Preparation And Experimental Anti-tumor Studies Of Microencapsulated Cells Releasing Human Tissue Inhibitor Of Metalloproteinase-2

Cell encapsulation in biocompatible and semipermeable polymeric membranes is an effective method for immunoprotection, regardless of the type of recipient (allograft, xenograft, etc.). The semipermeable nature of the membrane prevents high molecular weight molecules, antibodies and other immunologic moieties from coming into contact with the encapsulated cells and destroying them as foreign invaders, but permits the entry of nutrients and oxygen and the exit of therapeutic protein products. Microencapsulation has become one of the most important techniques for immobilizing cells for biomedical applications, and transplantation of cells or tissues, such as islets of Langerhans, hepatocytes, parathyroid cells, pituitary cells, and thymic epithelial cells, which are enclosed in a semipermeable membrane had been proposed as an alternative to hormone and enzyme replacement therapy.Microencapsulation of recombinant cell lines is viewed as a new delivery system for gene therapy. In this approach cell lines are engineered to secrete a desired gene product Enclosing these cells in immunoprotective devices would allow such nonautologous cells to be implanted into any host to deliver the desired gene product without triggering graft rejection. The advantages of this nonautologous method of gene delivery are:it does not require modification of the host's genome, thus providing additional measures of safety and cost saving;it provides ample material for quality assessment before implantation, a safety feature not available to most other forms of invivo delivery. Microencapsulated engineered cells secreting bioactive agent demonstrated good efficacy in tumor suppression in a series of experimental studies, showing high promise in cancer biotherapy.MMPs(matrix metalloproteinases) play key roles in the responses of cells to their microenvironment. By effecting proteolytic degradation or activation of cell surface and extracellular matrix(ECM) proteins they can modulate both cell-cell and cell-ECM interactions, which influence cell differentiation, migration, proliferation and survival. The proteolytic activity of MMPs is regulated by a number of physiological inhibitors. Specific inhibition is mediated by the TIMPs(tissue inhibitors of metalloproteinases). A tightly regulated balance between proteases and their inhibitors must exist at any developmental and physiological stage. A disruption in this balance is observed in many pathological conditions, including dififerent stages of tumor progression.TIMP-2 is a natural matrix metalloproteinase inhibitor that prevents the degradation of ECM proteins, with molecular weight of 21 kDa. It suppresses the hydrolytic activity of all activated members of the MMP family and in particular that of MT1-MMP, MMP-2, and MMP-9, which has been particularly implicated in tumor progression and angiogenesis. Moreover, TIMP-2 also possesses MMP-independent anti-angiogenic activity. The anti-neoplastic activity of TIMP-2 in tumor models had been demonstrated in several experimental approaches. It was reported that transfection of tumor cells with TIMP-2 cDNA results in the reduction of tumor growth, angiogenesis, and metastasis.The retrovirus-mediated TIMP-2 gene transfer in vivo showed similar result. A nude mouse model of colorectal liver metastasis showed that overexpression of TIMP-2 by hepatocytes protects from challenge by metastatic cells and, furthermore, reduces the growth of well-established metastases. fIn the present study, the recombinant eukaryotic expression plasmid pcDNA3/TIMP-2 was constructed by ligating human TIMP-2 gene, which was amplified by RT-PCR, and the pcDNA3 vector. The recombinant plasmid was transfected into CHO cells by liposome to establish stable TIMP-2-expressing cells. Thecells were then encapsulated in barium alginate microcapsules. The biologicalcharacteristics and anti-tumor activities of the microencapsulated engineered cells bothin vitro and in vivo were investigated to explore the feasibility of their use in cancerbiotherapy.Part one: Construction of eukaryotic expression plasmid of TIMP-2The total RNA extracted from human fetal lung fibroblast cell with TRIZOL was amplified by RT-PCR to obtain TIMP-2 cDNA. Cloning vector was constructed by ligating TIMP-2 cDNA with pMD18-T vector, and then transformed into E.coli strain DH 5a to amplify. Plasmids were extracted and identified by restriction endonuclease analysis and DNA sequencing. After digestion of the recombinant plasmid by restriction endonuclease EcoR I and Hind IE, gel-purified TIMP-2 cDNA was inserted into the EcoR I /Hind DI site of vector pcDNA3 to construct eukaryotic expression plasmid pcDNA3/TIMP-2. Restriction endonuclease analysis and DNA sequencing demonstrated that the targeting eukaryotic expression plasmid of TIMP-2 was successfully constructed. Part two: Establishment of stable TIMP-2-expressing cellsCHO cells were transfected with the recombinant plasmid pcDNA3/TIMP-2, using Lipofectamine reagent. Cells were treated with selection medium containing G418 to select stably transfected cells displaying neomycin resistance. Between 2 and 3 weeks into the selection process, resistant cells began to appear. They were separated out and cultured. RT-PCR was performed to investigate the expression of TIMP-2 at mRNA level. Western blot and ELISA assay were used to detect TIMP-2 in cell culture supernatant. The bioactivity of MMPs inhibition of the secreted TIMP-2 was examined using reverse zymography. RT-PCR demonstrated the expression of TIMP-2 mRNA. Western blot and ELISA assay showed the expression of TIMP-2 protein, whose bioactivity of MMPs inhibition was confirmed by reverse zymography. In conclusion, a stable expressing cell line of bioactive TIMP-2 was successfully established.Part three: Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metaIloproteinase-2Stable TIMP-2-expressing CHO/pcDNA3/TIMP-2 cells were encapsulated in barium alginate microcapsules and cultured in vitro. Morphological appearance of the microcapsules was observed under a light microscope. Cell viability was assessed using MTT assay. ELISA assay and reverse zymography were used to confirm the release of bioactive TIMP-2 from the microcapsules. Cryopreservation study of the microencapsulated cells was carried out using DMSO as preservative agent. The influence of the microencapsulated CHO/pcDNA3/TIMP-2 cells on proliferation and migration of endothelial cell and human colorectal carcinoma cell line SW620 was investigated. The microcapsules obtained appeared like a sphere with diameter of 300-600 fun, and clearly smooth surface. The microencapsulated cells survived well and kept proliferating over the 6 weeks observed. The secretion and bioactivity of TIMP-2 was not affected by the microcapsules. After preparation, the TIMP-2 production showed a gradual increase in company with the proliferation of the microencapsulated cells. The cryopreservation process did not damage the microcapsule morphology nor the viability of the cells inside. The growth and migration of endothelial cells were significantly inhibited by the microencapsulated CHO/pcDNA3/TTMP-2 cells.Part four: In vivo transplantation and anti-tumor studies of microencapsulated CHO/pcDNA3/TIMP-2 cellsThe microencapsulated CHO/pcDNA3/TIMP-2 cells were injected into the abdominal cavity of BALB/c mice. The microcapsules were retrieved and cultured at different time points after transplantation. Morphological appearance of the retrieved microcapsules was observed under a light microscope, and ELISA assay was used to assess their release of TIMP,-2. The microcapsules remained normal morphological features, with clearly smooth surface, and no sign of fibrotic overgrowth was found. The number of cells inside the microcapsules increased 4 weeks after transplantation, but the expression and release of TIMP-2 showed a gradual decrease.H22 cells were subcutaneously injected into BALB/c mice. After 3 days, the tumor-bearing mice were separated randomly into 3 groups, treated with microencapsulated CHO/pcDNA3/TIMP-2 cells, CHO/pcDNA3 cells, and saline, respectively. Tumor weight and microvessel density were recorded and analysed 21 days later. Results showed that tumor weight was significantly lower in microencapsulated CHO/pcDNA3/TIMP-2 cells group than in other 2 groups. The microvessel density in microencapsulated CHO/pcDNA3/TIMP-2 cells group was also significantly lower, showing the inhibition of angiogenesis of the microencapsulated CHO/pcDNA3/TIMP-2 cells. Conclusions:1. The recombinant eukaryotic expression plasmid of TIMP-2 was successfully constructed, and a stable expressing cell line of bioactive TIMP-2 was established.2. Barium alginate microcapsules with the appearance of a sphere, and diameter of 300-600 um were obtained using air-jet technique. The encapsulated cells survived well and kept proliferating. The microcapsules also have sufficiently large pore size to allow for the diffusion of bioactive TIMP-2.3. The cryopreservation process using DMSO as preservative agent did not damage the microcapsule morphology nor the viability of the cells inside.4. The microencapsulated CHO/pcDNA3/TIMP-2 cells inhibited the in vitro proliferation of endothelial cells stimulated with bFGF and endothelial cell migration.5. The microencapsulated CHO/pcDNA3/TIMP-2 cells showed high biocompatibi-lity and mechanical stability after 4-week in vivo transplantation.6. The microencapsulated CHO/pcDNA3/TIMP-2 cells inhibited tumor growth and angiogenesis in vivo in murine model.