1. Volume 6, Issue 5, Pages 627-636 (November 2002)
Development of Anti-tumor Immunity against a Non-immunogenic Mammary Carcinoma through in Vivo Somatic GM-CSF, IL-2, and HSVtk Combination Gene Therapy 
Dirk G. Brockstedt, Melissa Diagana, Ying Zhang, Kimvan Tran, Nicole Belmar, Melinda Meier, Adrienne Yang, Florence Boissiere, Augustine Lin, Yawen Chiang 
Molecular Therapy 
Volume 6, Issue 5, Pages (November 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Transgene expression in 4T1 cells after in vitro or in vivo transduction with adenoviral vectors. (A) The 4T1 cells were transduced in vitro at various MOI (VP/cell) of AV-TK (solid bars) or AV-empty (striped bars) as a control and 24 hours later assayed for HSVtk expression by intracellular staining. (B, C) The 4T1 cells were transduced in vitro with AV-GM/IL2 at various MOI and transgene expression was determined by ELISA. (D–G) Palpable 4T1 tumors of 40 to 60 mm3 were injected with 1 × 1010 VP of AV-TK and 48 hours later tumors were harvested and in vivo HSVtk transgene expression was determined by immunohistochemical staining with an anti-TK antibody (brown staining). (D, E) A 4T1 tumor injected with 1 × 1010 VP of AV-empty. (F, G) A 4T1 tumor injected with 1 × 1010 VP of AV-TK. Magnification: (D, F) ×4; (E, G) ×20.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
IL-2 and GM-CSF gene therapy augments the anti-tumor effect of HSVtk suicide gene therapy. (A, B) BALB/c mice were inoculated with 2.5 ×105 4T1 cells. Each group consisted of 18 mice. Palpable 4T1 tumors were injected intratumorally with adenoviral gene combinations, followed by 10 days of GCV or saline administration. (A) Tumor growth of subcutaneous 4T1 tumors. (B) Survival of 4T1-bearing mice after adenoviral treatment. (C) Surgical removal of remaining tumor tissue after intratumoral treatment with AV-TK/GCV plus AV-GM/IL2 increases survival. BALB/c mice bearing palpable 4T1 tumors were treated with AV-empty or AV-TK plus AV-GM/IL2 followed by 10 days of GCV treatment. On day 12 after viral treatment the remaining tumor tissue was surgically removed in half of the animals in each group. Non-operated mice (open symbols) and operated mice (filled symbols) were sacrificed when any signs of stress or labored breathing were observed.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Decreased formation of lung metastases in mice treated with AV-TK/GCV + AV-GM/IL2. BALB/c mice were injected with 2.5 × 105 4T1 cells. Palpable tumors were injected with adenoviral combinations when tumors reached 40 to 60 mm3 followed by 10 days of GCV or saline treatment. On day 12 post viral treatment the remaining tumor tissue was surgically removed in all the groups. Thereafter, animals were observed for 15 more days for the development of metastases. Animals were sacrificed and lungs were fixed in formalin for histopathological studies. Histological examination was performed on hematoxylin and eosin stained sections. We obtained 5-μm sections every 200 to 250 μm in order to obtain six levels per lung. (A–E) Histology of lung sections. Each picture represents the worst case of each group. (A) Buffer with GCV. (B) AV-TK with saline. (C) AV-TK with GCV. (D) AV-TK plus AV-GM/IL2 with saline. (E) AV-TK plus AV-GM/IL2 with GCV. (F) Percentage of metastases-free mice; numbers represent the number of metastases-free mice over the total number of mice. (G) Mean surface of metastases in μm2; metastases area was determined from six areas for each lung in order to obtain an index of metastatic load.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Combined cytokine therapy with HSVtk/GCV protects mice from a subsequent tumor challenge. The 4T1 tumor-bearing BALB/c mice were treated with AV-TK/GCV plus AV-GM/IL2 when tumors reached 40 to 60 mm3. Each group consisted of 18 mice. On day 12 after viral treatment the remaining tumor tissue was removed. Four days later mice were challenged on the contralateral site with a tumorigenic dose (1 × 104 cells) of either 4T1 (gray bars) or Line01 (striped bars) cells as a control. Non-tumor-bearing mice (sham surgery or no surgery) served as control with 10 mice per group. Mice were monitored for tumor growth.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Depletion of CD8+ but not CD4+ T lymphocytes abrogates the AV-TK/GCV + AV-GM/IL2 anti-tumor effect. To determine the role of CD4+ and CD8+ T lymphocytes in the inhibition and growth of lung metastases, mice were injected intravenously with 200 μg of depleting anti-CD4, anti-CD8, or both anti-CD4 and anti-CD8 6 and 3 days before AV-TK + AV-GM/IL2 treatment on day 0. Control mice received 200 μl of PBS. All the groups were treated with AV-TK/GCV plus AV-GM/IL2 when tumors reached 40 to 60 mm3. Each group consisted of 20 mice. On day 12 post viral treatment the remaining tumor tissue was removed. Mice were sacrificed when any signs of stress or labored breathing were observed.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Combined cytokine therapy with HSVtk/GCV induces a systemic 4T1-specific cytotoxic T lymphocyte response. Splenocytes from three 4T1-bearing animals were collected 16 days post intratumoral viral administration. Mice were treated intratumorally with either AV-TK alone or AV-TK plus AV-GM/IL2 followed by 10 days of GCV treatment. A standard 4-hour Cr51-release assay was used to measure cytotoxic activity against 4T1 tumor cells (A) and YAC cells (B) used as target cells to determine the NK cell activity of the spleen cell culture. (C) The lytic activity of a 4T1-specific T cell line was assessed to 4T1, CT26, and Line01 tumor cells. (D) To assess the effector cell population as well as the MHC restriction, the 4T1-specific T-cell line was pre-incubated with antibodies and their cytotoxic activity assessed in a 4-hour 51Cr-release assay at an E:T ratio of 4:1 against 4T1 tumor cells.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Combined AV-TK/GCV plus AV-GM/IL2 local treatment induces local and systemic ovalbumin-specific cellular immune response. C57BL/6 mice were injected with 2.5 × 105 B16 MO tumor cells expressing ovalbumin. Palpable tumors of 40 to 60 mm3 were injected with AV-empty or AV-TK plus AV-GM/IL2 followed by 9 days of GCV treatment. On day 9 after viral treatment, tumors and spleens of treated mice were harvested and the number of ovalbumin-specific cells was determined in an ELISPOT assay (A, B). Cells were cultured with (filled bars) or without (striped bars) the MHC class I-restricted epitope of ovalbumin, SIINFEKL, at 100 ng/ml. (A) Tumor-infiltrating cells, enriched for CD45+ lymphocytes from B16 MO bearing mice. (B) Splenocytes from tumor-bearing mice (note smaller scale). (C, D) Splenocytes from three B16 MO tumor-bearing animals were collected 9 days post intratumoral viral administration and restimulated for 7 days. Cytotoxic activity of the spleen cell culture was measured against EL-4 cells pulsed with the MHC class I-restricted epitope of ovalbumin or unpulsed EL-4 cells in a standard 4-hour 51Cr-release assay. (C) AV-TK/GCV + AV-GM/IL2 treatment. (D) AV-empty/GCV treatment. (E) To assess whether the ovalbumin-specific cytotoxic activity is mediated by CD4+ or CD8+ T lymphocytes, spleen cells from B16 MO tumor-bearing mice were pre-incubated with antibodies to CD4 or CD8 and their cytotoxic activity assessed in a 4-hour 51Cr-release assay against SIINFEKL pulsed EL-4 cells.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions