The Use of Laser Scanning Cytometry to Assess Depth of Penetration of Adenovirus p53 Gene Therapy in Human Xenograft Biopsies
1999; Elsevier BV; Volume: 155; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65506-x
ISSN1525-2191
AutoresMichael J. Grace, Lei Xie, Mary Lynn Musco, Shijun Cui, Maya Gurnani, Ruth DiGiacomo, Alice Chang, Stephen R. Indelicato, Jameel Syed, Robert C. Johnson, Loretta L. Nielsen,
Tópico(s)Molecular Biology Techniques and Applications
ResumoSCH58500 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 p53null SK-OV-3 human ovarian tumor cells or p53mut 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 β-galactosidase (Ad5/β-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 β-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/β-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 β-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. 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 p53null SK-OV-3 human ovarian tumor cells or p53mut 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 β-galactosidase (Ad5/β-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 β-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/β-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 β-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. p53 is a tumor suppressor gene frequently mutated in many human neoplasms.1Harris C Hollstein M Clinical implications of the p53 tumor-suppresser gene.N Engl J Med. 1993; 329: 1318-1327Crossref PubMed Scopus (1362) Google Scholar The cellular roles of p53 include activation of genes that inhibit cell cycle progression, promotion of DNA repair, and induction of programmed cell death (apoptosis).2Lane D Lu X Hupp T Hall P The role of p53 in the apoptotic response.Phil Trans R Soc London Ser. B. 1994; 345: 277-280Crossref PubMed Scopus (65) Google Scholar The introduction of wild-type p53 into transformed cells of a p53null or p53mut genotype is incompatible with the maintenance of a tumorigenic phenotype, usually inducing apoptosis (for review, see 3Nielsen L Maneval D p53 tumor suppressor gene therapy for cancer.Cancer Gene Ther. 1998; 5: 52-63PubMed Google Scholar). However, a key issue in the introduction of wild-type p53 genes into neoplastic cells is the delivery vehicle or vector. One emerging approach is to deliver the gene with a type 5 adenoviral vector (Ad5/p53).4Berkner K Development of adenovirus vectors for the expression of heterologous genes.BioTechniques. 1998; 6: 616-624Google Scholar To date, Ad5/p53 vectors have been used for a wide variety of preclinical proof-of-concept studies in the gene therapy of cancer,3Nielsen L Maneval D p53 tumor suppressor gene therapy for cancer.Cancer Gene Ther. 1998; 5: 52-63PubMed Google Scholar and ongoing phase I clinical trials support their safety in human cancer patients.5Schuler M Rochlitz C Horowitz J Schlegel J Perruchoud A Kommoss F Bollinger C Kauczor HU Dalquen P Fritz MA Swanson S Herrmann R Huber C A phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer.Human Gene Ther. 1998; 9: 2075-2082Crossref PubMed Scopus (155) Google Scholar, 6Nielsen L Pegram M Karlan B Elkas J Horowitz J Opportunities for p53 tumor suppressor gene therapy in ovarian and other peritoneal cancers.in: Seth P Adenoviruses: Basic Biology to Gene Therapy. R.G. Landes, Austin, TX1999: 295-303Google Scholar A natural question arising from these studies concerns the efficiency of gene delivery, to provide guidance for the design of clinical protocols. For ovarian cancer, it becomes critical to determine the depth of adenovirus drug penetration into tumor nodules dispersed throughout the peritoneal cavity after single and multiple doses. Intraperitoneal human tumor xenograft models with SK-OV-3 ovarian cells (p53null) or DU-145 prostate cells (p53mut) were used to study this issue. Tissue was analyzed for apoptosis with a new fluorescence laser scanning cytometry (LSC) technology to perform both automated quantitative analysis and positional mapping of apoptotic architecture within thin-tissue sections. This new assay was validated using the more traditional technique of immunohistochemistry (IHC) for β-galactosidase, p53, and the p53-induced protein, p21. LSC is a slide-based fluorescence analytical method analogous to flow cytometry. Thus, extensive quantitation of cellular or nuclear events is possible using LSC analysis.7Martin-Reay D Kamentsky L Weinberg D Hollister K Cibas E Evaluation of a new slide-based laser scanning cytometer for DNA analysis of tumors: Comparison with flow cytometry and image analysis.Am J Clin Pathol. 1994; 102: 432-438PubMed Google Scholar, 8Kamentsky L Burger D Gershman R Kamentsky L Luther E Slide-based laser scanning cytometry.Acta Cytol. 1997; 41: 123-143Crossref PubMed Scopus (165) Google Scholar In contrast to flow cytometry, the position of each fluorescent event is recorded as it is scanned on the slide, and electronic bit-map images of the scan are created. As a result, bit maps of scanned thin-tissue sections reveal the architectural context in which the fluorescent event has occurred. In tissue, LSC analysis is particularly useful for the measurement of nuclear-associated events. LSC has been used for the analysis of DNA content9Sasaki K Kurose A Miura Y Sato T Ikeda E DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry.Cytometry. 1996; 23: 106-109Crossref PubMed Scopus (74) Google Scholar, 10Kamada T Sasaki K Tsuji T Todoroki T Takahashi M Kurose A Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.Cytometry. 1997; 2: 290-294Crossref Scopus (29) Google Scholar, 11Gorczyca W Darzynkiewicz Z Melamed M Laser scanning cytometry in pathology of solid tumors: A review.Acta Cytol. 1997; 41: 98-108PubMed Google Scholar and with the immunophenotyping of malignant human biopsy tissue sections.12Clatch R Walloch J Zutter M Kamentsky L Immunophenotypic analysis of hematologic malignancy by laser scanning cytometry.Am J Clin Pathol. 1996; 105: 744-755PubMed Google Scholar, 13Clatch R Walloch J Multiparameter immunophenotypic analysis of fine needle aspiration biopsies and other hematologic specimens by laser scanning cytometry.Acta Cytol. 1997; 41: 109-122PubMed Google Scholar Recent reports have also demonstrated the use of LSC for the measurement of nuclear-associated proteins, nuclear cyclin B1 expression,14Gorczyca W Sarode V Melamed M Darzynkiewicz Z Laser scanning cytometric analysis of cyclin B1 in primary human malignancies.Mod Pathol. 1997; 10: 457-462PubMed Google Scholar p53,15Musco M Cui S Small D Nodelman M Sugarman B Grace M Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.Cytometry. 1998; 33: 290-296Crossref PubMed Scopus (42) Google Scholar and NF-κB.16Deptala A Bedner E Gorczyca W Darzynkiewicz Z Activation of nuclear factor kappa B (NF-κB) assayed by laser scanning cytometry (LSC).Cytometry. 1998; 33: 376-382Crossref PubMed Scopus (99) Google Scholar We have developed an LSC-based method for scanning 4 to 6-μm tissue sections for nuclear-associated apoptotic events, using terminal deoxynucleotidyltransferase (TdT) directed nick-end labeling (TUNEL) of fragmented DNA. Using this method, we analyzed human tumor xenografts and murine liver tissue from SCID mice treated with Ad5/p53 vector, Ad5/β-gal (Ad control) vector, or vehicle (buffer control) to quantitate and map the induction of apoptosis, and we correlated these results with IHC detection of p53 protein and p21 protein expression as a measure of vector penetration. It is likely that phase II/III clinical trials will incorporate an arm comparing traditional chemotherapy with chemotherapy combined with p53 gene therapy. Therefore, it is important to study possible interactions between p53 adenovirus and chemotherapeutic drugs in preclinical models before entering the clinic. Due to the clinical importance of paclitaxel (taxol) in treating ovarian cancer in solid tumors and our previous observation that paclitaxel enhances adenovirus type 5 (Ad5. transduction efficiency,17Nielsen L Lipari P Dell J Gurnani M Hajian G Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.Clin Cancer Res. 1998; 4: 835-846PubMed Google Scholar we decided to examine the effect of paclitaxel on adenovirus drug penetration after intraperitoneal dosing. The SK-OV-3 and DU-145 cell lines were obtained from American Type Culture Collection (Manassas, VA). SK-OV-3 human ovarian and DU-145 human prostate tumor cell lines were cultured in Eagle's minimal essential medium (MEM) with nonessential amino acids and Earle's balanced salt solution plus 10% fetal calf serum at 37°C and 5. CO2. SCH585000, an E-1-deleted adenovirus vector (Ad/p53), was constructed using the large fragment from dl327 and a plasmid containing the 1.4-kb full-length p53 cDNA with expression driven from the human cytomegalovirus promoter.18Wills K Maneval D Menzel P Harris M Sutjipto S Vaillancourt M Huang W Johnson D Anderson S Wen S Bookstein R Shepard H Gregory R Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer.Human Gene Ther. 1994; 5: 1079-1088Crossref PubMed Scopus (237) Google Scholar Recombinant virions were produced and purified as previously described.19Huyghe B Liu X Sutjipto S Sugarman B Horn M Shepard H Scandella C Shabram P Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography.Human Gene Ther. 1995; 6: 1403-1416Crossref PubMed Scopus (179) Google Scholar Viral particle concentrations were determined using anion-exchange high-pressure liquid chromatography20Shabram P Giroux D Gouldreau A Gregory R Horn M Huyghe B Liu X Nunnally M Sugarman B Sutjipto S Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles.Human Gene Ther. 1997; 8: 453-465Crossref PubMed Scopus (171) Google Scholar and A260nm measurement in 0.1% sodium dodecyl sulfate (w/v).21Maizel J White D Scharff M The polypeptides of adenovirus 1: evidence for multiple protein components in the virion and a comparison of types 2, 7a, and 12.Virology. 1968; 36: 115-125Crossref PubMed Scopus (611) Google Scholar SCH58000 was provided by Schering-Plough Biotechnology (Union, NJ). The β-galactosidase adenovirus vector construct was described previously18Wills K Maneval D Menzel P Harris M Sutjipto S Vaillancourt M Huang W Johnson D Anderson S Wen S Bookstein R Shepard H Gregory R Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer.Human Gene Ther. 1994; 5: 1079-1088Crossref PubMed Scopus (237) Google Scholar and was provided by Canji (San Diego, CA). Viral-construct infectivity was confirmed using a flow cytometry-based adenovirus infection assay.15Musco M Cui S Small D Nodelman M Sugarman B Grace M Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.Cytometry. 1998; 33: 290-296Crossref PubMed Scopus (42) Google Scholar The SK-OV-3 and DU-145 tumor xenograft models in SCID mice have been described previously.17Nielsen L Lipari P Dell J Gurnani M Hajian G Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.Clin Cancer Res. 1998; 4: 835-846PubMed Google Scholar Briefly, female SCID mice were injected intraperitoneally (i.p.) with either 1 × 106 SK-OV-3 ovarian tumor cells or 2.5 × 106 DU-145 prostate tumor cells on day 0. Tumors were allowed to establish for 3 to 4 weeks. For treatment, groups of n = 5 mice received adenovirus constructs administered i.p. in Ad control buffer (20 mmol/L NaH2PO4 pH 8.0; 130 mmol/L NaCl2; 2 mmol/L MgCl2; 2% sucrose). After sacrifice, tumor nodules were excised for analysis. Excised tumor nodules were uniformly small to medium sized. Three experiments were performed to evaluate adenovirus vector penetration. The first experiment evaluated the depth of penetration of an Ad5/β-gal construct in SK-OV-3 and DU-145 tumor-bearing mice. Each treatment dose of Ad5/β-gal contained 1 × 1010 viral particles. Tumor tissue was analyzed for β-galactosidase activity using IHC (Figure 1). In a second experiment, SK-OV-3 tumor-bearing SCID mice were treated i.p. with Ad buffer, Ad5/β-gal, or Ad5/p53 as either a single bolus or two consecutive doses 24 hours apart. Each dose of adenovirus construct contained 2.9 × 1010 viral particles. In a third experiment, SK-OV-3 tumor-bearing SCID mice received 10 mg/kg paclitaxel with the first bolus dose of buffer or Ad5/p53. In this experiment, the first dose contained 1 × 1010 virus particles of Ad5/p53; the second dose contained 2 × 1010 virus particles. Twenty-four hours after the last adenovirus dose, mice were sacrificed and tissues harvested for analysis. C.B.17/ICR-SCID mice were purchased from Taconic Farms (Germantown, NY). All mice were maintained in a VAF-barrier facility. Animal procedures were performed in accordance with the rules set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Schering-Plough Research Institute Animal Care and Use Committee. Paclitaxel was purchased from CalBiochem (San Diego, CA). For in vivo experiments, paclitaxel was dissolved in 1:1 absolute ethanol and Cremophor EL (Sigma Chemical Co., St. Louis, MO), then diluted 1:10 into 0.9. saline immediately before intraperitoneal injection. Excised tissue samples were either snap-frozen or fixed in 10. buffered formalin and processed overnight in a Miles VIP Tissue Processor (SAKura Finetek, Torrance, CA), then embedded in paraffin. Snap-frozen tissues were embedded using Tris-buffered saline medium (Triangle Biomedical Science, Durham, NC) and cut into 4- to 6-μm sections with a Microm HM505N cryostat (Carl Zeiss, Waldorf, Germany). paraffin-embedded tissues were cut into 5-μm sections with a Leitz model 1512 microtomed. Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% aqueous hydrogen peroxide for 15 minutes. Immunohistochemical staining was performed at 37°C, using a Ventana Immunostainer ES (Ventana Medical Systems, Tucson, AZ). Tissues were enzyme digested using protease 1 treatment for 4 to 8 minutes. For the detection of β-galactosidase activity, a 1:100 dilution of antibody for rabbit anti-β-galactosidase (5 Prime-3 Prime, Boulder, CO) was used. The antibody was incubated for 20 to 30 minutes at 37°C. For negative controls, primary antibody was substituted with nonimmune rabbit or mouse immunoglobulins (Vector Laboratories, Burlingame, CA) diluted to match the primary antibody protein concentration. The Ventana DAB detection system was used to detect specific antibody binding. The slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped using Permount (Fisher Scientific). Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated. Sections were treated with ethanol:acetic acid (2:1) at −20°C for 10 minutes, and antigens were retrieved by microwaving in 0.01 mol/L citric acid buffer (pH 6.0) for 10 minutes. Endogenous peroxidase activity was quenched by incubating the slides with 1.5. hydrogen peroxide in cold ethanol for 10 minutes. The slides were then blocked for nonspecific binding, using normal rabbit serum (NovoCastra, Newcastle, UK). The primary antibodies used were mouse anti-human p53 (NovoCastra) and mouse anti-human p21 (PharMingen, San Diego, CA). Both primary antibodies were incubated on slides for 1 hour at 23°C. For negative controls, primary antibodies were substituted with nonimmune mouse immunoglobulin (PharMingen) diluted to match the primary antibody protein concentration. Slides were then incubated for 30 minutes at 23°C, using biotinylated rabbit anti-mouse antibody diluted 1:500 in Dulbecco's modified phosphate buffered saline (DPBS; NovoCastra). Specific antibody binding was detected using ABC reagent (avidin/biotinylated horseradish peroxidase; NovoCastra) combined with diaminobenzadine (DAB) chromogen (Vector) for color development. The slides were then counterstained with hematoxylin (Sigma), washed in water once, washed in 95% ethanol three times for 30 seconds per wash, washed twice for 1 minute per wash with 100% ethanol, and then washed twice for 1 minute per wash, using Clear Rite (Richard-Allen, Kalamazoo, MI). Coverslips were added to the slides with Permount (Fisher Scientific, Pittsburgh, PA). Sections from snap-frozen SK-OV-3 tumor tissue and murine liver tissue were used for the first experiment. Sections from formalin-fixed, paraffin-embedded SK-OV-3 tumor tissue and murine liver tissue were used for the second experiment. Serial sections of 4 to 6 μm were used. Frozen tissue sections were pre-fixed by incubation in a solution of 10% buffered formalin at 4°C for 10 minutes and washed in DPBS. Prefixed frozen and formalin-fixed deparaffinized tissue sections were fixed by incubation in a solution of ethanol:acetic acid (2:1) at 20°C for 10 minutes and then washing for 5 minutes in DPBS. Apoptosis was detected with TUNEL and fluorescein-labeled dUTP (Boehringer-Mannheim, Indianapolis, IN) per kit instructions. For each set of serial sections, a TdT-negative control slide was used to set background nonspecific binding of fluorescein-dUTP. After TUNEL labeling, slides were incubated twice at 37°C for 10 minutes each, using TNT containing 1% bovine serum albumin. Slides were stained for nuclear localization by incubation at 23°C for 30 minutes, using 0.001% propidium iodide solution in DPBS-ethylenediaminetetraacetic acid/Triton X-100 solution containing a 1:7 dilution of RNase (Sigma). Slides were washed twice with DPBS and mounted using anti-fade medium. LSC analysis was performed using the CompuCyte (Cambridge, MA) brand LSC with analysis by WinCyte 2.1 PC-based software. A detailed description of LSC methodology has been previously published.8Kamentsky L Burger D Gershman R Kamentsky L Luther E Slide-based laser scanning cytometry.Acta Cytol. 1997; 41: 123-143Crossref PubMed Scopus (165) Google Scholar, 15Musco M Cui S Small D Nodelman M Sugarman B Grace M Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.Cytometry. 1998; 33: 290-296Crossref PubMed Scopus (42) Google Scholar, 22Luther E Kamentsky L Resolution of mitotic cells using laser scanning microscopy.Cytometry. 1996; 23: 272-278Crossref PubMed Scopus (74) Google Scholar The desired area of analysis was located visually using epifluorescent visual microscopy on the instrument, and the scan areas were set. Slides were scanned using a 20× objective and an Ar laser operating at 5 mW and using a 488-nm line. The focal plane was adjusted to the rear charged-coupled device camera, ie, the plane of the laser line. To avoid detector saturation, fluorescein and propidium iodide detector gain voltages were set in the LSC menu so that a maximum of 75% saturation was achieved for the brightest maximum pixel (max pixel) events scanned. Individual nuclei were detected using the orange- to long-red filter/detector cube configuration. Individual nuclei were contoured using long-red fluorescence and not exceeding a 100-pixel minimum-area threshold, centered on a scanned max pixel event. In general, this method contoured about 50% of a typical cell from the nucleus out. Contouring extensively outside the nuclear area, ie, greater than 67% of the cell, was kept to a minimum to avoid the contouring of multiple nuclei as a single event. Area versus max pixel and CompuSort (CompuCyte) relocation was used to help discriminate inadvertent multiple-nuclei contouring. TUNEL-positive events were detected using the fluorescein filter/detector configuration. The strategy for quantitative analysis was to analyze a single representative tumor and a single representative liver section for each mouse in a treatment group. For the Ad5/p53 study, tissues from four mice per group were used; in the Ad5/p53+ paclitaxel study, tissues from five mice per group were used. For LSC studies, only tissue from the SK-OV-3 human tumor xenograft model was used. For each tumor or liver section, at least four serial sections were made for analysis, with sections about 10 μm apart. The first section was used as the TUNEL-nonspecific binding (NSB) control in which the enzyme TdT was left out, but the fluorescein-dUTP label was included. This control section was used to establish the background green fluorescence within the scanned tissue. The analytical gate to be used to define a positive apoptotic event was then set at between 0.5 and 1.0 log-order above the mean max pixel for the TUNEL-NSB-scanned population. After analysis of the control section, the remaining serial sections for each tissue were analyzed for quantitation. TUNEL-positive nuclei and total nuclei scanned for all serial scans of a single tissue were summed, and a total percentage of TUNEL-positive nuclei was calculated for the single tissue. Weight-average percentages were used to calculate the group mean and percentage coefficient of variance. Statistical analysis was performed on the weight-average percentages by the single-tailed Student's t-test. From each scan an x-y positional bit-map of total nuclei was generated to image the tissue; TUNEL-positive nuclei were then false-color-imaged with Adobe Photoshop (Adobe Systems, San Jose, CA) and overlaid onto the total nuclei to create a comprehensive bit map showing the localization of apoptosis within the tissue analyzed. Nuclei with green fluorescence more than 0.5 log above the TdT-negative control were false colored red on the bit maps to best contrast against nonapoptotic nuclei (light blue). Tumors from Ad5/β-gal-treated mice were first analyzed for vector penetration by β-galactosidase protein IHC. Figure 1 shows IHC staining (brown) for the presence of β-galactosidase protein in representative SK-OV-3 xenograft tissue (Figure 1, B and C) and DU-145 xenograft tissue (Figure 1D) from Ad5/β-gal-treated mice, but not from buffer control treatment (Figure 1A). The two-dimensional penetration of the β-gal adenovirus vector shows staining limited usually to the first 1 to 10 cells from the edge of the tumor. In some mice, the hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity (data not shown). Because the SK-OV-3 tumor cell is a p53null phenotype, we were able to perform IHC staining for the presence of human p53 protein to assess depth of penetration of the Ad5/p53 vector. As shown in Figure 2 (C and D), tumor sections from Ad5/p53-treated mice showed strong staining for p53 protein at the edge of the tumor section, with expression limited to about 10 cells in depth. p21 is a downstream p53-regulated protein involved in the progression of cell cycle. IHC staining of serial sections for p21 protein revealed very strong staining along the tumor edge, with the same depth of penetration as seen by β-galactosidase and p53 IHC (Figure 2, F and G). SK-OV-3 tumor cells are p21wt, and therefore light background staining for p21 was detected in central regions of the tumor. Simultaneous administration of paclitaxel did not change the depth of adenovirus particle penetration into tumor nodules for p53 protein expression (Figure 2E) or p21 protein expression (Figure 2H). LSC methodology was developed to detect apoptotic cell nuclei for quantification and to confirm depth of penetration of the functional endpoint (apoptosis) of p53 protein expression as a result of Ad5/p53 vector delivery. As described in Materials and Methods, individual cells within the tissue were contoured using a low concentration of propidium iodide to locate each nucleus in the orange–long-red wavelengths. In control experiments, we have determined that, at the gain settings used for the fluorescein (green) detector configuration, no spectral overlap from 488-nm excitation of propidium iodide is detected. Our first goal with LSC analysis was to correlate the fluorescence TUNEL assay with the IHC observations for p53 and p21 proteins. Additional tissue sections were also analyzed using IHC Apoptag (Oncor, Gaithersburg, MD) to correlate the LSC-based TUNEL assay (data not shown). We observed that apoptotic nuclei could be easily discriminated from nonapoptotic nuclei by fluorescein intensity and using an analytical gate set by the NSB control as described in Materials and Methods. In Figure 3, representative tissue scans of tumors taken from a buffer control-treated mouse (A), an Ad5/β-gal-treated mouse (B), and an Ad5/p53-treated mouse (C–E. demonstrate the depth of penetration for apoptosis. Ad5/p53 treatment resulted in significant fluorescence from nick-end labeling of DNA ends (red color dots) associated with nuclei located on the edge of the tumor (D), with penetration of between 1 and 10 cells into the tumor. Apoptotic nuclei were minimal, diffuse, and nonlocalized in tumor tissues taken from buffer control and Ad5/β-gal-treated mouse. Excised tumor nodules were small to medium sized, and no significant necrosis was observed. Serial sections from the same Ad5/p53-treated tumor are shown in Figure 3, C–E. Sections shown in panels C and E were taken from either end of the tumor, but were sectioned within 10 cell layers of the tum
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