Suppression of Tumor Growth Following Intralesional Therapy with TRAIL Recombinant Adenovirus  Thomas S. Griffith, Elizabeth L. Broghammer  Molecular Therapy  Volume 4, Issue 3, Pages 257-266 (September 2001) DOI: 10.1006/mthe.2001.0439 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Death of ALVA 31 cells, but not normal prostate epithelial cells (PrEC), after Ad5-TRAIL infection results from increased production of TRAIL protein. Microtiter plates were seeded with 2 × 104 ALVA 31 (A) or PrEC (B) cells/well and allowed to adhere for at least 6 h before infection with Ad5-TRAIL or Ad5-βgal at the indicated number of pfu/cell for 4 h. Cells were washed with PBS, and then incubated with medium alone or medium containing recombinant TRAIL (rTRAIL) at the indicated concentrations. Cell viability was determined after 20 h by crystal violet staining. Each value represents the mean of three wells. For clarity, S.D. bars were omitted, but were < 5% for all data points. Similar results were observed in three independent experiments. (C) Production of TRAIL protein in ALVA 31 cells following Ad5-TRAIL infection induces apoptosis. We infected 24-well plates containing 5 × 105 cells/well with Ad5-TRAIL (1000 pfu/cell) for 4 h, prepared cell lysates at the indicated times after infection, and determined TRAIL protein levels, caspase-8 activation, and PARP cleavage by western blot analysis. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Ad5-TRAIL local therapy significantly suppresses tumor xenograft outgrowth. Male CB.17 SCID mice were injected subcutaneously with 106 ALVA 31 (A) or 5 × 106 RT-4 (B) cells on day 0. Animals were then given an injection of PBS, Ad5-βgal (109 pfu), or Ad5-TRAIL (109 pfu) at the site of tumor implantation on day 1. Tumor size (mm2) was monitored over time, and the number of tumor-bearing animals per number of total animals in the group is shown. For both tumor types, animals treated with Ad5-TRAIL had significantly smaller (P ≤ 0.05) tumors than either the PBS- or Ad5-βgal-treated animals. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Ad5-TRAIL therapy does not select for TRAIL- or adenovirus-resistant tumor cells. Tumors were excised from individual mice from each group in Fig. 2A, minced, and placed into culture for 5 d. (A) ALVA 31 cells cultured from tumors or parental ALVA 31 cells were labeled with 51Cr and then added to each well (104 cells/well) of microtiter plates containing the indicated concentrations of rTRAIL. Culture supernatants were collected after 8 h and analyzed for released 51Cr. Each value represents the mean of three wells. For clarity, standard error bars were omitted from the graph, but were < 5% for all data points. (B) ALVA 31 cells cultured from tumors or parental ALVA 31 cells were seeded in 24-well plates (105 cells/well) and allowed to attach for at least 6 h before infecting with Ad5-GFP (1000 pfu/cell for 4 h). The number of Ad5-GFP-infected cells was determined after 24 h by flow cytometry, with open histograms representing uninfected cells and filled histograms representing Ad5-GFP-infected cells. The number indicates the percentage of GFP-positive cells. All histograms represent 104 gated cells, and viability was > 95% as assessed by propidium iodide exclusion. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Ad5-TRAIL therapy does not select for TRAIL- or adenovirus-resistant tumor cells. Tumors were excised from individual mice from each group in Fig. 2A, minced, and placed into culture for 5 d. (A) ALVA 31 cells cultured from tumors or parental ALVA 31 cells were labeled with 51Cr and then added to each well (104 cells/well) of microtiter plates containing the indicated concentrations of rTRAIL. Culture supernatants were collected after 8 h and analyzed for released 51Cr. Each value represents the mean of three wells. For clarity, standard error bars were omitted from the graph, but were < 5% for all data points. (B) ALVA 31 cells cultured from tumors or parental ALVA 31 cells were seeded in 24-well plates (105 cells/well) and allowed to attach for at least 6 h before infecting with Ad5-GFP (1000 pfu/cell for 4 h). The number of Ad5-GFP-infected cells was determined after 24 h by flow cytometry, with open histograms representing uninfected cells and filled histograms representing Ad5-GFP-infected cells. The number indicates the percentage of GFP-positive cells. All histograms represent 104 gated cells, and viability was > 95% as assessed by propidium iodide exclusion. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Ad5-TRAIL therapy significantly suppresses the growth of established tumors. Male CB.17 SCID mice were injected subcutaneously with 106 ALVA 31 cells on day 0. Animals were then given an injection of PBS, Ad5-βgal (109 pfu), or Ad5-TRAIL (109 pfu) at the site of tumor implantation on day 1 or day 10. Tumor size (mm2) was monitored over time. Animals treated with Ad5-TRAIL at day 1 or day 10 had significantly smaller (P < 0.05) tumors than either PBS- or Ad5-βgal-treated animals. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Kinetics of recombinant adenovirus transgene expression in vivo. (A) Fourteen-day-old subcutaneous ALVA 31 tumors, approximately 70 mm2 in size, were injected with 109 pfu Ad5-luc, and then harvested at various times after infection and assayed for luciferase activity. (B) Ten-day-old subcutaneous ALVA 31 tumors, approximately 40 mm2 in size, were injected with 109 pfu Ad5-TRAIL, and then harvested at various times after infection and homogenized. The anti-TRAIL monoclonal antibody M181 and protein G-Sepharose were used to precipitate TRAIL protein in the homogenates. Precipitates were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with an anti-TRAIL antiserum and anti-rabbit-HRP monoclonal antibody. (C) Multiple Ad5-TRAIL injections suppress established tumor growth greater than single injections. Male CB.17 SCID mice were injected subcutaneously with 106 ALVA 31 cells on day 0. Animals were then given a single injection of PBS, Ad5-βgal (109 pfu), or Ad5-TRAIL (109 pfu) at the site of tumor implantation on day 1 or day 10, or injections on day 10 and 17, and tumor size (mm2) was monitored over time. Ad injections at day 10 and 17 were given after the tumor measurements were taken. Animals treated with Ad5-TRAIL had significantly smaller (P < 0.05) tumors than either the PBS- or Ad5-βgal-treated animals for all treatment groups. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Kinetics of recombinant adenovirus transgene expression in vivo. (A) Fourteen-day-old subcutaneous ALVA 31 tumors, approximately 70 mm2 in size, were injected with 109 pfu Ad5-luc, and then harvested at various times after infection and assayed for luciferase activity. (B) Ten-day-old subcutaneous ALVA 31 tumors, approximately 40 mm2 in size, were injected with 109 pfu Ad5-TRAIL, and then harvested at various times after infection and homogenized. The anti-TRAIL monoclonal antibody M181 and protein G-Sepharose were used to precipitate TRAIL protein in the homogenates. Precipitates were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with an anti-TRAIL antiserum and anti-rabbit-HRP monoclonal antibody. (C) Multiple Ad5-TRAIL injections suppress established tumor growth greater than single injections. Male CB.17 SCID mice were injected subcutaneously with 106 ALVA 31 cells on day 0. Animals were then given a single injection of PBS, Ad5-βgal (109 pfu), or Ad5-TRAIL (109 pfu) at the site of tumor implantation on day 1 or day 10, or injections on day 10 and 17, and tumor size (mm2) was monitored over time. Ad injections at day 10 and 17 were given after the tumor measurements were taken. Animals treated with Ad5-TRAIL had significantly smaller (P < 0.05) tumors than either the PBS- or Ad5-βgal-treated animals for all treatment groups. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Ad5-TRAIL-treated tumors exhibit areas of apoptotic death. Male CB.17 SCID mice were injected subcutaneously (s.c.) with 106 ALVA 31 cells and allowed to grow for 7 d. PBS (A), Ad5-βgal (109 pfu; B), or Ad5-TRAIL (109 pfu; C) were injected into the tumor, and the tumor was excised 24 h later and processed for histologic examination. Numerous pycnotic nuclei are evident in the Ad5-TRAIL-treated tumor, which is consistent with cellular death by apoptosis. Molecular Therapy 2001 4, 257-266DOI: (10.1006/mthe.2001.0439) Copyright © 2001 American Society for Gene Therapy Terms and Conditions