Inhibition of Tumorigenicity and Metastasis of Human Bladder Cancer Growing in Athymic Mice by Interferon- Gene Therapy Results Partially from Various Antiangiogenic Effects Including Endothelial Cell Apoptosis
چکیده
We determined whether the IFNgene could suppress angiogenesis, tumor growth, and metastasis of human bladder transitional cell carcinoma. The highly tumorigenic and metastatic 253J B-V human bladder transitional cell carcinoma (TCC) cell line (resistant to the antiproliferative effects of IFN) was infected in vitro with adenoviral -galactosidase (Ad-LacZ), murine adenoviral IFN(Ad-mIFN), or human adenoviral IFN(Ad-hIFN) and implanted into the bladders of athymic nude mice. Ad-mIFNand Ad-hIFNwere used because of the species specificity of IFN. The transient production of mIFNand hIFNfrom the infected 253JB-V tumor cells significantly inhibited tumorigenicity and spontaneous lymph node metastasis. Subsequently, the 253J B-V cells were implanted into the subcutis of athymic nude mice, and established tumors were treated by direct intratumoral injection with AdmIFN, Ad-hIFN, Ad-LacZ, or PBS. By in situ hybridization (ISH) and immunohistochemical analysis (IHC), expression of hIFNand mIFNmRNA and protein within the tumors was demonstrated after Ad-hIFNand Ad-mIFNgene therapy, respectively. The therapy also induced necrosis in both the Ad-mIFNand AdhIFN-treated tumors. IHC revealed decreased tumor cell proliferation and the sequestration of activated macrophages within the tumors after Ad-mIFNtherapy. In addition, the expression of the proangiogenic factors bFGF, and MMP-9 protein (by IHC) was significantly down-regulated by Ad-hIFNgene therapy. Double-immunofluorescent IHC for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and CD-31 demonstrated tumor and endothelial cell apoptosis in those tumors treated with Ad-hIFNgene therapy. Tumor-induced angiogenesis, as determined by the microvessel density, was decreased in tumors treated with both Ad-mIFNand Ad-hIFN. These data suggest that the inhibition of tumorigenicity and the metastasis of the 253J B-V cells after infection with Ad-IFNis caused by the inhibition of angiogenesis and the activation of host effector cells. INTRODUCTION TCC is the fifth most common solid malignancy in the United States. Over 52,000 cases will be diagnosed and more than 12,000 deaths will be secondary to this cancer in the year 2000 (1). Seventy to 80% of patients with bladder cancer present with low-grade, noninvasive tumors confined to mucosa, with these cases initially managed by endoscopic resection. Superficial tumors recur in 60–70% of patients, and about 30% of these tumors progress to a higher grade or stage (2). The high recurrence rate and the unpredictability of the progression patterns have led to the widespread use of various intravesical therapies in an attempt to qualitatively change the ongoing urothelial Received 9/18/01; revised 12/28/01; accepted 1/2/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Core Grant CA-16672, CA71861-01 (to C. P. N. D.), NIH SPORE Grant 1P50 CA91846 tobacco settlement funds as appropriated by the Texas State Legislature (to W. F. B.), American Cancer Society Grant RPG-98-332-01 (to Z. D.), the Detweiler Traveling Fellowship from the Royal College of Physicians and Surgeons of Canada (to J. I. I.), and the Royal College of Surgeons in Ireland Postgraduate Travelling Fellowship and Trinity College Dublin Postgraduate Travelling Scholarship (to P. S.). 2 To whom requests for reprints should be addressed, at Department of Urology, Box 446, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3250; Fax: (713) 794-4824; E-mail: cdinney@mdanderson.org. 3 The abbreviations used are: TCC, transitional cell carcinoma of the bladder; Ad-IFN, adenoviral vector carrying IFN; Ad-hIFN, human adenoviral IFN; Ad-mIFN, murine adenoviral IFN; AdLacZ, adenoviral -galactosidase; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; MMP-9, matrix metalloproteinase-9; mIFN, murine IFN; hIFN, human IFN; IL-8, interleukin-8; NK, natural killer; CMV, cytomegalovirus; MOI, multiplicity/ multiplicities of infection; IHC, immunohistochemical analysis; PCNA, proliferative cell nuclear antigen; Tdt, terminal deoxynucleotidyl transferase; TUNEL, Tdt-mediated dUTP-biotin nick-end labeling; ISH, in situ hybridization; MLEC, murine lung endothelial cell, HMEC, human microvascular endothelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; pfu, plaque-forming unit(s); poly(dT), polydeoxythymidylic acid; iNOS, inducible nitric oxide synthase. 1258 Vol. 8, 1258–1270, April 2002 Clinical Cancer Research Research. on September 12, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from carcinogenesis (3). Intravesical therapy is an attractive option because relatively high doses of therapeutic agents can be delivered, establishing direct contact with the bladder tumor, with minimal systemic side effects. Intravesical chemotherapy, or more successfully intravesical Bacille Calmette-Guérin, often can prolong the progression-free interval after initial endoscopic resection. However, up to 50% of these patients will have disease progression despite Bacille Calmette-Guérin, and up to 30% of patients with recurrent superficial TCC will die of metastatic bladder cancer within 15 years (3). The standard therapy for patients who progress to invasive TCC is radical cystectomy. Unfortunately, disease recurs in up to 50% of these patients despite surgery (4). The growing awareness of the critical role of angiogenesis in tumor growth and metastasis has prompted a major effort to develop antiangiogenic therapies. Preclinical studies indicate that gene transfer strategies are a means to apply antiangiogenic therapy against solid tumors (5–7). TCC appears to be an ideal target to evaluate gene therapy because of the relative ease with which the vector can be delivered directly into the bladder (8). The profound bystander effect induced by Ad-IFNmakes this vector and cytokine an attractive choice for intravesical gene therapy (9). The IFNs are a family of regulatory natural glycoproteins that regulate cell growth and differentiation; inhibit the expression of oncogenes; up-regulate apoptosis; and activate lymphocytes, NK cells, and macrophages (10–13). The continuous exposure of various human cancer cells to noncytostatic concentrations of human IFN down-regulated the expression of bFGF (8, 14–16), IL-8 (17, 18), and MMP-9 (19–21). Furthermore, chronic administration of IFNs can inhibit the in vivo expression of bFGF, VEGF, and MMP-9 by human TCC, with subsequent inhibition of tumor-induced neovascularization and tumor growth (22). Although IFNs have been clinically effective against certain hematopoietic neoplasms (23, 24), and although chronic systemic administration of low-dose IFN has produced regressions of such vascular tumors as pulmonary hemangiomas (25), infantile hemangiomas (26), Kaposi’s sarcoma (27), and malignant hemangiopericytoma (28), IFNs have not been clinically effective against most solid neoplasms (29–32). Pharmacokinetic studies have demonstrated that the half-life of IFNs in the circulation of patients is in the order of minutes (33–35). The resulting lack of sustained IFN levels may be responsible for the failure of IFN to inhibit tumor growth, in part, by its antiangiogenic effect. The poor outcome of many patients with superficial TCC is a sobering testimony to the double-edged sword of attempted bladder preservation in the absence of a truly effective intravesical therapy. Intravesical gene therapy provides a promising approach for patients with refractory superficial bladder cancer, and adenoviral-mediated IFNgene therapy would appear to be a suitable agent for this purpose (2, 3, 5–10, 13, 15–18, 20–22, 36, 37). However, our preliminary data and early clinical experience indicate that the removal of the glycoaminoglycan layer is necessary for the effective treatment of superficial (and perhaps invasive) disease by intravesical gene therapy approaches, and we are exploring methods for enhancing gene transfer after intravesical instillation. The present study tested the hypothesis that the growth and metastasis of human TCC can be inhibited by increasing the local expression of IFNwithin the tumor by gene transfer of either mIFNor hIFN. Demonstrating this principle provides the rationale for intravesical IFNgene therapy. Because IFNappears to be more stable than IFNin an acidotic environment, we focused our study on IFNgene therapy (37–39). Because of the species specificity of IFN, we used both mIFNand hIFNto investigate the host response as well as the direct effects of therapy on the human tumor cells. We found that the orthotopic growth and metastatic potential of 253JB-V cells engineered to transiently and constitutively produce either hIFNor mIFNwas suppressed, and that the direct intratumoral delivery of IFNgene therapy inhibited tumor growth by activating tumoricidal host effector cells with mIFNand by suppressing tumor-induced angiogenesis with hIFN. MATERIALS AND METHODS Mice. Male athymic BALB/c nude mice (age, 6 to 8 weeks) were obtained from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). All of the mice were maintained in a laminar air-flow cabinet under pathogen-free conditions and used at 10–12 weeks of age. Mice were housed in facilities approved by the American Association for the Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, the Department of Health and Human Services, and NIH. Tumor Cell Culture. 253J B-V human TCC tumor cells were maintained as a monolayer culture in MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100 units/ml penicillin, 100 g/ml streptomycin, and vitamins (CMEM). MLECs were immortalized by SV40 transformation and maintained in CMEM. HMECs (Cascade Biologicals, Portland, Or) were maintained in medium 131 with microvascular growth supplements (Cascade Biologicals). The 253J B-V cells were harvested by a 1-min treatment with 0.025% trypsin-0.01% EDTA solution. The culture flask was tapped to detach the cells. The cells were washed in CMEM and resuspended in HBSS in preparation for implantation into the mice. Production of Adenoviral Vectors. The full coding region of the mIFNcDNA (provided by Dr. T. Taniguchi, University of Osaka, Osaka, Japan) was subcloned into the plasmid pxCMV to derive the shuttle vector pECIFN. The shuttle vector and plasmid pJM17 were cotransfected into 293 cells (American Type Culture Collection) by liposome-mediated transfection with Lipofectin (Life Technologies, Inc.) to generate the replication-deficient adenoviral vector AdCMVmIFN. Dr. W. Zhang (Baxter Healthcare Co., Round Lake, IL) provided the plasmids pxCMV, pJM17, and Ad5-LacZ (a recombinant adenovirus encoding the Escherichia coli lacZ gene was used as a control). The human CMV promoter drives both the lacZ and IFN genes. AdCMVmIFN(Ad-mIFN) was identified by PCR and infection of cells. The sequences of the PCR primers were 5 -CTTGGTTCTTATGCGAGCGG-3 and 5 CCACAACTAGAATGCAGTG-3 , which are located outside the two ends of the insert on the shuttle vector pECIFN. One 1259 Clinical Cancer Research Research. on September 12, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from clone of Ad-mIFNwas subcloned three times, resulting in a clone of wild-type virus-free Ad-mIFN. The presence of wild-type adenovirus was identified by plaque assay on HeLa cells at 10 MOI and by PCR using primers specific for the E1a region (U: 5 -TGA GAC ATA TTA TCT GCC ACG-3 and L: 5 -CCT CTT CAT CCT CGT CAC-3 ) that were deleted in the recombinant adenovirus. Only preparations that did not contain wild-type virus were used. Ad-LacZ, and Ad-mIFNwere propagated in 293 cells grown in CMEM. The viruses were released by three freeze/ thaw cycles and purified by a two-step CsCl gradient centrifugation. After dialysis at 4°C against 10 mM Tris/HCl (pH 7.5), 1 mM MgCl2, and 10% glycerol, aliquots of the vectors were stored at 80°C. The titers of the vectors were assessed on 293 cells by plaque assay and were 5 to 20 10 pfu/ml for most preparations. Ad-hIFN(provided by Dr. Arbans K. Sandhu, University of Pennsylvania, Philadelphia, PA) was propagated in the same manner. Susceptibility of 253J B-V Cells to Adenoviral Infection. The 253J B-V cells were trypsinized and harvested, and 1 10 cells were plated in each well of a 24-well polystyrene culture plate. After overnight incubation, the 253J B-V cells were infected with Ad-mIFN, Ad-hIFN, or Ad-LacZ as described previously (40). Staining for -galactosidase protein, which indicated successful infection with Ad-LacZ, was performed by aspirating the medium, then rinsing gently with cold PBS using 1 ml/well. The cells were overlayed with a fixative containing 0.5% glutaraldehyde in PBS. They were then incubated at 4°C for 5 min. The fixative was then rinsed gently twice with PBS at room temperature. After the PBS was rinsed off, the cells were exposed to the -galactosidase stain, as described previously (40). A MOI of 10:1 was shown to be optimal for both Ad-mIFNand Ad-LacZ, demonstrating minimal cytotoxicity and maximal effect on IFNproduction. The optimal MOI for the Ad-hIFNwas 5:1. No increase in cytotoxicity was seen when the MOI was increased to 10:1. ELISA Analysis. The 253J B-V cells were trypsinized and harvested, and 1 10 cells were plated in six-well polystyrene culture plates. After overnight incubation, the 253JB-V cells were infected with 0, 5, 10, 20, 25, 30, and 40 MOI of Ad-hIFN. Ad-LacZ was used as a control using the same titers. After incubation for 24, 48, 72, 96, and 120 h, the cell supernatant was collected and analyzed by ELISA (Fujirebio Inc., Tokyo, Japan) for accumulated hIFNprotein concentration. ELISA for Angiogenic and Metastatic Factor Expression. 253J B-V, MLEC, and HMEC cells were plated in six-well plates. Each cell type was infected with Ad-LacZ, Ad-mIFN, and Ad-hIFNat a MOI of 10:1. Cell supernatants we collected 24, 48, and 72 h after viral infection and analyzed by ELISA (Quantikine immunoassay kits; R&D Systems, Minneapolis, MN) for expression of bFGF, IL-8, and VEGF protein. Cell numbers in each group were determined by MTT assay. RNA Isolation and Northern Blot Analysis. The 253J B-V cells were trypsinized and harvested, and 1 10 cells were placed in 50-mm polystyrene culture plates and incubated overnight. Ad-hIFNat 10:1 MOI was used to infect the cells. Ad-LacZ was used as a control at 10 MOI. The mRNA was extracted from these cells at 24, 48, and 72 h using the FastTrack kit (Invitrogen, San Diego, CA). For Northern blot analysis, 1 g of mRNA was fractionated on 1% denaturing formaldehyde/agarose gels, capillary-transferred to GeneScreen nylon membrane (DuPont Co., Boston, MA), and UV crosslinked with 120,000 J/cm using a UV Stratlinker 1800 (Stratagene). Hybridization using cDNA probes was performed as described previously (16). The DNA probes used were cDNA fragments corresponding to hIFN, bFGF, and MMP-9. Rat glyceraldehyde-3-phosphate dehydrogenase cDNA fragments were used as a loading control. Growth-inhibitory Effects of Adenoviral Vectors on 253J B-V Cells. The 253J B-V tumor cells were plated at 5 10 cells/well in a 96-well plate. Cells were treated with Ad-hIFNand Ad-LacZ, both at a 10:1 MOI, and with PBS using equal volumes. After incubation for 1–5 days, the cells were treated with 50 l of 1% MTT solution and incubated for 2 h. After suctioning of the MTT solution, 100 l of 10% DMSO was added to each well; and after the colorimetric reaction had occurred, the absorbance of the plates was read on the spectrophotometer at 450 m. There was insignificant growth inhibition of the 253J B-V cells when exposed to either Ad-mIFNor Ad-hIFNcompared with Ad-LacZ (data not shown). In Vitro Infection of 253J B-V Cells for Orthotopic Implantation. The 253J B-V cells were trypsinized and harvested, and subsequently 1 10 cells were plated in 50-mm culture flasks. After a 12-h incubation, the tumor cells were infected using Ad-hIFN, Ad-mIFN, and Ad-LacZ at a MOI of 10:1. After a 12 h incubation, the cells (253J B-V-mIFN, 253J B-V-hIFN, and 253J B-V-LacZ) were trypsinized and harvested in preparation for orthotopic implantation. Orthotopic Implantation of 253J B-V and in Vitroinfected 253J B-V Cells. Each mouse was anesthetized with sodium pentobarbital (25 mg/kg) i.p. and placed in the supine position. A lower midline incision was performed, and the bladder was exposed. The 253J B-V, 253J B-V-mIFN, 253J B-V-hIFN, and 253J B-V-LacZ cells (1 10 cells in 50 l of HBSS) were implanted into the wall of the bladder in the area of the bladder dome using 30-gauge needles on disposable 1-ml syringes. A successful implantation was indicated by a bleb in the bladder wall serosa. The abdominal wound was closed in one layer with metal wound clips (Autoclips; Clay Adams, Parsippany, NJ). Twenty-eight days later, the mice were killed by cervical dislocation after anesthesia with sodium pentobarbital, and the bladders and lymph nodes were excised and placed into formalin or OCT compound (Miles Laboratories, Elkhort, IN). Heterotopic Implantation of 253J B-V Cells and in Vivo Therapy. Mice were anesthetized with sodium pentobarbital (25 mg/kg) i.p. The 253J B-V cells (1 10 cells in 200 l of HBSS) were subsequently injected s.c. into each mouse’s right flank using 30-gauge needles on disposable 1-ml syringes. A successful injection was indicated by a raised skin bleb. Once the tumors reached 5 mm in diameter, they were intratumorally treated weekly for 3 weeks with Ad-mIFN, Ad-hIFN, and Ad-LacZ using 1.0 10 pfu/injection or with PBS. Assuming tumor cell number is 1 10 in a 1 cm tumor, we estimated tumor cell number to be 1.25 10 in a 5 5 5-mm tumor, also assuming that this volume would approximate our tumors 1260 Inhibition of Tumorigenicity and Metastasis of 253J B-V Research. on September 12, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from when measured at a 5-mm diameter at the skin surface. A 30-gauge needle on a disposable 1-ml syringe was used to instill 200 l per treatment. Tumor diameters were measured weekly with calipers. Mice in all of the treatment groups maintained stable body weight throughout the entire treatment, without any obvious toxic effects. Mice were killed by using sodium pentobarbital (25 mg/kg) i.p. and subsequent cervical dislocation. The s.c. tumors were excised and weighed. The tumor samples were analyzed by H&E staining, IHC, and ISH. IHC. Frozen tissue sections were used by placing harvested tumors in OCT compound and snap-frozen in liquid nitrogen. Frozen sections (8–10 m) were subsequently placed on Superfrost-plus slides (Fisher Scientific) and fixed in cold acetone. Paraffin-embedded tissue sections were obtained by placing harvested tumors in 10% formalin with subsequent paraffin embedding after 48 h. Paraffin sections (3–5 m) were subsequently placed on Superfrost-plus slides (Fisher Scientific) and were deparaffinized in xylene, rehydrated in graded ethanol, and transferred to PBS. The slides were rinsed twice with PBS, and endogenous peroxidase was blocked using 3% hydrogen peroxide in PBS for 12 min. The tissues were washed three times with PBS and incubated for 20 min at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Frozen tissue samples were incubated for 18 h at 4°C with a 1:100 dilution of a monoclonal rat anti-CD31 antibody (PharMingen, San Diego, CA) or a 1:70 dilution of rat antiscavenger macrophage receptor antibody (Serotec, Raleigh, NC). Paraffinembedded tissue samples were incubated for 18 h at 4°C with a 1:200 dilution of rabbit-polyclonal anti-bFGF antibody (Sigma Chemical Co., St. Louis, MO), a 1:750 dilution of rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a 1:50 dilution of rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA), a 1:50 dilution of rabbit polyclonal anti-MMP-9 antibody (Calbiochem, La Jolla, CA), a 1:400 dilution of rabbit anti-hIFNantibody (Lee Biomolecular Research Laboratories, San Diego, CA), a 1:400 dilution of rabbit anti-mIFNantibody, a 1:50 dilution of monoclonal mouse anti-PCNA antibody (Dako, Carpinteria, CA), or a 1:200 dilution of rabbit polyclonal anti-iNOS antibody (BD Transduction Laboratories, Chino, CA). The samples were rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution of peroxidase-conjugated antirabbit IgG or antirat IgG. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics). The sections were then washed three times with distilled water. A positive reaction was indicated by a brown staining. The stained sections were examined in a Zeiss photomicroscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a three-chip charge-coupled device color camera (model DXC-960 MD; Sony Corp., Tokyo, Japan). The intensity of the staining was quantified in six different areas of tumor samples for bFGF, VEGF, IL-8, MMP-9, hIFN, mIFN, and iNOS by an image analyzer using the Optimas software program (version 5.2; Media Cybernetics, Carlsbad, CA) to yield an average measurement (22). Images covering a range of staining intensities were captured electronically, a color bar (montage) was created, and a threshold value was set in the red, green, and blue mode of the color camera. All of the subsequent images were quantified based on this threshold. The integrated absorbance of the selected fields was determined based on the equivalence to the mean log inverse gray-scale value multiplied by the area of the field. The samples were not counterstained for this analysis; therefore, absorbance was attributable solely to the product of the IHC reaction. For PCNA, densities of proliferative cells were expressed as the average of the five highest areas identified within a single 200 field. The area of highest neovascularity on the slides analyzed for CD31 was identified under low power ( 40). Individual microvessels were quantified according to the procedure described by Weidner et al. (41) The image was projected and recorded by digitizing the image in a cooled chargecoupled device Optotronics Tec 470 camera (Optotronics Engineering, Goleta, CA) linked to a computer and a digital printer (Sony Corporation). The results were expressed as the highest number of microvessels identified within a single 200 field. After image analysis, the slides were counterstained with Gill’s hematoxylin (Biogenex Laboratories, San Ramon, CA). TUNEL Assay. Cell death in tumors was determined by the TUNEL method (42). Paraffin-embedded tissue sections (5 m thick) were deparaffinized and rehydrated as above. The slides were rinsed twice in distilled water with BRIJ (DW/BRIJ) and treated with proteinase K (1:500, 20 g/ml) for 15 min; endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 min. The tissue sections were then washed three times with DW/BRIJ and incubated for 10 min at room temperature with Tdt buffer. Excess Tdt buffer was drained, and the samples were incubated for 18 h at 4°C with terminal transferase and biotin-16-dUTP. The samples were rinsed four times with TB buffer (0.03 M sodium chloride, 0.03 M sodium citrate) and incubated for 30 min at 37°C in a 1:400 dilution of peroxidase-conjugated streptavidin. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics). The sections were washed three times with PBS, counterstained with Gill’s hematoxylin, and again washed three times with PBS. For TUNEL, densities of apoptotic cells were expressed as the average of the five highest areas of greatest intensity identified within a single 200 field. ISH. A specific antisense oligonucleotide cDNA probe, complementary to the mRNA transcripts of hIFNidentified on the basis of published reports of the cDNA sequence, was designed as described previously (43). The cDNA probe was synthesized with six biotin molecules at the 3 end via direct coupling using standard phosphoramidine chemistry as described previously (Research Genetics; 44, 45). The lyophilized probes were reconstituted to a 1g/ l stock solution in Brigati probe diluent (Research Genetics). ISH was performed as described previously (46). The Microprobe manual staining system (Fisher Scientific) was used to stain tissue sections (4 m) of formalin-fixed, paraffin-embedded tumor specimens mounted on ProbeOn slides (Fisher Scientific). The slides were placed in the Microprobe slide holder, dewaxed, and dehydrated with Autodewaxer and Autoalcohol (Research Genetics), followed by enzymatic digestion with pepsin. Hybridization of the probe was performed for 90 min at 45°C, and the samples were then washed three times with 2 SSC (0.15 M NaCl and 0.015 M sodium citrate) for 2 min at 45°C. The samples were incubated with alkaline phosphataselinked enhancer (Biomeda Corp., Foster City, CA) for 1 min at 45°C and then incubated at 45°C for 40 min with chromagen 1261 Clinical Cancer Research Research. on September 12, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from substrate FastRed (Research Genetics). In this assay a red stain indicated a positive reaction. The integrity of mRNA in each sample was verified by using a poly(dT)20 probe. All of the specimens analyzed produced an intense histochemical reaction, which indicated that the mRNA was well preserved. To avoid variability in probe concentration, all specimens were stained in a single session for each probe. Stained sections were examined in the same manner as for IHC above. Measured areas of 1 mm were located at the edge or center of the tumor. In each area, the cytoplasmic staining was quantified to derive an average value (13). The intensity of staining was compared by integrated absorbance of poly(dT)20. Immunofluorescence Double Staining of Endothelial Cells and Apoptotic Cells. For IHC, frozen tissue sections (8 m thick) were fixed and treated as above for CD31 IHC. Samples were washed four times with PBS and incubated for 1 h with a fluorescent-labeled antirat secondary antibody, Texas Red (1:200; Jackson ImmunoResearch Laboratory, Inc., West Grove, PA). After sections were washed twice with PBS, TUNEL was performed using a commercial kit according to the manufacturer’s protocol (Promega, Madison, WI). The tissue sections were fixed in 4% Triton X-100 in PBS for 5 min at room temperature. After the slides were rinsed twice with PBS for 5 min, they were incubated with equilibration buffer from kit for 10 min. The equilibration buffer was removed, and reaction buffer containing equilibration buffer, nucleotide mix, and Tdt enzyme was added to the tissue sections and incubated in a humid chamber at 37°C for 1 h, although avoiding exposure to light. EDTA was added to the slides for 5 min to stop the reaction. To identify all nuclei, the slides were soaked with a 1:2000 dilution of Hoechst solution in a Coplin jar for 10 min. The slides were examined under an Olympus Inverted System IX70 microscope (Melville, NY) and images were captured using a digital camera. CD31-positive endothelial cells were detected by localized red fluorescence. Fluoroscein-12-dUTP was incorporated at the 3 -OH ends of fragmented DNA, resulting in localized green fluorescence within the nucleus of apoptotic cells. The apoptotic endothelial cells had a yellow fluorescence because of the overlapping green and red. Statistical Analysis. The significance of the differences in the in vivo data were evaluated by the Mann-Whitney U test (47).