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.
Received: 2002-12-10 Accepted:
To explore the anti-tumor immunity against CT26 colon tumor of the
microencapsulated cells modified with murine interleukine-12 (mIL-12)
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.
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
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;
Alginate microcapsules have been used extensively for different
applications, particularly for the encapsulation of pancreatic islet cells
and insulin delivery. 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.
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.
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 0.5×109 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
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
The BALB/C mice were inoculated subcutaneously in the right-behind armpit
with CT26 cells (2