNF-α expressions and the cytotoxicity of T
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lymphocytes tests
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Tumor tissue was collected after the final treatment and IL-12 level was measured in the four groups (NS, DMP, DMP-pVax, and DMP-pIL12) of the subcutaneous model and peritoneal
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model by Flow Cytometry. DMP-pIL12 treated mice had significantly higher level of IL-12 in
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tumor tissue in both models (A). Tumor tissue of the subcutaneous model and ascites of the peritoneal model (NS, DMP, DMP-pVax, and DMP-pIL-12) were collected after two doses treatment, and we measured the expression of TNF-α (B) and IFN-γ (C) by ELISA in the tumor tissue and ascites. DMP-pIL12 treated mice had significantly higher level of IFN-γ and TNF-α in tumor tissue and ascites (Mean ± SEM, n=3) (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, C). (D) T lymphocytes derived from spleens of DMP-pIL12, DMP-pVax, DMP and saline treated mice were tested against Ct26 cells at different E: T ratios (X axis label is ratio of effector cells (E) to 51Cr-labeled target cells (T).) by a standard 4 h 51Cr release assay as described in Materials
ACCEPTED MANUSCRIPT and Methods. T lymphocytes from the spleens of DMP-pIL12 treated mice showed higher
Cell apoptosis, cell proliferation and tumor angiogenesis detections
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Figure 7.
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SEM, n=3, p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax) .
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cytotoxicity against Ct26 cells than did T lymphocytes from DMP-pVax, DMP or NS (Mean ±
(A) Cell apoptosis were assessed by counting the number of TUNEL-positive cells in the field
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(five high power fields per slide), and DMP-pIL12 was superior to other controls in increasing cell apoptosis. DMP-pIL12 significantly increased apoptosis (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax). (B) Cell apoptosis were assessed by counting the
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number of Ki67-positive cells in the field (five high power fields per slide), and DMP-pIL12
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was superior to other controls in inhibiting cell proliferation. DMP-pIL12 significantly inhibited cell proliferation(p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax). (C)
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Angiogenesis were assessed by counting the number of CD31-positive vessels in the field
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(five high power fields per slide). DMP-pIL12 significantly inhibited MVD (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax).
Figure 8. Toxicity assessment in vivo with pathological section. Histological examinations of HE-stained vital organ sections. No significant pathological changes were detected.
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Graphical Abstract
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Schedule of cancer gene immunotherapy with IL-12. First, a novel gene carrier was prepared with a self-assembly method. MPEG-PLA and DOTAP were assembled into a new gene carrier, DOTAP/MPEG-PLA Micelles (DMP). Then pIL12 plasmid was carried into cancer cells by DMP. And the cancer cells expressed and secreted the IL-12 protein. IL-12 could activate the immune cell, then immune cells secrete some tumor-killing factor to kill cancer cell.
S1549-9634(17)30067-9 doi: 10.1016/j.nano.2017.04.006 NANO 1565
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
8 July 2016 4 April 2017 10 April 2017
Please cite this article as: Liu Xiaoxiao, Gao Xiang, Zheng Songping, Wang Bilan, Li Yanyan, Zhao Chanjuan, Muftuoglu Yagmur, Chen Song, Li Ying, Yao Haiyan, Sun Hui, Mao Qing, You Chao, Guo Gang, Wei Yuquan, Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.04.006
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 Modified nanoparticle mediated IL-12 immunogene therapy for colon cancer Xiaoxiao Liu1,7,*, Xiang Gao1,*,#, Songping Zheng1,*, Bilan Wang2, Yanyan Li6, Chanjuan
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Zhao2, Yagmur Muftuoglu3, Song Chen3, Ying Li3,8, Haiyan Yao4, Hui Sun5, Qing Mao1, Chao You1, Gang Guo1 and Yuquan Wei1 1
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Department of Neurosurgery and Institute of Neurosurgery, State Key Laboratory of
Biotherapy, West China Hospital, West China Medical School, Sichuan University and
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Collaborative Innovation Center for Biotherapy, Chengdu, 610041, PR China. 2
Department of Pharmacy, West China Second University Hospital of Sichuan University,
Chengdu, 610041, PR China. 3
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Department of Pharmacology, Yale School of Medicine, Yale University, New Haven, CT
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06520, USA. 4
Southern Connecticut State University, New Haven, Connecticut, United States of America.
5
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Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven,
6
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Connecticut, United States of America. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Fudan
University, Shanghai, China. 7
Department
of
Medical
Oncology,
Cancer
Center,
State
Key
Laboratory
of
Biotherapy/Collaborative Innovation Center for Biotherapy, and West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, PR China. 8
Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University,
Dalian 116023, China *These authors are considered equal first authors.
ACCEPTED MANUSCRIPT #
Correspondence: Xiang Gao
Tel: +86 28 8542 2136; Fax: +86 28 8550 2796
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Email: [email protected]
This work was supported by the National Natural Science Foundation of China
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(NSFC81502165) and the Sichuan University Outstanding Young Scholars Research Fund (2016SCU04A04).
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Conflict of interest: The authors declare no confltics of interest.
ACCEPTED MANUSCRIPT Abstract For the past few years, immunotherapy has recently shown considerable clinical benefit in
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CRC therapy, and the application of immunologic therapies in cancer treatments continues to increase perennially. Interleukin-12, an ideal candidate for tumor immunotherapy, could
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activate both innate and adaptive immunities. In this study, we developed a novel gene delivery system with a self-assembly method by MPEG-PLA and DOTAP(DMP) with
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zeta-potential value of 38.5 mV and size of 37.5 nm. The supernatant of lymphocytes treated with supernatant from Ct26 transfected pIL12 with DMP could inhibit Ct26 cells growth ex vivo. Treatment of tumor-bearing mice with DMP-pIL12 complex has significantly inhibited
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tumor growth at both the subcutaneous and peritoneal model in vivo by inhibiting
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angiogenesis, promoting apoptosis and reducing proliferation. The IL-12 plasmid and DMP
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complex may be used to treat the colorectal cancer in clinical as a new drug.
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Key word: Colorectal cancer, Immunotherapy, Interleukin-12, Nanoparticles.
ACCEPTED MANUSCRIPT Background Colorectal cancer (CRC), one of the most prevalent cancers, is a leading cause of cancer
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mortality worldwide1. An estimated 1.4 million cases are diagnosed every year, and more than 690,000 patients died from the disease in 2012 according to global cancer statistics 2. Due to
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significant strides in traditional treatments, such as surgical management, chemotherapy and radiation therapy, the average survival for advanced CRC now approaches 30 months3,4.
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However, almost half of all patients develop liver or peritoneal metastases over the course of this disease. Even with treatment, the median survival for metastatic CRC patient is still relatively low, and current treatment paradigms appear to have reached their maximum
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benefit. Therefore, for decades, considerable efforts have been devoted to developing more
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effective anti-tumor agents and better strategies for targeting tumors5-7. Over the past few years, immunotherapy has shown considerable clinical benefit in
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treating CRC, and the application of immunologic therapies in cancer treatments continues to
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increase every year. Cytokine-based immune therapy, which could induce or augment the anti-tumor immune response, has proven effective in a variety of models 3,9. Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine, was proven to be an ideal candidate for tumor immunotherapy, due to its ability to activate both innate and adaptive immune responses9-11. Specifically, IL-12 has been demonstrated to be one of the most potent anti-tumor cytokines in experimental animal models, and it proves effective in eradicating experimental tumors, eliciting long-term anti-tumor immunity, and inhibiting tumor formation and metastases12-14. However, clinical trials testing the therapeutic potential of administering IL-12 have been halted because of serious potential systemic toxicity and lower-than-anticipated efficacy15,16.
ACCEPTED MANUSCRIPT Conversely, administration of IL-12 via gene therapy is an ideal method for harnessing the power of this cytokine because expression of IL-12 can be maintained at low levels and will
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eventually subside to basal levels. To optimize the delivery of IL-12 gene therapy, more efficient gene carriers with lower levels of toxicity are strongly needed.
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While replication-deficient adenovirus (Ad) offers several important advantages as a vector for gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal
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transduction efficiency, and adverse systemic side effects17-19. Its application for gene therapy introduces serious concerns about endogenous virus recombinations, oncogenic effects, and immunological reactions. However, non-viral gene delivery systems such as the nanoparticles
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described in this work show great potential for overcoming physical and biological barriers to
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achieving therapeutic levels of transgene expression at target sites20-24. In this study, we developed a novel gene delivery system with a self-assembly method by poly(ethylene
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methoxy
glycol)-poly(lactide)
(MPEG-PLA)
and
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1,2-dioleoyl-3-trimethylammonium-propan (DOTAP). Further, we show that transfected cancer cells express and secrete high levels of IL-12, which activates the immune system. In addition, potential toxicity is dramatically reduced, and the resulting steady expression of IL-12 creates a stronger anti-tumor immune response. Also presented in this work are the pharmaceutical properties, in vitro biological activity, in vivo anti-tumor effects, anti-tumor mechanisms, and a preliminary toxicity evaluation of DOTAP/MPEG-PLA-pIL12 (DMP-pIL12) .
ACCEPTED MANUSCRIPT Methods Materials
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1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, U.S.); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
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tetrazolium bromide (MTT) from Sigma (USA); Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) from Gibco BRL (USA); methanol and acetic acid
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(HPLC grade) from Fisher Scientific (UK); Mice lymphocyte separation medium from Dakewe (China) and dimethyl sulfoxide (DMSO) and acetone from KeLong Chemicals (China). Antibodies purchased include: rat anti-mouse CD31 polyclonal antibody (BD USA),
rabbit
anti-mouse
Ki67
antibody
(Abcam,
USA),
and
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PharmingenTM,
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rhodamine-conjugated secondary antibody (Abcam, USA). A plasmid encoding p35 and p40 subunits of murine IL12 (pIL12) was constructed at the BGH site of the pVAX vector
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(Invitrogen Corp., Carlsbad, CA, U.S.), an expression vector encoding kanamycin resistance
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under the control of a separate T7 promoter/enhancer. pVax served as a negative control. All plasmid DNA were extracted using the EndoFree Plasmid Giga kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Preparation of DOTAP/MPEG-PLA (DMP), characterization of DMP and agarose gel electrophoresis of naked plasmid DNA and complexes are described in the supplementary materials.
Cell culture and transfection experiments Murine Ct26 colon carcinoma cells was purchased from American Type Culture
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Chemical Co., St. Louis, MO), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in a humidified atmosphere containing 5 % CO2 at 37 °C.
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DMP and pEGFP plasmid were incubated respectively in RPMI 1640 medium without FBS for five min. After that, they were mixed and incubated for twenty min. The Ct26 cells in
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6 well cell culture plates were washed twice used RPMI 1640 medium without FBS. Pre-mixed solution was put into 6 well cell culture plates for 4 h. Then pre-mixed solution replaced by RPMI 1640 medium containing 10 % FBS. Subsequent operations were took in
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corresponding time points. DMP, containing 4 μg pEGFP, was used to transfect Ct26 cells for
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48 h. Transfected cells were photographed using an Array Scan VTI HCS Reader (Thermo Fisher Scientific Inc., Waltham, MA, U.S.). Transfection efficiencies at both ratios (pEGFP
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versus DMP at 1 : 100 and 1 : 150) were determined using FACS Calibur flow cytometry (BD
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Biosciences, San Jose, CA, U.S.).
Measurement of IL-12 expression Ct26 cells were transfected with DMP-pIL12 liposomes and cultured for 72 h before IL-12 expression was measured by RT-PCR, flow cytometry, and ELISA. IL-12 mRNA was measured by Reverse Transcription polymerase chain reaction (RT-PCR) using the PrimeScript TM RT reagent Kit (Takara Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning, China) and 2×Taq MasterMix kit (Beijing CoWin Biotech Co., Ltd., Beijing, China). The RT-PCR primers for IL-12 gene were 5’-TCC TGC TTC ACG CCT TCA-3’ (forward) and 5’-
ACCEPTED MANUSCRIPT AGT CCA GTC CAC CTC TAC AAC ATA-3’ (reverse). Transfection efficiency was examined by flow cytometry. Cells were permeabilized using a BD Cytofix/Cytoperm™
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Fixation/permeabilization Kit and stained with PE Rat Anti-Mouse IL-12 (p40/p70) overnight. The level of IL-12 expression in the cell culture supernatant at 24 h, 48 h, and 72 h were
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measured by ELISA kits (eBioscience, Inc., San Diego, CA, U.S.) According to manufacturer’s instructions, the plate was incubated with capture antibody overnight at 4 °C.
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The plates were washed in accordance with the requirements. Then, standards and samples were put into the plates at room temperature for 2 h. The detection antibody was added after washing the plates. The work of color and termination were done after 1 h. Finally, read
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absorbance at 450 nm with a reference filter of 570 nm.
Measurement of cell viability and secretion of IFN-γ
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Ct26 cells were culture in a 6 well cell culture plates, with a concentration of 2 * 105 cells
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per well. After Ct26 cells were transfected with NS (NS: normal salt), DMP, DMP-pVax (pVax: the vector plasmid), or DMP-pIL12 for 72 h, supernatant was collected from each group.
These samples were further applied to culture lymphoctytes derived from murine spleen. After 24 h, the viability of lymphocytes from each group (NS, DMP, DMP-pVax, and DMP-pIL12) was tested by the MTT method. Briefly, 20 µl of 5 mg/ml MTT was added into each well and incubated at 37 °C for 3 h. The lymphocytes were fixed on the 96 well cell culture plates by centrifuging at 3000 rpm. Media was removed and 150 µl of DMSO was added. The cell plate was shaked for 15 min in dark. Lastly absorbance at 570 nm was read by
ACCEPTED MANUSCRIPT microplate reader (Thermo, USA). Another experiments were carried out at the same time and supernatant was collected from each group of lymphocytes and further used to culture CT26
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cells and with 20 µM oxaliplatin as positive control. After 24 h, the viability of CT26 cells from each group was tested, again using the MTT method what we described above except
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using centrifuge. In addition, supernatant was collected from each group of lymphocytes after
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3 h, 8 h, and 24 h of culture to measure the expression of IFN-γ by ELISA as described.
In vivo study of the anti-tumor properties of DMP-pIL12
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This section is expounded in the supplementary materials.
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Cytotoxicity Assay
A 4 h 51Cr release assay was performed as previously described by other reports25,26. In
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the peritoneal tumor model, splenocytes from each group (NS, DMP, DMP-pVax, or
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DMP-pIL12) were harvested after the mice received two treatments. Briefly, spleen lymphocytes obtained from the four groups, as the effector cells, were treated with ammonium chloride-potassium lysis buffer to deplete erythrocytes. Ct26 (1 * 106), as the target cells, were labeled with 100 μCi 51Cr for 1 h at 37 °C, washed, and resuspended at a concentration of 1 * 105 cells/ml. A total of 200 μL of effector cells (E) and 51Cr-labeled target cells (T) were assigned at different E:T ratios to each well of a 96 well plate and incubated for 4 h at 37 °C. Supernatant (100 μL) from each group was then harvested; cellular activity was calculated by this formula: % cytotoxicity = [(experimental release − spontaneous release) / (maximum release − spontaneous release) ] × 100.
ACCEPTED MANUSCRIPT TUNEL assay of Tumor Samples Apoptotic cells in tumor tissues were detected in paraffin sections using the Dead End
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Fluorometric system (Promega, Madison, WI, U.S.), a terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, according to the manufacturer's
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instructions. All sections were observed and digitally photographed under a DM 2500 fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany), and the
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quantification of TUNEL-positive cells was assessed according to previous reports21.
Measurement of tumor cell proliferation and microvessel density
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Immunohistochemistry identifying expression of the Ki67 antigen and CD31, marker for
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assessing microvessel density (MVD), were carried out with rabbit anti-mouse Ki67 and rabbit anti-mouse CD31 antibodies using the labeled streptavidin-biotin method as previously
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described21. Quantification of MVD was estimated by counting the number of microvessels in
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five random fields at ×200 magnification. A single microvessel was defined as a discrete cluster or single cell stained positive for CD31. All sections were observed or counted by two investigators or pathologists in a blinded fashion.
Assessment of systemic toxicity induced by treatment with DMP-pIL12 To evaluate toxicity caused by treatment with DMP-pIL12, vital organs (heart, liver, spleen, lungs, and kidneys) of mice were collected and fixed in 4 % paraformaldehyde. After 24 h, samples were embedded in paraffin wax and sliced into 4-μm sections. Sections were then hydrated and stained with hematoxylin and eosin (Beyotime Biotechnology (Shanghai)
ACCEPTED MANUSCRIPT Co., Ltd., Shanghai, China) (H&E) according to the manufacturer’s instructions for histomorphometric analysis. Representative images of tissues were taken with an Olympus
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light microscope.
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Statistical analysis
All data were analyzed using GRAPHPAD PRISM software (GraphPad, San Diego, CA).
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Data from multiple groups were analyzed using ANOVA, and multiple comparisons between the groups were performed using the Newman–Keuls method after ANOVA. Survival data were plotted using Kaplan–Meier curves and analyzed using the log-rank test. Tumor volumes
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[(smaller diameter)2 (larger diameter) * 0.52] and assessment of vessel density were analyzed
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using the Student’s t test. All values were presented as the mean±the standard error of
Results
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measurement. P < 0.01 was considered to be statistically significant for all experiments.
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DMP successfully incorporates DNA DMP and DMP-pIL12 were produced using the described self-assembly method (Figure 1A). As shown in Figure 1B, the zeta-potential and size of DMP were 38.5 mV and 37.5 nm, respectively (Figure 1C). TEM images show that, morphologically, DMP adopts a bilayer spheroidal structure at around 25 nm in diameter (Figure 1D). Gel retardation assays were used to characterize the ability of the DMP liposome to carry DNA. As shown in Figure 1E, free DNA not entrapped in the DMP appeared as a bright band (lanes 1 to 3), and no other band of free DNA was observed from lanes 13 to 15. This suggests that pIL-12 DNA was completely incorporated into the DMP and that the complexes were prepared successfully
ACCEPTED MANUSCRIPT without free DNA (pIL12) at the ratio of 100 times the amount of DMP to DNA. And loading efficiency in different weight ratio of 1 : 25, 1 : 50 and 1 : 100 are 70.3 ± 5.2 % , 91.3
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± 2.3 % and 100 %, respectively.
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DMP-pIL12 has high transfection efficiency in Ct26 cells
In the ex vivo cell transfection assays, DMP had high GFP transfection efficiency in Ct26
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cells after incubation for 24 h as shown in Figure 2A. The specific transfection rate demonstrated by flow cytometry analysis was 39.2 % and 54.4 % when DMP was mixed with GFP at a ratio of 1 : 100 and 1 : 150, respectively. (Figure 2B).
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Ct26 cells were transfected with NS, DMP, DMP-pVax, or DMP-pIL12, and we then
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measured the expression of IL-12 in these group cells. The level of IL-12 mRNA in the DMP-pIL12 group was significantly higher than in the control groups (Figure 2C). We also
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tested the transfection efficacy of DMP carrying IL-12 and found the 1 : 150 ratio to be
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optimal, which resulted in a transfection rate of 50.7 % and acceptable cell toxicity as shown in Figure 2D. Therefore, we used this ratio in the remaining studies. Further, the level of IL-12 expression in the cell culture supernatant was significantly higher than in the control groups after 24 h, 48 h, and 72 h of culture (Figure 2E).
DMP-pIL12 increases the proliferation and anti-tumor effects of lymphoctyes Ct26 cells were transfected with NS, DMP, DMP-pVax, or DMP-pIL12 for 72 h, and supernatant from these groups were collected for culture of lymphocytes. After 24 h of culture, viability of lymphocytes cultured with supernatant from the DMP-pIL12 group was
ACCEPTED MANUSCRIPT significantly higher than the lymphocytes cultured with supernatant from the control groups (Figure 3A). Supernatant from the four groups of lymphocytes were then collected to culture
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fresh Ct26 cells for 24 h. Supernatant from lymphocytes cultured with supernatant from DMP-pIL12-transfected Ct26 cells significantly inhibits the growth of fresh Ct26 cells
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(Figure 3B). We then analyzed the mechanisms behind this observation and found higher levels of IFN-γ expression in this supernatant than in the corresponding control samples.
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(Figure 3C-E)
DMP-pIL12 causes in vivo anti-tumor effects
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DMP and its complexes loaded with pVax and pIL-12 were used to treat Ct26 colon
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cancer in BALB/c mice model via intra-tumor injection. DMP-pIL12 treatment showed dramatic anti-tumor effects than other complexes (DMP-pVax, DMP, and NS) as shown in
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Figure 4 (A, C, D), and no significant differences were observed among mice in the
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DMP-pVax, DMP, and NS groups. In addition, DMP-pIL12-treated mice had slightly lower body weights than mice in the control groups (Figure 4B). We also observed similar anti-tumor effects in the Ct26 peritoneal model of colon cancer. Figure 5A and Figure 5B shows that the average weight of DMP-pIL12-treated mice and tumor were significantly lower than that of mice in the DMP-pVax, DMP, and NS groups. The mice administered DMP-pIL12 had significantly fewer and smaller tumor nodules than mice in the control groups (Figure 5C and 5D). In addition, administration of DMP-pIL12 also reduced the occurrence of ascites (Figure 5E).
ACCEPTED MANUSCRIPT Administration of DMP-pIL12 increases the expression of IL-12 and secretion of IFN-γ and TNF-α
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We next explored the potential mechanisms responsible for the observed anti-tumor effects of DMP-pIL12 and tested the expression of IL-12 by flow cytometry and ELISA.
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After treatments, tumors from both mouse models were harvested, and we found that mice administered DMP-pIL12 had significantly higher levels of expression of IL-12 in the tumor
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tissue than mice from control groups (Figure 6A and Supplementary Figure 1A and 1B), but no difference in serum (Supplementary Figure 1C and 1D). The anti-tumor effects of DMP-pIL12 could also be explained by the increase of IFN-γ
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and TNF-α expression in ascites and tumor tissues after treatments (Figure 6B, Figure 6C and
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Supplementary Figure 2). Specifically, the mean TNF-α concentration in DMP-pIL12- treated tumors in the subcutaneous model was significantly higher than that in DMP-pVax-treated
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tumors, in which only a very small concentration of TNF-α was expressed. We also observed
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similar results in the peritoneal model (Figure 6B). These observations suggest that increased expression of IL-12 results in expression of TNF-α at the tumor site. In addition, we observed the highest levels of IFN-γ in the subcutaneous and peritoneal models as a result of treatment with DMP-pIL12 (Figure 6C). DMP-pIL12 treated mice had no difference at level of TNF-α and IFN-γ in serum in subcutaneous model. And DMP-pIL12 treated mice had lower level of IFN-γ in serum in peritoneal model (Supplementary Figure 3).
DMP-pIL12 increases the cytotoxicity of T lymphocytes IL-12 plays an important role in the activation of T lymphocytes. Here, the effect of
ACCEPTED MANUSCRIPT DMP-pIL12 on T lymphocytes was investigated using the standard
51
Cr-release assay. T
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the indicated ratios of effector cells to target cells (Figure 6D).
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lymphocytes from mice treated with DMP-pIL12 caused the highest level of cytotoxicity at
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DMP-pIL12 induces apoptosis of cancer cells, inhibits tumor cell proliferation, and suppress tumor angiogenesis
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Anti-tumor mechanisms of DMP-pIL12 were studied by TUNEL, the Ki67 test, and CD31 staining. Treatment with DMP-pIL12 caused a significantly increase in apoptosis within tumor cell populations compared to other groups as determined by the TUNEL assay
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(Figure 7A). Furthermore, expression of IL-12 inhibits proliferation of tumor cells. Treatment
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with DMP-pIL12 significantly inhibits cancer cell proliferation compared to other groups as determined by Ki67 staining (Figure 7B). In addition, the DMP-pIL12 treatment group also
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showed anti-angiogenesis effects in tumors compared to DMP-pVax, DMP, and NS as
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determined by CD31 staining (Figure 7C). These results suggest that DMP-pIL12 induces apoptosis of cancer cells, inhibits tumor cell proliferation, and suppresses tumor angiogenesis, thus causing the anti-tumor effects observed in this study.
Treatment with DMP-pIL12 does not cause toxicity to vital organs To examine potential toxicity caused by treatment, organs (heart, liver, spleen, lungs, and kidneys) from treated mice were harvested and stained with H&E for histopathological analyses. Sections of all vital organs from mice treated with DMP-pIL12 showed normal histological morphology, and no toxicity caused by DMP-pIL12 was found (Figure 8). In
ACCEPTED MANUSCRIPT addition, no obvious systemic toxicity was observed, as determined by measurements of
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appearance, body weight, fecal output, and urinary excretion.
Discussion
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Cancer gene therapy, the treatment of the cancer using antisense, small interfering RNA or other DNA at the site of tumor development, has gained worldwide attention in recent
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years due to reports of successful therapeutic intervention using this approach27,28. Interleukin-12 is a naturally occurring cytokine that shows promise for treating cancer by inducing specific anti-tumor immune responses29. Local administration directly into the tumor
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site has proven to be much safer than systemic delivery30. The purpose of this present study
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was to overcome the low transfection efficiency of liposomal delivery of IL-12 gene therapy
cancer.
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and to explore the improved efficacy of IL-12 gene therapy for the treatment of colorectal
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In the present study, we have developed a novel delivery system applied to gene therapy in colon cancer. The DMP gene vector presented in this work shows improved properties for gene delivery both in ex vivo and vivo, which contributes to the striking anti-tumor effects of pIL-12-based complexes. Treatment of tumor-bearing mice with DMP-pIL12 has significantly inhibited tumor growth at both the subcutaneous and peritoneal model. Intensively increased expression of IL-12, IFN-γ and TNF-α was found in tumor tissue/ascites of mice treated with DMP-pIL12 while compared with other groups such as DMP-pVax group, which might be beneficial from the higher transfection efficiency of DMP and resulted in induction of tumor cell apoptosis, inhibition of tumor cell proliferation and tumor angiogenesis as detected by
ACCEPTED MANUSCRIPT immunohistochemical staining, and also the high cytotoxicity of T lymphocytes to tumor cells. The DMP-based gene delivery system developed here improves the effectiveness of gene
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expression in both in ex vivo tumor cells and in vivo tumor tissues, giving promise to the use of this experimental therapeutic in clinical treatment of CRC.
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Cancer immunotherapy using cytokines has a high potential to activate or suppress the immune response against tumors31,32. However, the short half-life of cytokines in vivo is a
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major impediment to cytokine therapy33. Nanocarrier enables sustained cytokine release to enhance the long-term memory T-cell response. Several clinical trials have suggested that nanocarrier can be safely used for repeated subcutaneous injection and they enhance strong
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antigen specific humoral and cellular immunological responses34. So nanocarrier is a
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promising delivery system for cancer immunotherapy. In our research, we proved that DMP had high transfection effect with improved IL-12 expression, and also low toxicity.
that
enhances
Th1
immune
response,
maturation
of
cytotoxic
T
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polypeptides
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Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked
lymphocytes9,12,35. The present study shows that DMP-pIL12 allowed for efficient cellular uptake of pIL12 at the tumor site, and lead to local production of high levels of IL-12. We also found that DMP-pIL12 stimulated lymphocytes activity, and the subsequent higher level of TNF-α and IFN-γ production than the controls. The current results are supported by previous findings: IL-12 could stimulate the production of TNF-α and IFN-γ, which serves to mediate the destruction of cancerous cells by inducing an anti-proliferative state36-39. Besides, TNF-α is also closely related to the cell apoptosis, inflammation and immunity40-42.
ACCEPTED MANUSCRIPT We then investigated the change of angiogenesis (CD31), tumor cell apoptosis (TUNEL), and Ki67 expression in tumors of each group to determine the antitumor mechanisms of
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DMP-pIL12. Histopathology analysis showed presence of severe apoptosis and reduced tumor cells proliferation, which was further confirmed by TUNEL assay, reduced expression
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of Ki67. DMP-pIL12 suppressed tumor-associated angiogenesis more than the other three complexes. Reports have also indicated that IFN-γ could be anti-angiogenic and block new
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blood vessel formation. What’s more, pathological angiogenesis plays a crucial role in tumor growth, dissemination, and the formation of ascites43. The high expression levels of IL-12 and IFN-γ after DMP-pIL12 administration contributed to the inhibition of tumor vasculature in
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the peritoneal cavity, may explain why DMP-pIL12 has a dramatic impact on ascites
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formation in the current animal model of disseminated colon cancer. IL-12 was also reported to be able to stimulate T lymphocytes, and we found
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DMP-pIL12 stimulated high cytotoxic factors released from T lymphocytes against Ct26 cells
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as measured by 51Cr-release CTL activity in our study. This was supported by previous study that T lymphocytes provide a possibilities for developing effective cancer immunotherapies by improving T lymphocytes responses and making them less susceptible to tumor microenvironment44-47. The safety and toxicity of DMP-pIL12 were also evaluated. The results suggested that the mice were in good physical health, and DMP-pIL12, DMP-pVax, DMP were all safe formulations as indicated by i.p. and intro-tumor administration. Therefore, we concluded that the novel DMP-pIL12 complexes are promising anticancer agents with good safety profiles and might be potential candidates for clinical use for CRC treatment.
ACCEPTED MANUSCRIPT To conclude, this study has proven that pIL12 can be safely delivered via DMP directly and efficiently into tumors, and that the effects of the IL-12 cytokine are safe and effective for
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treating colon cancer lesions. Hopefully, the results of the study will confirm the efficacy of DMP-pIL12 and pave the way for future studies into the safety of delivering the IL-12 into
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colon cancer tissue.
Biodegradable DOTAP/MPEG-PLA gene carriers were prepared and investigated for
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efficacy. DMP complexes with pDNA at a 100 : 1 or higher weight ratio allow for efficient delivery of pDNA and high transfection efficiency both in ex vivo and vivo. pIL-12-DMP deters the growth of colon cancer by inhibiting angiogenesis, promoting apoptosis, and
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reducing cell proliferation. The IL-12 plasmid and DMP complex can be used as a new drug
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to more effectively treat colorectal cancer in a clinical setting.
ACCEPTED MANUSCRIPT References (1)DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, et al. Cancer
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T
treatment and survivorship statistics, 2014. CA Cancer J Clin. 2014, 64, 252-71. (2)Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics,
SC
2012. CA Cancer J Clin. 2015, 65, 87-108.
Mol Aspects Med. 2014, 39, 61-81. (4)Carballal
S,
MA NU
(3)Di Franco S, Todaro M, Dieli F, Stassi G. Colorectal cancer defeating? Challenge accepted!
Rodriguez-Alcalde
D,
Moreira
L,
Hernandez
L,
Rodriguez
L,
Rodriguez-Moranta F, et al. Colorectal cancer risk factors in patients with serrated polyposis
ED
syndrome: a large multicentre study. Gut. 2015.
PT
(5)Halama N, Zoernig I, Berthel A, Kahlert C, Klupp F, Suarez-Carmona M, et al. Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by
CE
Anti-CCR5 Therapy in Cancer Patients. Cancer Cell. 2016, 29, 587-601.
AC
(6)Linnekamp JF, Wang X, Medema JP, Vermeulen L. Colorectal cancer heterogeneity and targeted therapy: a case for molecular disease subtypes. Cancer Res. 2015, 75, 245-9. (7)Salazar R, Ciardiello F. Optimizing Anti-EGFR Therapy in Colorectal Cancer. Clin Cancer Res. 2015, 21, 5415-6. (8)Moon JJ, Huang B, Irvine DJ. Engineering nano- and microparticles to tune immunity. Adv Mater. 2012, 24, 3724-46. (9)Del Vecchio M, Bajetta E, Canova S, Lotze MT, Wesa A, Parmiani G, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res. 2007, 13, 4677-85. (10)Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of
ACCEPTED MANUSCRIPT angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995, 87, 581-6. (11)Weiss JM, Subleski JJ, Wigginton JM, Wiltrout RH. Immunotherapy of cancer by
RI P
T
IL-12-based cytokine combinations. Expert Opin Biol Ther. 2007, 7, 1705-21. (12)Yoo JK, Cho JH, Lee SW, Sung YC. IL-12 provides proliferation and survival signals to
SC
murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway. J Immunol. 2002, 169, 3637-43.
MA NU
(13)Strasly M, Cavallo F, Geuna M, Mitola S, Colombo MP, Forni G, et al. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk. J Immunol. 2001, 166, 3890-9.
ED
(14)Yao L, Sgadari C, Furuke K, Bloom ET, Teruya-Feldstein J, Tosato G. Contribution of
PT
natural killer cells to inhibition of angiogenesis by interleukin-12. Blood. 1999, 93, 1612-21. (15)Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects single-dose
interleukin-12
CE
of
exposure
on
interleukin-12-associated
toxicity
and
AC
interferon-gamma production. Blood. 1997, 90, 2541-8. (16)Melero I, Mazzolini G, Narvaiza I, Qian C, Chen L, Prieto J. IL-12 gene therapy for cancer: in synergy with other immunotherapies. Trends Immunol. 2001, 22, 113-5. (17)Duffy MR, Parker AL, Bradshaw AC, Baker AH. Manipulation of adenovirus interactions with host factors for gene therapy applications. Nanomedicine (Lond). 2012, 7, 271-88. (18)Coughlan L, Vallath S, Gros A, Gimenez-Alejandre M, Van Rooijen N, Thomas GJ, et al. Combined
fiber
modifications
both
to
target
alpha(v)beta(6)
and
detarget
the
coxsackievirus-adenovirus receptor improve virus toxicity profiles in vivo but fail to improve antitumoral efficacy relative to adenovirus serotype 5. Hum Gene Ther. 2012, 23, 960-79.
ACCEPTED MANUSCRIPT (19)Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, et al. Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses.
RI P
T
2010, 2, 2290-355.
(20)Cheng X, Darong Y, Lin M, Bingan Lu, Libao C, Qiuhong L, et al. Encapsulating Gold
SC
Nanoparticles or Nanorods in Graphene Oxide Shells as a Novel Gene Vector. ACS Appl. Mater. Interfaces, 2013, 5 (7), 2715–2724.
MA NU
(21)Gao X, Wang S, Wang B, Deng S, Liu X, Zhang X, et al. Improving the anti-ovarian cancer activity of docetaxel with biodegradable self-assembly micelles through various evaluations. Biomaterials. 2015, 53, 646-58. C,
Yongmao
L,
Copolymer
Z,
Bing
Grafted
X,
Zhiqiang
Luminescent
C,
Wenguang
Carbon
Dots
L.
As
a
PT
Polycation-b-Polyzwitterion
Xinyun
ED
(22)Lu
Multifunctional Platform for Serum-Resistant Gene Delivery and Bioimaging. ACS Appl.
CE
Mater. Interfaces, 2014, 6 (22), 20487–20497.
AC
(23)Chen L, She X, Wang T, He L, Shigdar S, Duan W, et al. Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles. Nanoscale. 2015, 7, 14080-92. (24)Christina
AH,
Maryam
T.
Substrate-Mediated
Gene
Delivery
from
Glycol-Chitosan/Hyaluronic Acid Polyelectrolyte Multilayer Films. ACS Appl. Mater. Interfaces, 2013, 5 (3), 524–531 (25)Lu Y, Wei YQ, Tian L, Zhao X, Yang L, Hu B, et al. Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor. J Immunol. 2003, 170, 3162-70.
ACCEPTED MANUSCRIPT (26)Liu JY, Wei YQ, Yang L, Zhao X, Tian L, Hou JM, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood.
RI P
T
2003, 102, 1815-23.
(27)Brenner MK, Gottschalk S, Leen AM, Vera JF. Is cancer gene therapy an empty suit?
SC
Lancet Oncol. 2013, 14, e447-56.
(28)Han J, Wang Q, Zhang Z, Gong T, Sun X. Cationic bovine serum albumin based
MA NU
self-assembled nanoparticles as siRNA delivery vector for treating lung metastatic cancer. Small. 2014, 10, 524-35.
(29)Qu Y, Chen L, Lowe DB, Storkus WJ, Taylor JL. Combined Tbet and IL12 gene therapy
ED
elicits and recruits superior antitumor immunity in vivo. Mol Ther. 2012, 20, 644-51.
PT
(30)Zhao X, Bose A, Komita H, Taylor JL, Kawabe M, Chi N, et al. Intratumoral IL-12 gene therapy results in the crosspriming of Tc1 cells reactive against tumor-associated stromal
CE
antigens. Mol Ther. 2011, 19, 805-14.
AC
(31)Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science. 1993, 260, 547-9.
(32)Mager LF, Wasmer MH, Rau TT, Krebs P. Cytokine-Induced Modulation of Colorectal Cancer. Front Oncol. 2016, 6, 96. (33)Vignali DA, Kuchroo VK. IL-12 family cytokines: immunological playmakers. Nat Immunol. 2012, 13, 722-8. (34)Tahara Y, Akiyoshi K. Current advances in self-assembled nanogel delivery systems for immunotherapy. Adv Drug Deliv Rev. 2015, 95, 65-76.
ACCEPTED MANUSCRIPT (35)Schulz EG, Mariani L, Radbruch A, Hofer T. Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12. Immunity. 2009, 30,
RI P
T
673-83.
(36)Brunda MJ, Gately MK. Interleukin-12: potential role in cancer therapy. Important Adv
SC
Oncol. 1995, 3-18.
(37)Yuzhalin AE, Kutikhin AG. Interleukin-12: clinical usage and molecular markers of
MA NU
cancer susceptibility. Growth Factors. 2012, 30, 176-91.
(38)Diouf I, Fievet N, Doucoure S, Ngom M, Andrieu M, Mathieu JF, et al. IL-12 producing monocytes and IFN-gamma and TNF-alpha producing T-lymphocytes are increased in
ED
placentas infected by Plasmodium falciparum. J Reprod Immunol. 2007, 74, 152-62.
PT
(39)Sangro B, Melero I, Qian C, Prieto J. Gene therapy of cancer based on interleukin 12. Curr Gene Ther. 2005, 5, 573-81.
CE
(40)Noguchi S, Mori T, Igase M, Mizuno T. A novel apoptosis-inducing mechanism of
AC
5-aza-2'-deoxycitidine in melanoma cells: Demethylation of TNF-alpha and activation of FOXO1. Cancer Lett. 2015, 369, 344-53. (41)Griffin GK, Newton G, Tarrio ML, Bu DX, Maganto-Garcia E, Azcutia V, et al. IL-17 and TNF-alpha sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation. J Immunol. 2012, 188, 6287-99. (42)Liu Y, Jiao F, Qiu Y, Li W, Lao F, Zhou G, et al. The effect of Gd@C82(OH)22 nanoparticles on the release of Th1/Th2 cytokines and induction of TNF-alpha mediated cellular immunity. Biomaterials. 2009, 30, 3934-45. (43)Pasquet M, Golzio M, Mery E, Rafii A, Benabbou N, Mirshahi P, et al. Hospicells
ACCEPTED MANUSCRIPT (ascites-derived stromal cells) promote tumorigenicity and angiogenesis. Int J Cancer. 2010, 126, 2090-101.
RI P
T
(44)Zabala M, Lasarte JJ, Perret C, Sola J, Berraondo P, Alfaro M, et al. Induction of immunosuppressive molecules and regulatory T cells counteracts the antitumor effect of
SC
interleukin-12-based gene therapy in a transgenic mouse model of liver cancer. J Hepatol. 2007, 47, 807-15.
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(45)Zhang L, Morgan RA, Beane JD, Zheng Z, Dudley ME, Kassim SH, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015, 21,
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2278-88.
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(46)Michelin MA, Montes L, Nomelini RS, Abdalla DR, Aleixo AA, Murta EF. Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.
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Oncol Lett. 2015, 10, 1523-1526.
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(47)Cavallo F, Quaglino E, Cifaldi L, Di Carlo E, Andre A, Bernabei P, et al. Interleukin 12-activated lymphocytes influence tumor genetic programs. Cancer Res. 2001, 61, 3518-23.
ACCEPTED MANUSCRIPT Figure Legends: Figure 1. Preparation and physicochemical properties of DMP.
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(A) preparation of DMP and pIL12 complex: First, a novel gene carrier was prepared with a self-assembly method. MPEG-PLA and DOTAP were assembled into a new gene carrier,
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DOTAP/MPEG-PLA Micelles (DMP). Then pIL12 plasmid was carried into cancer cells by DMP. (B) Zeta-potential of DMP; (C) Particle size of DMP; (D) Morphological
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characteristics of DMP by TEM observation; (E) Gel retardation assay of DNA and complexes. Lane 0 and 17, DNA marker; lanes 1 to 3, naked pIL12; lane 4 to 6, DMP; lane 7 to 9, weight ratios of pIL12 with DMP (1 : 25); lane 10 to 12, weight ratios of pIL12 with
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DMP (1 : 50); lane 13 to 15, weight ratios of pIL12 with DMP (1 : 100). pIL12 was
without free DNA.
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completely incorporated into DMP at a weight ratio of 1 : 100 and complexes were prepared
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Figure 2. Transfection efficiency measurement
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Transfection efficiency measurement of DMP. DMP, containing 4 μg pEGFP, were used to transfect Ct26 cells for 48 h. The transfection efficiencies at both weight ratio (pEGFP versus DMP as 1 : 100 and 1 : 150) were determined using (A) TEM and (B) Flow Cytometry. (C) The expression of IL-12 mRNA level detected by RT-PCR in NS, DMP, DMP-pVax and DMP-pIL12 transfected Ct26 cell groups; (D) IL-12 expression detected by Flow cytometry when IL-12 was mixed with DMP at a ratio of 1 : 150; (E) Elisa detection of the IL-12 in the cell supernatant of different group (NS, DMP, DMP -pVax and DMP -pIL12). (Mean ± SEM, n=3, p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, for 24 h, 48 h and 72 h, DMP-pIL12 versus NS, DMP, DMP-pVax, C)
ACCEPTED MANUSCRIPT Figure 3. MTT test of cell activity and expression of IL12 induced the secretion of IFN-γ of cultured lymphocytes test.
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When Ct26 cells were transfected with NS, DMP, DMP-pVax or DMP-pIL12 for 72 h, the supernatant from different treatments were added into lymphocytes and treated for 24 h, and
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the lymphocytes activity was tested by MTT test. (A) When the lymphocytes were treated for 24 h, the supernatant were added into Ct26 cells and treated for 24 h, and the Ct26 cell
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activity was tested by MTT test (B). (Mean±SEM, n=6) (*: p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); *: p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B). The lymphocytes were treated with cell culture supernatant collected from NS, DMP, DMP-pVax
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or DMP-pIL12 group, and the corresponding supernatants after 3 h (C), 8 h (D) and 24 h (E)
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culture in each group were then collected for measure of IFN-γ. IL-12 DMP-pIL12-mediated IFN-γ expression was substantially higher than that of other groups. (Mean ± SEM, n=3) (p <
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0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP,
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DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, E)
Figure 4. Anti-colon cancer effect of DMP-pIL12 in subcutaneous tumor model A) tumor growth curves; B) Body weight of different groups; C) Tumor weight; D) Tumor photos of of NS, DMP, DMP-pVax and DMP-pIL12 treatment groups. (Mean ± SEM, n=6) (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, C)
ACCEPTED MANUSCRIPT Figure 5. Anti-colon cancer effect of DMP-pIL12 in peritoneal colon cancer model. (A)Body weight; (B)Tumor weight of different group; (C) Images of mouse and the
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corresponding tumor; (D) The number of tumor node in different group; (E) ascites content of NS, DMP, DMP-pVax and DMP-pIL12 treated mice. (Mean ± SEM, n=6) (p < 0.01,
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DMP-pIL12 versus NS, DMP, DMP-pVax, A); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, D); p < 0.01,
Figure 6.
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DMP-pIL12 versus NS, DMP, DMP-pVax, E)
IL-12 expression, IFN-γ, TNF-α expressions and the cytotoxicity of T
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lymphocytes tests
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Tumor tissue was collected after the final treatment and IL-12 level was measured in the four groups (NS, DMP, DMP-pVax, and DMP-pIL12) of the subcutaneous model and peritoneal
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model by Flow Cytometry. DMP-pIL12 treated mice had significantly higher level of IL-12 in
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tumor tissue in both models (A). Tumor tissue of the subcutaneous model and ascites of the peritoneal model (NS, DMP, DMP-pVax, and DMP-pIL-12) were collected after two doses treatment, and we measured the expression of TNF-α (B) and IFN-γ (C) by ELISA in the tumor tissue and ascites. DMP-pIL12 treated mice had significantly higher level of IFN-γ and TNF-α in tumor tissue and ascites (Mean ± SEM, n=3) (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, B); p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax, C). (D) T lymphocytes derived from spleens of DMP-pIL12, DMP-pVax, DMP and saline treated mice were tested against Ct26 cells at different E: T ratios (X axis label is ratio of effector cells (E) to 51Cr-labeled target cells (T).) by a standard 4 h 51Cr release assay as described in Materials
ACCEPTED MANUSCRIPT and Methods. T lymphocytes from the spleens of DMP-pIL12 treated mice showed higher
Cell apoptosis, cell proliferation and tumor angiogenesis detections
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Figure 7.
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SEM, n=3, p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax) .
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cytotoxicity against Ct26 cells than did T lymphocytes from DMP-pVax, DMP or NS (Mean ±
(A) Cell apoptosis were assessed by counting the number of TUNEL-positive cells in the field
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(five high power fields per slide), and DMP-pIL12 was superior to other controls in increasing cell apoptosis. DMP-pIL12 significantly increased apoptosis (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax). (B) Cell apoptosis were assessed by counting the
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number of Ki67-positive cells in the field (five high power fields per slide), and DMP-pIL12
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was superior to other controls in inhibiting cell proliferation. DMP-pIL12 significantly inhibited cell proliferation(p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax). (C)
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Angiogenesis were assessed by counting the number of CD31-positive vessels in the field
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(five high power fields per slide). DMP-pIL12 significantly inhibited MVD (p < 0.01, DMP-pIL12 versus NS, DMP, DMP-pVax).
Figure 8. Toxicity assessment in vivo with pathological section. Histological examinations of HE-stained vital organ sections. No significant pathological changes were detected.
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Graphical Abstract
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Schedule of cancer gene immunotherapy with IL-12. First, a novel gene carrier was prepared with a self-assembly method. MPEG-PLA and DOTAP were assembled into a new gene carrier, DOTAP/MPEG-PLA Micelles (DMP). Then pIL12 plasmid was carried into cancer cells by DMP. And the cancer cells expressed and secreted the IL-12 protein. IL-12 could activate the immune cell, then immune cells secrete some tumor-killing factor to kill cancer cell.