Therapeutic Effect Of HER2/Neu-IL12-C.Novyi-NT Treatment To HER2/Neu Positive Breast Cancer Mice

Objective To evaluate the feasibility of attenuated C.novyi (C.novyi-NT) as a vector for gene therapy; prepared chimeric gene of HER2/neu-IL12modified C.novyi-NT (HER2/neu-IL12-C.novyi-NT) for the therapy of HER2/neu-positive breast cancer in mice model.Methods (1) The distribution of C.novyi-NT in normal mice. The suspension of C.novyi-NT0.5ml (5×107) was injectied intravenously to normal mice of Kunming strain, they were killed in1,2,4,7days respectively after injection. The liver, kidney and lung were taken and homogenated. After anaerobic inoculating, the number of bacteria was calculated in the organs.(2) The distribution of C.novyi-NT in tumor-bearing mice. EMT6breast cancer bearing mice model were prepared, then C.novyi-NT spore suspension0.5ml (5x10’) was intravenously injected into the tumor-bearing mice. The mice were sacrificed at1,2,4and7days respectively after injection. The tumors as well as their organs were taken and the following process was performed as described in the method of (1).(3) Oncolytic effect of C.novyi-NT. C.novyi-NT spores were injected intravenously into the tumor bearing mice as the same quantity and process as elucidated in method (2). After paraffin slice from the organs were prepared, HE and Gram’s dyeing were performed respectively for observing the size of necrosis area in the former and the bacterial distribution in latter.(4) Security evaluating. C.novyi-NT suspension was intravenously injected to normal mice (5×107/each), their feeding, drinking and general condition was observed daily. The mice were killed at the7th days, their organs were taken and the following process was performed as the metod (1).(5) Preparation of HER2/neu-IL12-C.novyi-NT.The method of ultrasound DNA delivery (UDD) was used to transform the recombinant shuttle plasmid of HER2/neu-IL12chimeric gene (pEH1) to C.novyi-NT, and the HER2/neu-IL12-C.novyi-NT was selected by erythromycin.(6) Preparation of HER2/neu-EMT6. PE1was used to transfer the recombinant plasmid of pcDNA6-HER2/neu into EMT6breast cancer cells, the HER2/neu-EMT6were selected by blasticidin. Then, HER2/neu-positive breast cancer bearing mice were prepared by injecting the HER2/neu-EMT6modified cells in the right prothorax of mice.(7) Theraputic effect of UER2/neu-IL12-C.novyi-NT.24HER2/neu-positive breast cancer bearing mice were divided randomly into4groups, group Ⅰ:C.novyi-NT; groupⅡ:pEH1; group Ⅲ:HER2/neu-IL12-C.novyi-NT; group Ⅳ:PBS. At the7th day after the tumor cell inoculted when the size of the tumor was greater than200mm3in diameter, the reagents as above were injected into the tumor directly. At the days of4,7,9,11,13,15after tumor cells inoculated, the size of tumor was measured and growth curve of the tumors were drew. At the15th days, all the mice were killed, the tumor and spleen were taken and weighed, and the spleen index was calculated.Results (1) C. novyi-NT has the expected characteristics of targeting location in tumor and moreover, has an obvious oncolytic effect alone.(2) Preliminary security evaluation revealed that C.novyi-NT can not make any hazard in mice.(3) By the method of UDD, the recombinant shuttle plasmid was transfered into C. novyi-NT, and the HER2/neu-IL12-C.novyi-NT was successfully prepared.(4)The size of tumors in groups Ⅰ-Ⅲ were signifcantly smaller than that of PBS control group (group Ⅳ)(p<0.01); while, it was even much smaller in the HER2/neu-IL12-C.novyi-NTused group (group Ⅲ) than the other two groups(group Ⅰ and Ⅱ)(p<0.01).Conclusion As a vector of gene therapy, C. novyi-NT has good feature of restricted tumor targeting, security as well as certain oncolytic effects; HER2/neu-IL12-C.novyi-NT exerted satisfactory theraputic effect in the mice model of HER2/neu-positive breast cancer.