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ISSN 1007-9327 CN 14-1219/R  World J Gastroenterol  2003 May;9(5):951-955

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy

Shu Zheng, Zuo-Xiang Xiao, Yue-Long Pan, Ming-Yong Han, Qi Dong


Shu Zheng, Zuo-Xiang Xiao, Yue-Long Pan, Ming-Yong Han, Qi Dong, Cancer Institute, Zhejiang University, Hangzhou 310009, Zhejiang Province, China
Supported by National Natural Science Foundation of China, No. 40621130
Correspondence to: Dr. Shu Zheng, Cancer Institute, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, Zhejiang Province, China.  zhengshu@zju.edu.cn
Telephone: +86-571-87784501    Fax: +86-571-87214404
Received: 2002-12-10    Accepted: 2003-01-16

Abstract

AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene.

METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated.

RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-g, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice.

CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy.

Zheng S, Xiao ZX, Pan YL, Han MY, Dong Q. Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy. World J Gastroenterol  2003; 9(5): 951-955
http://www.wjgnet.com/1007-9327/9/951.asp

INTRODUCTION
Alginate microcapsules have been used extensively for different applications, particularly for the encapsulation of pancreatic islet cells and insulin delivery[1]. This method has also been used for the encapsulation of cells that release growth hormone, b-endorphin, endostatin and other agents for gene therapy[2-5]. The alginate membranes allow the free exchange of nutrients and oxygen between the implanted cells, and could prevent the escape and elimination of encapsulated cells. More important, this approach provides a prolonged sustained delivery of recombinant protein produced by the cells, thus maintaining high levels of the agent.
    In recent years, interleukine-12 (IL-12) has received considerable interest in cancer biologic therapy. In vivo IL-12 was found to have a potent antitumor efficacy in a variety of murine tumor models[6,7]. Local or systemic treatment with recombinant IL-12 protein (rIL-12) was shown to inhibit the growth of established subcutaneous tumor and tumor metastasis[8-10]. However, systemic administration of rIL-12 caused severe dose-dependent toxicity and led to an interruption of the first human trial[11]. In contrast, the local transfer of cytokine genes as a means for gene therapy could circumvent such systemic toxicity and provide effective and persistent local cytokine levels for immune cells activation[12-15]. Some studies using an ex vivo IL-12 gene therapy yielded encouraging results, showing that murine fibroblasts or tumor cells transduced in vitro with IL-12 cDNA, using a retroviral vector, were able to induce antitumor immune responses in the absence of apparent toxicities[16]. This strategy, however, has many obstacles precluding successful clinical application: e.g. autologous somatic cells or tumor cells are difficult to culture and transfect, and selection for transfected cells requires prolonged culture and the attendant costs of these process are expensive. To avoid these potential disadvantages, an alternative approach to obtain prolonged local cytokine secretion is adopted to use microencapsulated engineered cells to secrete IL-12.
    In the present study, NIH3T3 cells engineered to continuously secrete high levels of mIL-12 were encapsulated with alginate. The ability of this system to secrete biologically active mIL-12 capable of inhibiting the tumor growth of a murine colon carcinoma xenograft in the mouse was investigated.

MATERIALS AND METHODS
Mice and cell lines
Male BALB/C mice aged between 6 and 8 weeks were purchased from Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China) and housed in a specific pathogen-free condition for all experiments. Mouse fibroblasts (NIH3T3) and the murine colon adenocarcinoma cell line (CT26) were donated by the Institute of Immunology, Zhejiang University (Hangzhou, China). Cells were cultured in RPMI-1640 medium (GIBCO-BRL, Gaithersburg, MD, USA) supplemented with 10 % heat-inactivated fetal calf serum (FCS; HyClon, Logan, UT, USA), 2 mM glutamine, penicillin 100 U/ml, and streptomycin 100 mg/ml.

Expression plasmids and transfection of NIH3T3 cells
Murine p35 and p40 subunits of mIL-12 were subcloned into pcDNA3.1 plasmids containing a cytomegalovirus (CMV) immediate-early enhancer promoter and a G418 selected gene. NIH3T3 cells were stably transfected with these expression plasmids using LF2000TM (Ivitrogen, Life Technologies, USA). To obtain stably transfected clones (NIH3T3-mIL-12), transfected cells were grown in G418 containing medium (400 g/L, Ivitrogen, Life Technologies, USA) for 14 days, and resistant clones were propagated separately. With subsequent determination of mIL-12 expression by ELISA kit (R&D systems, Inc., USA).

Microencapsulation of NIH3T3-mIL-12
NIH3T3-mIL-12 cells were encapsulated within microspheres composed of Ba2+-alginate. Briefly, cells were resuspended in sodium alginate-saline (1.5 % wt/vol, purified by Syringe Driven Filter Unit) (Sigma, St Louis, MO, USA) to a final ratio of 0.5109 cells/L of alginate. The suspension was sprayed through an air jet-head droplet-forming apparatus, into a solution of 4.9 % Barium chloride (pH 7.4, Sigma), where they were allowed to gel for 10 min, washed three times with PBS, then cultured in the conditioned medium described above. The number of cells encapsulated and the viability of the cells in the microcapsules were evaluated weekly using a modified MTT assay.

In vitro release of mIL-12 from encapsulated NIH3T3-mIL-12 cells
Microencapsulated NIH3T3-mIL-12 cells were suspended in the conditioned medium described above at a density of 2105 cells/well. The medium was collected every 2 hrs. and assayed for mIL-12 using ELISA assay (Endogen, Woburn, MA, USA). Medium from NIH3T3-mIL-12 monolayer cells was used as a positive control.

Murine studies
The BALB/C mice were inoculated subcutaneously in the right-behind armpit with CT26 cells (2