The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies

SCH58500 is an agent for gene therapy of cancer, consisting of a replicatio n-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppress or gene (Ad5/p53). An important question about the use of Ad5/p53 gene ther apy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetrat ion of an Ad5/p53 vector by dosing the vector in an experimental human xeno graft/SCID model. To assess depth of penetration, we developed a novel meth odology for scanning tissue sections by laser scanning cytometry (LSC). SCI D mice were given intraperitoneal injections of either P53(null) SK-OV-3 hu man ovarian tumor cells orp53(mut) DU-145 human prostate tumor cells to est ablish xenograft solid tumors. Mice were then dosed once or twice at 24-hou r intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenov irus construct expressing P-galactosidase (Ad5/beta-gal), or a buffer contr ol. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53, Twenty-four h ours after each last dose, the human solid tumor xenograft and relevant mou se tissue were removed from each mouse for the analysis of Ad5/p53 penetrat ion, Immunohistochemistry CMC) for beta-galactosidase protein revealed a de pth of penetration of between 1 and 10 cells from the tumor surface. In som e mice, hepatocytes in the periportal regions of liver lobules were also po sitive, indicating systemic absorption of adenovirus from the peritoneal ca vity, IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenograf ts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver-tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LS C analysis demonstrated a high level of apoptosis in the tumors that had be en removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis wa s significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/ p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5 /beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%) Sca n bit maps from the extensive LSC analyses confirmed that apoptosis was pre sent to about the same depth (1-10 cells) as had been identified by MC for beta-galactosidase,: p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measu red by IHC for p53 or p21 protein. However, the combination therapy did cau se an elevation in the number of tumor cells undergoing apoptosis.