Volume 9, Issue 3, Pages 347-354 (March 2004) Candidate Genes Associated with Tumor Regression Mediated by Intratumoral Il-12 Electroporation Gene Therapy  Shulin Li, Xueqing Xia, Francesca M Mellieon, Jianguo Liu, Stacy Steele  Molecular Therapy  Volume 9, Issue 3, Pages 347-354 (March 2004) DOI: 10.1016/j.ymthe.2003.11.022 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Intratumoral Il-12 EGT induces high levels of Mig, Stat1, and IRF7 mRNA in tumors. For intratumoral EGT, Il-12 or control plasmid DNA (10 μg) was injected into the tumor followed by two electric pulses. The electric parameters were 450 V/cm and two pulses for a duration of 20 ms. Ten micrograms of RNA for each tumor sample was used to determine the level of gene expression by Northern blot. “pIL-12” and “pCtrl” represent tumors injected with Il-12 plasmid DNA and empty plasmid DNA, respectively. Each lane represents a tumor sample isolated from a separate animal. Three mice were treated with control plasmid DNA and another three mice with plasmid DNA encoding Il-12. (A) Induction of Mig by intratumoral Il-12 EGT. (B) Induction of Stat1 and IRF7 by intratumoral Il-12 EGT. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Intramuscular Il-12 EGT also increases the levels of Mig, Stat1, and IRF7 expression in tumors. For intramuscular EGT, plasmid DNA (10 μg) with and without the Il-12-expressing cassette was injected into muscle followed by electric pulses, as described under Materials and Methods. The electric parameters used for intramuscular delivery were 350 V/cm and two pulses for a duration of 20 ms. Northern blot analysis and data labeling were the same as described for Fig. 1. A total of eight animals (n = 4) was used, four for control and four for Il-12 treatment. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Intratumoral Il-12 EGT induces a higher level of Stat1 protein in tumors than intramuscular treatment. Stat1 protein levels in tumors were compared in intratumoral and intramuscular Il-12 EGT by Western blot analysis, using a polyclonal anti-mouse Stat1 antibody. (A) Stat1 proteins are primarily induced in the tumors treated with intratumoral Il-12 EGT. (B) Stat1 is detected in tumors receiving intramuscular Il-12 EGT only when the membrane was overexposed on a film. The data labeling is the same as in Fig. 1. A nonspecific binding protein was also detected in all samples from intramuscular and intratumoral Il-12 EGT. The detection of this nonspecific binding protein indicates that the lack of Stat1 in some of the control mice (pCtrl) is not due to missed or reduced sample loading. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Analysis of IRF7 localization in the tumor cells after intratumoral or intramuscular Il-12 EGT. Tumor samples from the tumor-bearing mice treated with intratumoral and intramuscular Il-12 EGT were obtained on day 10. Frozen tumor sections were stained with anti-mouse IRF7 polyclonal antibody and the representative image was taken under a microscope at 40× magnification for (A) to (D). (E) A 10-fold reduction of the image. Arrows point to the positively stained anti-IRF7 antibody in the nuclei. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Analysis of the distribution of Il-12 protein after different routes of administration of Il-12 plasmid DNA using electroporation. The purpose of this experiment is to compare the levels of Il-12 expression and the difference in distribution of Il-12 between tumor and serum. Tumor and serum samples (n = 5) were obtained on day 3 after administration of Il-12 DNA plasmid. The procedures for intratumoral and intramuscular EGT are the same as described in the legends to Figs. 1 and 2, respectively. The level of gene expression was determined using ELISA as detailed under Materials and Methods. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 The effects of interference of Il-12-mediated candidate genes on gene expression and immune cell infiltration. (A) Interference of Stat1 inhibits the expression of antiangiogenic gene IP10. Northern blot analysis of the expression of IP10 was performed in Stat1−/− and wild-type mice treated with and without Il-12 gene in the muscles. Ten micrograms of plasmid DNA of pCtrl or pIL-12 was injected intramuscularly via electroporation, as described in the Fig. 2 legend. (B and C) Injection of Mig (pMig) into the tumor via electroporation increases infiltration of CD4+ T cells into the tumor. Thirty micrograms of plasmid DNA with and without Mig was injected into tumors (n = 5) once a week for a total of two administrations. Three days after the second administration, tumor-bearing mice were sacrificed and infiltration of CD4+ and CD8+ T cells was analyzed from tumor sections using immunostaining, as described under Materials and Methods. Molecular Therapy 2004 9, 347-354DOI: (10.1016/j.ymthe.2003.11.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions