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Activation of iCaspase-9 in Neovessels Inhibits Oral Tumor Progression

¹ Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences, and Endodontics, and
² Department of Oral and Maxillofacial Surgery, University of Michigan School of Dentistry, 1011 N. University, Rm. 2309, Ann Arbor, MI 48109-1078, USA; and
³ Center for Molecular Imaging, Department of Radiology, University of Michigan School of Medicine, Ann Arbor

View larger version (100K): [in a new window]   Figure 1. Activation of iCaspase-9 in neovascular endothelial cells is sufficient to decrease tumor microvessel density. (A) Graph depicting the number of microvessels in tumors populated with OSCC-3 (oral squamous cell carcinoma cells) and stably transduced HDMEC-iCasp-9 or control cells (HDMEC-LXSN) retrieved from mice treated with 3 consecutive daily intraperitoneal injections of 2 mg/kg AP20187. Each mouse received one scaffold seeded with HDMEC-iCasp-9 + OSCC-3, and one control scaffold seeded with HDMEC-LXSN + OSCC-3. We evaluated 5 tumors from 5 independent mice per condition, and the data presented in the graph represent mean values (± SD) of 10 microscopic fields per tumor. Statistical significance (asterisk) was determined at p 0.05, with the microvessel density for the HDMEC-LXSN group used as control. (B,C) Photomicrographs of representative histological sections depicting immunochemistry with Factor VIII antibody to identify blood vessels (arrows). Tumor microvessels are relatively small and can be identified only by immunohistochemistry, presumably due to the rapid growth of xenografted OSCC-3 tumors (scale bar, 50 µm for B-C).

View larger version (80K): [in a new window]   Figure 2. Characterization of luciferase expressing squamous cell carcinoma cells by in vivo bioluminescence. (A) Luciferase activity of two clones of UM-SSC-17B-luc and controls, i.e., UM-SCC-17B-neo (transduced with empty vector), or substrate only without cells (-). Cells were seeded in six-well plates (50,000 cells/well, triplicate wells/condition) and cultured for 48 hrs. Statistical significance (asterisk) was determined at p 0.05, with the luciferase activity obtained for the UM-SCC-17B-neo cells used as control. (B) Luciferase activity of 0-100,000 UM-SSC-17B-luc (M2) cells cultured for 48 hrs in six-well plates (triplicate wells/condition), demonstrating that the intensity of bioluminescence in vitro is directly correlated to the number of cells. Statistical significance was determined at p 0.05, with the baseline luciferase activity obtained for substrate only (no cells) used as control. (C,D) Determination of saturation times for in vivo bioluminescence imaging of 1 representative mouse (N = 1) that received 1 scaffold containing HDMEC-iCasp-9 and OSCC3-luc (lower, righthand side), and 1 scaffold containing HDMEC-LXSN and OSCC3-luc (upper, lefthand side). This mouse was treated with 2 mg/kg AP20187 for 3 consecutive days. Images were acquired 1–16 min post-injection of luciferin. The graph presented in panel (C) depicts a time-course for bioluminescence intensity after injection of luciferin in the mouse depicted in panel (D). (E) In vivo bioluminescence imaging of 1 representative mouse (N = 1) bearing a xenografted tumor (HDMEC-LXSN and OSCC-3-luc) from day 14 through day 35 post-implantation, for evaluation of luciferase expression over time.

View larger version (83K): [in a new window]   Figure 3. In vivo bioluminescence analysis of the effect of iCaspase-9 activation on tumor progression. (A) Representative images of mice injected with either Ad-iCaspase-9 (no promoter) or Ad-VEGFR2-iCaspase-9 (with endothelial cell-specific promoter), and treated with either the dimerizer drug AP20187 (2 mg/kg) or the negative control phosphate-buffered saline (PBS) solution. In vivo bioluminescence imaging was performed 3 days after tumor implantation (baseline), 20 days post-implantation (day of adenovirus injection), 21 days post-implantation (just prior to treatment with AP20187), and 25 days post-implantation (just prior to death). (B–E) Each graph depicts, presented in the left side of this Fig. (panel A), the quantification of bioluminescence over time for the corresponding mouse (N = 1).

View larger version (31K): [in a new window]   Figure 4. Tumor progression is inhibited by intratumoral injection of the adenovirus Ad-VEGFR2-iCasp9 and administration of AP20187. (A) Graph depicting bioluminescence values (± SD) of mice bearing tumors (UM-SCC-17B-luc cells) injected with either Ad-VEGFR2-iCasp-9 or Ad-iCasp-9 (no promoter control), and treated with either 2 mg/kg AP20187 or negative control phosphate-buffered saline (PBS). Statistical significance (asterisk) was determined at p 0.05, with the bioluminescence values obtained for the Ad-iCasp-9 + PBS group in each individual time point used as controls. (B) Graph demonstrating the percent change in bioluminescence from day 21 (beginning of treatment with AP20187) to day 25 (death). Statistical significance was determined at p 0.05, with the bioluminescence values obtained for the Ad-VEGFR2-iCasp-9 + PBS group as control. We evaluated 5 tumors per condition from 5 independent mice, and the data presented in the graphs (panels A and B) represent mean values (± SD) of 10 microscopic fields per tumor. (C,D) Photomicrographs of representative histological sections after immunochemistry with Factor VIII antibody to identify tumor blood vessels (arrows). Tumors (UM-SCC-17B-luc) were retrieved from mice that received Ad-VEGFR2-iCaspase-9, and were treated with either PBS (C) or 2 mg/kg AP20187 (D) (scale bar, 50 µm for C–D).