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 replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts 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. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was 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%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.
Grace
M J
MJ
Department of Biotechnology, Schering-Plough Research Institute, Union, New Jersey 07083, USA. michael.grace@spcorp.com
Xie
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Musco
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Cui
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Gurnani
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DiGiacomo
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Chang
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Indelicato
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Syed
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Johnson
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Nielsen
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D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D001706:Biopsy; D002467:Cell Nucleus / Q000648:ultrastructure; D005260:Female; D016158:Genes, p53 / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007150:Immunohistochemistry; D007274:Injections, Intraperitoneal; D007834:Lasers; D008099:Liver / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology* / Q000628:therapy*; D017239:Paclitaxel / Q000494:pharmacology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D016159:Tumor Suppressor Protein p53; D001616:beta-Galactosidase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D017239:Paclitaxel