Volume 8, Issue 2, Pages 238-248 (August 2003) Recombinant vaccinia virus expressing IL-2 generates effective anti-tumor responses in an orthotopic murine model of head and neck carcinoma  Santanu Dasgupta, Pulak K Tripathi, Malaya Bhattacharya-Chatterjee, Bert O'Malley, Sunil K Chatterjee  Molecular Therapy  Volume 8, Issue 2, Pages 238-248 (August 2003) DOI: 10.1016/S1525-0016(03)00148-5 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Survival of mice prevaccinated subcutaneously with SCC VII/SF cells prior to tumor implantation (protocol I). (▵) Vaccinated with PBS; (♦) prevaccination with irradiated and rvv-IL-2-infected SCC VII cells; (▪) intratumoral vaccination with rvv-IL-2; (•) prevaccination plus intratumoral injection. Each group consisted of at least 15 mice. Data were analyzed by Kaplan Meier test. Survival was significantly longer (P < 0.003) in mice treated with prevaccination plus intratumoral injection compared to mice treated with either intratumoral or subcutaneous immunization alone. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Tumor growth after treatment by protocol I. (A) Tumors were measured externally in two dimensions [(d1 + d2)/2] on day 7 after treatment with rvv. Each group contained at least 8 mice. (▵) vaccinated with PBS; (▴) prevaccinated with irradiated and rvv-infected SCC VII cells; (□) intratumoral vaccination with rvv; (○) prevaccination plus intratumoral vaccination with rvv. Rvv-lacZ (empty symbols) and rvv-IL-2 (filled symbols) were used for the preparation of the vaccines. Values are in means ± SE. (B) Tumor weights on day 10 of mice treated by protocol I. (▪) Intratumoral vaccination with rvv-IL-2; (•) prevaccination plus intratumoral vaccination with rvv-IL-2. Each group consisted of at least five mice. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Survival of the mice prevaccinated subcutaneously with irradiated and rvv-infected SCC VII/SF cells followed by intratumoral rvv vaccination (protocol I). (○) Rvv-lacZ; (•) rvv-IL-2. Mice vaccinated with rvv-IL-2 vaccine survived significantly longer (P < 0.0001) than those treated with lacZ vaccine. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Survival of mice with established tumors (protocol II). (▵) Vaccinated with PBS; (▴) subcutaneous vaccination with irradiated and rvv-infected SCC VII cells; (□) intratumoral vaccination with rvv; (○) subcutaneous plus intratumoral vaccination with rvv vaccines. Rvv-lacZ (empty symbols) and rvv-IL-2 (filled symbols) were used for the preparation of the vaccines. Each group consisted of 15 mice. Mean survival of the mice receiving combined subcutaneous and intratumoral rvv-IL-2 vaccination was significantly (P < 0.02) longer than that of either the group that received subcutaneous or those that received intratumoral vaccination. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Tumor growth in mice with established tumors after vaccination. (□) Intratumoral vaccination with rvv; (○) subcutaneous vaccination with irradiated and rvv-infected SCC VII cells plus intratumoral vaccination with rvv. Rvv-lacZ (empty symbols) and rvv-IL-2 (filled symbols) were used for the preparation of the vaccines. The ovals indicate day of vaccination; arrowheads indicate days mice were sacrificed for the determination of tumor weights. Values are means ± SE of three mice. Tumor weights in groups of mice treated with subcutaneous plus intratumoral rvv-IL-2 vaccine were significantly smaller compared to those that received intratumoral vaccination only. Day 9, P < 0.02; day 12, P < 0.04; day 16, P < 0.04. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Apoptosis of tumors. Apoptotic indices were determined with fresh frozen tumor sections using a fluorescein-labeled TUNEL cell death detection kit. For each group, multiple tumor sections were stained and positive cells were counted in high-power fields under a fluorescence microscope. □, vaccination with PBS; ▪, subcutaneous vaccination with irradiated and rvv-lacZ infected SCCVII/SF cells; ▪, intratumoral vaccination with rvv-lacZ; , subcutaneous plus intratumoral rvv-lacZ vaccination; ▪, subcutaneous vaccination with irradiated and rvv-IL-2 infected SCCVII/SF cells; ▪, intratumoral vaccination with rvv-IL-2; ▪, subcutaneous plus intratumoral rvv-IL-2 vaccination. Values are Mean ± SE. Apoptotic indices were significantly higher (P < 0.0001) in tumors from mice immunized with rvv-IL-2 vaccine compared to those treated with rvv-lacZ vaccine or those mock vaccinated with PBS. Apoptotic index was higher in the group treated with subcutaneous plus intratumoral rvv-IL-2 vaccination (P < 0.003) compared to the group treated by intratumoral rvv-IL-2 vaccine. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Apoptosis of tumors. Apoptotic indices were determined with fresh frozen tumor sections using a fluorescein-labeled TUNEL cell death detection kit. For each group, multiple tumor sections were stained and positive cells were counted in high-power fields under a fluorescence microscope. □, vaccination with PBS; ▪, subcutaneous vaccination with irradiated and rvv-lacZ infected SCCVII/SF cells; ▪, intratumoral vaccination with rvv-lacZ; , subcutaneous plus intratumoral rvv-lacZ vaccination; ▪, subcutaneous vaccination with irradiated and rvv-IL-2 infected SCCVII/SF cells; ▪, intratumoral vaccination with rvv-IL-2; ▪, subcutaneous plus intratumoral rvv-IL-2 vaccination. Values are Mean ± SE. Apoptotic indices were significantly higher (P < 0.0001) in tumors from mice immunized with rvv-IL-2 vaccine compared to those treated with rvv-lacZ vaccine or those mock vaccinated with PBS. Apoptotic index was higher in the group treated with subcutaneous plus intratumoral rvv-IL-2 vaccination (P < 0.003) compared to the group treated by intratumoral rvv-IL-2 vaccine. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Immunohistochemical detection of tumor-infiltrating immune cells in vaccinated mice. Fresh frozen tumor sections (5 μm) were stained with anti-mouse antibodies specific for CD4+ (left) and CD8+ (middle) lymphocytes and macrophages (right) as described under Materials and Methods. Arrows indicate the immune cells in tumor beds. (A) Vaccinated with PBS; (B) intratumoral rvv-IL-2 vaccination; (C) intratumoral vaccination with rvv-IL-2 plus subcutaneous vaccination with irradiated and rvv-IL-2-infected SCC VII/SF cells. Original magnification ×400. The numbers of CD4+ T cells (P < 0.0001) and CD8+ T cells (P < 0.003) as well as of macrophages (P < 0.0001) were significantly higher in mice treated with subcutaneous plus intratumoral rvv-IL-2 vaccine compared to mice treated with intratumoral rvv-IL-2 alone. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 8 Distribution of macrophages in the spleen and tumor-draining lymph nodes of control and IL-2-vaccinated mice. Immune cells from spleen and regional lymph nodes were collected on day 9 and stained with PE-conjugated antibody for macrophage (Mac3). Isotype-matched PE-conjugated antibody (Iso Mac3) was used as negative control in the flow cytometric analysis. The positive staining is shown in the corresponding right graph for each group. Quadrants are set according to the baseline signal given by the control antibody. (A) Vaccinated with PBS; (B) subcutaneous injection with irradiated and rvv-LacZ-infected SCC VII cells plus intratumoral rvv-LacZ vaccination, (C) intratumoral vaccination with rvv-IL-2; (D) subcutaneous vaccination with irradiated and rvv-IL-2-infected SCC VII cells plus intratumoral rvv-IL-2 vaccination. Molecular Therapy 2003 8, 238-248DOI: (10.1016/S1525-0016(03)00148-5) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions