Immune Response Of Heparanase Gene Modified Dendritic Cell-based Vaccine On Gastric Cancer Cells

Background:Gastric cancer is the second most common neoplasm world widely, representing approximately 10% of newly diagnosed malignancies and accountings for more than 12% of cancer death. Invasion and secondary spread through the blood and lymphatic system are characteristic features of gastric cancer and other malignant tumors and is the most important cause resulting in patients'death. Most gastric cancer patients have developed local or remote metastasis when they visit doctors and lose operation opportunity. Accordingly, combined therapy becomes the most important therapy for gastric cancer, especially for those experienced metastatic events. As an ingredient of combined therapy of gastric cancer, biotherapy can not only treat cancer independently, but also can be combined with surgery, radiotherapy and chemotherapy. It has advantages of higher therapeutic effects, better specificity and minor side effects and attracts more and more attentions recently. Tumor active immunity is a part of tumor biotherapy and is an important research field in tumor treatment. Dendritic cells initiate and regulate immune response. It is believed to be the most potent professional antigen presenting cells and has the most powerful antigen presenting capacity. Along with the intensive understandings to its biological behaviors, it attracted more and more attentions as a new method involving cancer immune therapy. Dendritic cell-based vaccine is emerging as a representative of new vaccines and manifesting good clinical applications. It is very important to select a suitable antigen in the cancer immunotherapy. Heparanase is a tumor metastasis-related gene recently cloned by four research groups. It had pivotal roles in the progress of tumor invasion and metastasis. High expression of heparanase was found in most kinds of human malignant tumors. There existed close relationship between heparanase expression and cancer prognosis. Since metastasis and invasion is the characteristic of most kinds of cancers, heparanase is expected to be a new common tumor associated antigen (TAA) of most kinds of human malignant tumors and is emerging as an ideal target for tumor gene therapy and biological therapy. Cytotoxicity T lymphocytes (CTL) are the most effective cells defending tumor cells. But it is difficult for cancer patients to generate effective CTL response. Specific anti-tumor immune response is expected to be generated by combination TAA with DCs since TAA would be presented with high performance. Those specific CTLs would kill tumor cells expressing the very kind of TAA, whereas no killing effects would occur to the cells which have no expression of those TAA. Heparanase, a newly found tumor metastatic-associated gene, can be transfer to DCs as a kind of TAA and generate heparanase specific DC vaccine. In the context, CTL can be activated by heparanase specific DC vaccines. Immune killing effects would be expected to be generated to gastric cancer cells that expressed heparanase, especially those metastatic gastric cancer cells. Genetically modified DC is the most popular kind of DC vaccines. By means of molecular methods, exogenous gene can be transduced into DCs. Genetically modified DCs by genes coding for TAA to DCs has potential advantages. First, the genetically modified DCs may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DCs ensures that the gene product is endogenously processed leading to the generation of MHC class I restricted CTLs which is the effector arm of cell-mediated immune response against tumor cells. Many studies found specific lysis can be achieved using CTL pulsed with TAA loaded DC and this predicted that gene modified DCs is a prospected means of anti-tumor method. Methods:Using surgically resected samples, expression of heparanase in gastric cancer and its corresponding normal tissues was detected by RT-PCR and immunohistochemical methods. Microvessel density (MVD) and c-met were measured by immunohistochemical methods to study the relationship between expression of heparanase and MVD and c-met in gastric cancer. Human type 5 replication-defected adenovirus encoding full length of heparanase cDNA (Ad5-Hpa) was constructed using homologous gene recombination method. High titer virus was achieved by duplicated in HEK293 cellsand purified by CsCl; dendritic cells originated from peripheral blood mononuclear cells (PBMNCs) of healthy HLA-A2 positive donors were cultured and induced to maturation by cytokine combinations of rhGM-CSF, rhIL-4 and rhTNF-α. The cells were then verified by three methods, namely: cell morphology, expression of different CD molecules and MLR; the maturated DCs were then transferred by Ad5-Hpa to generate heparanase gene modified DC vaccine. Expression of heparanase in those DC vaccines was identified by immunohistochemical methods. HLA-A2 positive T lymphocytes were activated by co-cultured with those genetically modified DC vaccine repeatedly. CTL-mediated cell lysis to gastric cancer cell lines KATO-III and SGC-7901 was assayed in vitro by standard 51Cr releasing assay. IFN-γreleasing was measured by ELISA when heparanase specific CTL co-cultured with KATO-III cells. Results:(1)Heparanase mRNA was detected in 37 out of 47 (74.5%) gastric tumor samples by RT-PCR, which was significantly higher than that in its adjacent tissues (P<0.01). The positive rate of heparanase mRNA in gastric carcinomas was correlated with tumor diameter, invasive depth, regional lymph nodes metastasis and TNM stage. Further study also showed that MVD in heparanase positive group was significantly higher than that in its counterpart (50.7±14.9 vs. 31.1±9.5, P<0.01). Expression of heparanase mRNA correlated with expression of c-met (r=0.714, P<0.01) and may be modulated by c-met through NF-κB pathway. (2)Ad5-Hpa was achieved by homologous gene recombination methods. After duplicated and purified, high titer virus of 5×1010pfu/ml was achieved for gene transfer. (3)Maturated DCs were obtained from peripheral blood-oriented MNCs co-cultured with cytokine combinations of rhGM-CSF,rhIL-4 and rhTNF-α.; Genetically modified DC vaccine was prepared by transferred heparanase gene to maturated DCs with Ad-Hpa. Measured by immunohistochemical method, enhanced expression of heparanase was detected after transferred with Ad5-Hpa. CTLs primed by heparanase gene modified DC vaccine exhibited potent specific lysis to KATO-III gastric cancer cells which are heparanase and HLA-A2 positive measured by standard 51Cr releasing assay, meanwhile, IFN-γreleasing increased in this group. There were no notable killing effects to SGC-7901 gastric cancer cells, which are HLA-A2 dismatched and autologous lymphocytes whichwere also heparanase positive expression. Conclusion:Expression of heparanase contributes to metastasis and angiogenesis of gastric cancer, and correlates with poor clinicalpathologic characteristics in its prognosis. Activation of c-met maybe involved in the modulation of heparanase through NF-κB pathway. Heparanase may be used as a good target for gastric cancer immunotherapy. Ad5-Hpa was successfully constructed by homologous recombination. High titer virus of 5×1010pfu/ml was achieved for gene transfer. Maturated DCs were achieved from adherent cells of PBMNCs co-cultured with different cytokine combination. DC vaccine genetically modified by heparanase gene (Hpa-DC vaccine) was successfully achieved by transferred Ad5-Hpa to DCs. Hpa-DC vaccine can activate CTL and induce potent immune responses against HLA-matched gastric cancer cells in vitro, whereas there were no killing effects to self-lymphocytes. This founding shows that heparanase gene modified DC vaccine has characteristic of higher effects and good safety, and provides experiment evidences for the future clinical applications.