Cell lines
GI-101A human ductual breast adenocarcinoma cells were kindly provided by A. Aller (Rumbaugh-Goodwin Institute for Cancer Research, Inc., FL, USA) and cultured in RPMI 1640 supplemented with 5 ng/ml β-estradiol and 5 ng/ml progesterone (Sigma Aldrich, Taufkirchen, Germany), 1 mM sodium pyruvate, 10 mM HEPES, 20% FBS, 100 Units/ml penicillin, and 100 μg/ml streptomycin (PAA Laboratories, Cölbe, Germany). African green monkey kidney fibroblasts (CV-1) were obtained from the American Type Culture Collection (ATCC-No. CCL-70) and cultured in DMEM supplemented with 10% FBS. The murine endothelial cell line 2H-11 (ATCC-No. CRL-2163) as well as mouse brain endotheliomas bEnd.3 (kindley provided by G. J. Hämmerling, Deutsches Krebsforschungszentrum, Heidelberg, Germany) were obtained in DMEM with 10% FBS. Human umbilical vein endothelial cells (HUVEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in M199 medium supplemented with 10% FBS, 10 ng/ml human EGF and 50 μg/ml endothelial cell growth supplement (Sigma Aldrich). The human kidney cell line 293FT was obtained from Invitrogen GmbH (Karlsruhe, Germany) and cultured in DMEM supplemented with 10% FBS, 0.1 mM non-essential amino acids, 6 mM L-glutamine, and 1 mM sodium pyruvat. All cells were maintained at 37°C and 5% CO2.
Viruses and plasmids
The construction of the attenuated vaccinia virus strain GLV-1h68 was described previously by Zhang et al. [16]. Briefly, three expression cassettes (encoding for Renilla luciferase-GFP fusion protein, β-galactosidase and β-glucuronidase) were recombined into the F14.5L, J2R and A56R loci, respectively, of the LIVP strain viral genome. Viruses were propagated in CV-1 cells and purified through sucrose gradients.
The RFP-expressing GI-101A cell line was constructed using the ViraPower™ Gateway Cloning and Lentiviral Expression System Kit (Invitrogen GmbH, Germany) in accordance with the manufacture's instructions. The mRFP-encoding plasmid pCR-TK-Sel-mRFP was provided by Q. Zhang (Genelux Corporation, San Diego) and used as a template for PCR amplification of the mRFP gene using primers containing attB recombination sites for gateway cloning (forward-attB1-mRFP: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGCCTCCTCCGAGG-3', reverse-attB2-mRFP: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGAATTCGCCCTTTCATTAGG-3'). The mRFP-containing lentiviral vectors were generated by gateway recombination between the pDONR-221-mRFP entry vector and the pLenti6/V5-DEST destination vector. The mRFP-containing replication-incompetent Lentiviruses for transduction of GI-101A cells were produced in 293FT cells using Lipofectamine™2000 for transfection with the ViraPower™ Packaging Mix and the pLenti6/V5-DEST-mRFP expression plasmid. Stable-expressing GI-101A-RFP clones were selected using 10 μg/ml blasticidin.
Tumor inoculation and administration of the virus
All animal experiments were carried out in accordance with protocols approved by the Regierung von Unterfranken, Germany (permit number: 55.2-2531.01-17/08).
Six-week-old female athymic nude Foxn1
numice were obtained from Harlan Winkelmann GmbH (Borchen, Germany). Six-week-old female B6.12956-Rag2
tm1FwaN12 mice and Tac:NIHS-Lyst
bg
Foxn1
nu
Btk
xldmice were ordered from Taconic Inc. (Hudson, NY, USA). GI-101A breast cancer cells (5 × 106/100 μl PBS) were subcutaneously (s.c.) injected into the abdominal right flank and tumor volume was calculated as (length × width2)/2. For all experiments, tumors were grown up to 200-400 mm3 in size (4-6 weeks) before viral administration. A single viral dose of 1 × 106 or 5 × 106 plaque forming units (p.f.u.) in 100 μl PBS was injected either intraveneously (i.v.) via the tail vein or via the retro-orbital (r.o.) sinus vein. For r.o. injection, animals were anesthetized using 75 mg/kg ketamine (Pfizer, Karlsruhe, Germany) and 20 mg/kg xylazine (Bayer, Leverkusen, Germany).
Immunohistochemistry
For histological studies, tumors were excised and snap-frozen in liquid N2, followed by fixation in 4% paraformaldehyde/PBS pH 7.4 for 16 h at 4°C. Fixed tumors were rinsed in PBS and embedded in 5% (w/v) low-melt agarose (AppliChem, Darmstadt, Germany). Tissue-sectioning (100 μm) was performed using the Leica VT1000S Vibratome (Leica, Heerbrugg, Switzerland) and the labelling procedures were previously described in detail elsewhere [18].
Fluorescence microscopy
The fluorescence-labelled preparations were examined using the MZ16 FA Stereo-Fluorescence microscope (Leica) equipped with the digital DC500 CCD camera and the Leica IM1000 4.0 software (1300 × 1030 pixel RGB-color images) as well as the Leica TCS SP2 AOBS confocal laser microscope equipped with an argon, helium-neon and UV laser and the LCS 2.16 software (1024 × 1024 pixel RGB-color images). Digital images were processed with Photoshop 7.0 (Adobe Systems, Mountain View, CA) and merged to yield overlay images.
Fluorescence intensity measurements
Fluorescence intensity of the CD31- and MHCII-labelling in 100-μm-thick Vibratome sections of control tumors and infected areas of GLV-1h68-colonized tumors was measured on digital images (× 50 objective, × 1 ocular, tissue region 2700 μm by 2150 μm) of specimens stained for CD31 or MHCII immunoreactivity. On the fluorescence microscope, the background fluorescence was set to a barely detectable level by adjusting the gain of the CCD camera before all the images were captured with identical settings. RGB-images were converted into 8-bit gray scale images (intensity range 0 - 255) using Photoshop 7.0. The fluorescence intensity of the CD31-labelling represented the average brightness of all vessel-related pixels and was measured using Image J software http://rsbweb.nih.gov/ij. For CD31-labelling the mean value was calculated for nine images (three images of three different control and GLV-1h68-infected tumors) and presented with standard deviation.
The extent of the viral distribution in GLV-1h68-colonized tumors was measured by the GFP fluorescence signal on digital images (× 10 objective, × 1 ocular, image size 14 mm by 11.1 mm) of two whole tumor cross-sections (100 μm) of five or six different tumors. The whole area of the tumor cross-section was determined by Hoechst-labelling of cell nuclei. Both, GFP and Hoechst fluorescence images were converted into 8-bit gray scale images (intensity range 0 - 255) using Photoshop 7.0. The background fluorescence of GFP images was set to the fluorescence intensity of < 20 using Image J software. A fluorescence intensity of 20 was thus established as the threshold for distinguishing pixels of the GFP signal from those of the background. The area of pixels (inch2) on GFP images (fluorescence intensity > 20) as well as on Hoechst images (fluorescence intensity > 0) was measured by Image J and the proportion of infected tissue was calculated for two images from each tumors (n = 6). Mean values + standard deviations are shown.
Measurements of microvessel density and vessel diameter
The vascular density was determined in microscopic images (× 200 objective, × 1 ocular, tissue region 680 μm by 540 μm) of CD31-labelled tumor sections. On the fluorescence microscope, for each image the CD31 fluorescence was set to a clearly detectable level by individually adjusting the gain of the CCD camera before the images were captured. All images were decorated with five horizontal lines at identical positions using Photoshop 7.0 and all vessels which intersected these lines were counted to yield the vascular density. The vascular density was calculated for nine images (three images of three different control and GLV-1h68-infected tumors) and presented as mean values with standard deviations.
The vessel diameter was measured on digital images (× 200 objective, × 1 ocular) of CD31-labelled 100-μm-thick tumor cross-sections using Leica IM1000 4.0 software. Images of control and infected tumors (GLV-1h68-infected area) were obtained with individual exposure times to get optimal CD31 signals and exclude signal-dependent variability of vessel diameter. Seven horizontal lines were drawn across each image and the diameter of all blood vessels that intersected these lines was measured (5 images per tumor). Mean values + standard deviations are shown.
Antibodies, reagents and treatment of animals
Endothelial cells were labelled with monoclonal rat anti-mouse CD31 antibody (BD PharMingen, San Diego, CA) or hamster anti-mouse CD31 antibody (Chemicon, International, Temecula, CA). Pericytes were labelled with Cy3-conjugated monoclonal mouse anti-mouse α-smooth muscle actin (SMA) (Sigma Aldrich). Basement membrane was labeled using polyclonal rabbit anti-mouse collagen IV antibody (Abcam, Cambridge, UK). Immune cells were labeled using rat anti-mouse MHCII antibody (B, dendritic cells, monocytes, macrophages) and rat-anti mouse CD45 antibody (common leukocyte antigen) (eBioscience, San Diego, CA).
The Cy3- or Cy5-conjugated secondary antibodies (donkey) were obtained from Jackson ImmunoResearch (West Grove, PA).
Phalloidin-TRITC (Sigma Aldrich) was used to label actin and Hoechst 33342 to label nuclei in tissue sections.
For the labelling of functional blood vessels in tumors, mice were anesthetized using 75 mg/kg ketamine and 20 mg/kg xylazine, followed by the injection of 100 μg of biotinylated-Lycopersicum esculentum lectin (Vector Laboratories, Burlingame, CA) via the tail vein of the mice. Two minutes later the chest was opened, and the vasculature was perfused at a pressure of 120 mmHg with fixative (4% paraformaldehyde/PBS pH 7.4) from a cannula inserted into the left ventricle. After fixation, tumors were removed and prepared for histology. Tumor cross-sections (100 μm) were labelled with Cy3-conjugated streptavidin (Sigma Aldrich) to visualize the lectin-labelled tumor vasculature.
Nonspecific rat-IgG from Jackson ImmunoResearch was used in extravasation studies and injected intravenously into tumor-bearing mice (11 mg/kg body weight). After 6 h incubation, the treated tumors were excised and used for histological analysis. Surface plot profiles of the IgG extravasation pattern were prepared using ImageJ software.
For immunosuppression a stock solution of cyclophosphamide monohydrate (42 mg/ml) (Sigma Aldrich) was prepared in water and sterile filtered. Immediately before use, the stock solution was diluted 1:1 in 1.8% NaCl to yield a final concentration of 21 mg/ml. CPA was administered by intraperitoneal injection twice per week throughout the entire duration of the study. The treatment was started 10 days p.i. with an initial dose of 140 mg/kg body weight followed by 100 mg/kg body weight. The dose and schedule was based on previously published studies of CPA immunosuppression in mice and hamsters [19].
Viral replication in vitro
For viral replication assays, tumor cells as well as endothelial cells were seeded in triplicates into 24-well plates to reach a confluency of 80% after a culture period of 12-16 h. Before infection, cell layers were starved with individual starvation media containing 1% FBS for 24 h and were finally infected with GLV-1h68 at m.o.i. of 0.01. After 1 h of incubation, the infection medium was replaced by fresh starvation medium and cells were cultured for further 6, 24 and 48 h, respectively. At the indicated time points, cells and supernatants were harvested and after three thaw-freeze cycles, serial dilutions of the lysates were titered by standard plaque assays on CV-1 cells.
Co-culture experiments
To mimic in vivo conditions, we cultured endothelial cells on growth factor reduced Matrigel Matrix (BD Biosciences, Heidelberg, Germany), which is a soluble basement membrane extract. For co-culture experiments, we coated 24 well plates with 100 μl of Matrigel for 30 min at 37°C. Endothelial cells (1 × 104 cells/well) were seeded into 24 well plates and allowed to assemble into tube-like structures. Three hours later GI-101A-RFP tumor cells were seeded into these wells and co-cultures were incubated for 12-15 h. Co-cultures were infected with GLV-1h68 at m.o.i. of 0.5 for 1 h, before the medium was replaced with virus-free medium. The degree of infection was microscopically determined after 24 h.
Microarray performance and statistical analysis
Total RNA from both infected and uninfected GI-101A xenografts at days 21 and 42 post VACV infection was extracted using Trizol reagent (Sigma Aldrich) according to the manufacturer's instructions. Total RNA was amplified into anti-sense RNA (aRNA) as previously described [20, 21] and the quality of both, total RNA and secondarily amplified RNA was tested with the Agilent Bioanalyzer 2000 (Agilent Technologies, Palo Alto, CA). Confidence about array quality was based on the principle of reference concordance as previously described [22]. Mouse reference RNA was prepared by homogenization of the following mouse tissues (lung, heart, muscle, kidneys and spleen) and RNA was pooled from 4 mice. Pooled reference and test aRNA was isolated and amplified in identical conditions during the same amplification/hybridization procedure to avoid possible inter-experimental biases. Both, reference and test aRNA was directly labeled using ULS aRNA Fluorescent labeling Kit (Kreatech, Netherlands) with Cy3 for reference and Cy5 for test samples.
Whole genome mouse 36 k oligo arrays were printed in the Infectious Disease and Immunogenetics Section of the Department of Transfusion Medicine (IDIS), Clinical Center, National Institute of Health, Bethesda using oligos purchased from Operon (Huntsville, AL). The Operon Array-Ready Oligo Set (AROS™) V 4.0 contains 35,852 longmer probes representing 25,000 genes and about 38,000 gene transcripts and also includes 380 controls. The design is based on the Ensembl Mouse Database release 26.33b.1, Mouse Genome Sequencing Project, NCBI RefSeq, Riken full-length cDNA clone sequence, and other GenBank sequence. The microarray is composed of 48 blocks and one spot is printed per probe per slide. Hybridization was carried out in a water bath at 42°C for 18-24 hours and the arrays were then washed and scanned on a Gene Pix 4000 scanner at variable PMT to obtain optimized signal intensities with minimum (< 1% spots) intensity saturation.
Resulting data files were uploaded to the mAdb databank http://nciarray.nci.nih.gov and further analyzed using BRBArrayTools developed by the Biometric Research Branch, National Cancer Institute [23]http://linus.nci.nih.gov/BRB-ArrayTools.html and Cluster and Treeview software [24]. The global gene-expression profiling consisted of 16 experimental samples. Global expression data were filtered using automated filtering option of BRBArray software. Therefore, genes involved in pathways such as "adhesion molecules on lymphocytes, B lymphocytes cell surface molecule, cytokines and inflammatory response, monocytes and its surface molecules, neutrophiles and its surface molecules, T cytotoxic cell surface molecules, T helper cell cytotoxic molecules" as listed by the Biocarta database were included. Genes that belonged to at least one of those pathways, that were present in more than 10 experimental samples (≥ 60%) and with a fold change of two in at least one sample passed the filter. Gene ratios were average corrected across experimental samples. Subsequent cluster analysis applying uncentered correlation algorithm with genes involved in selected pathways as listed above allowed experimental samples to cluster according to their biological similarity. Treeview program was used for visualization of array data [25].
Statistics
A two-tailed Student's t test was used for statistical analysis. P values of < 0.05 were considered statistically significant.