PC14PE6-RFP human lung adenocarcinoma cells were stably transduced with the full-length dsRed2 cDNA as described by Kienast el al.  and kindly provided to us by F. Winkler (University of Heidelberg, Neurooncology, Heidelberg, Germany) in 2008. The PC14PE6-RFP cells used in this study were authenticated by the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) by STR profiling to be identical with the parental cell line PC14 (Riken, Japan) in 2012. PC14PE6-RFP cells were cultured in DMEM supplemented with 1x MEM non-essential amino acids, 2 mM GlutaMAX, 10% FBS, 100 Units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C and 5% CO2.
Construction of the attenuated Vaccinia virus strains GLV-1h68 and GLV-1h108 was described previously by Zhang et al.  and Frentzen et al. , respectively. 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 virus genome. In case of GLV-1h108 the glaf-1 coding sequence was recombined into the J2R locus of the parental GLV-1h68 virus strain. Viruses were propagated in CV-1 cells and purified through sucrose gradients.
Tumor inoculation and administration of the virus
All animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Explora Biolabs (San Diego, USA, protocol number EB11-025) or the government of Unterfranken (Würzburg, Germany, protocol number AZ 55.2-2531.01-17/08).
Six-week-old female athymic nude Foxn1
mice were obtained from Harlan Laboratories (Netherlands and Indianapolis). PC14PE6-RFP tumor cells (4 × 105/100 μl PBS) were subcutaneously (sc) injected into the abdominal right flank. Tumor volume was calculated as length × width2 × 0.52. For all experiments, tumors were grown up to 100–200 mm3 in size (13–14 days) before viral administration. A single viral dose of 1 × 107 plaque forming units (pfu) in 100 μl PBS was injected intravenously (iv) via the tail vein.
Fluorescence live-animal imaging
Tumor cell growth and viral infection were monitored directly by the RFP expression of tumor cells and GFP expression of Vaccinia virus-infected cells and quantified with the Maestro EX imaging system (CRI, Woburn, MA) using appropriate filters for RFP (tumor; excitation: 503–555 nm, emission: 580 nm cut-in) and GFP (virus; excitation: 445–490 nm, emission: 515 nm cut-in). Images were evaluated and quantified using the Maestro Version 2.10.0 software.
For flow cytometric analysis of tumors and exudates, four end-stage PC14PE6 tumor-bearing mice (28 dpim) were sacrificed by CO2 inhalation. Effusions were punctured and 400 μl of the exudates were collected. Preparation of tumors were performed as previously described . In both effusion and tumor preparations lysis of erythrocytes using an isotonic ammonium cloride lysis buffer was performed and DNA was digested using 5 MU/ml DNase I.
Blocking of unspecific binding-sites and antibody-labeling using anti-mouse CD45-PECy7 (eBioscience, San Diego, CA, USA) was performed as decribed elsewhere . Immediately before use, dead cells were labeled with propidium iodide (PI) solution.
Labeled cells were subsequently analyzed, using the Accuri C6 Cytometer and FACS analysis software CFlow Version 220.127.116.11 (Accuri Cytometers, Inc. Ann Arbor, MI USA).
In vivo MRI
In vivo MRI measurements of tumor-bearing mice were performed at room temperature on a 7 Tesla Bruker Biospec System (Bruker BioSpin GmbH, Reinstetten, Germany) using a 35 mm diameter home-built quadrature birdcage coil. For in vivo imaging the animals received inhalation anesthesia (1-2% isoflurane) during the measurement and were placed in a home-built measurement container according to safety regulations.
T1 weighted (T1w) spin echo experiments were performed at different time points following an injection (iv) of 0.1 mmol/kg body weight Gadopentetate-Dimeglumine (Gd-DTPA, Magnevist, Bayer Schering Pharma AG, Berlin, Germany) of exemplary mock infected animals. Furthermore, for the shown data, a T2 weighted (T2w) spin echo experiment was performed for anatomical correlation approximately half an hour after injection of the contrast agent.
For n = 4 additional animals (n = 2 mock/ GLV-1h68) multi spin echo (MSE) experiments were performed for the evaluation of tissue T2 times (without contrast agent). Data processing of the MRI data was performed in MATLAB (The MathWorks Inc., Natick, USA) using home-written software routines. The chosen MRI sequence parameters are provided in the Additional file 1.
Protein isolation and hVEGF/mVEGF ELISA of tumor samples
Quantitative evaluation of VEGF concentrations in tumor tissues was performed using a human (Thermo Scientific, EHVEGF) and mouse specific VEGF ELISA kit (abcam, ab100751) according to the manufacturer’s protocol. Tumors were isolated 7 dpi, lysates were prepared as described previously . Absorbance was measured using a Tecan sunrise absorbance reader (Tecan, Crailsheim, Germany). Protein concentrations for each sample were interpolated from a VEGF-specific standard curve.
RT-PCR of ß-actin
Tissue samples were analyzed for the presence of PC14PE6-RFP cells by RT-PCR. Brains, lungs, and livers of PC14PE6-bearing mice (28 dpim) were homogenized in TRIzol Reagent (Invitrogen) to isolate total RNA. Samples were further treated as described elsewhere . Primer sequence for human ß-actin: (forward: 5′-CCT CTC CCA AGT CCA CAC AG-3′and reverse: 5′- CTG CCT CCA CCC ACT C-3′) and for murine ß-actin: (forward: 5′-CGT CCA TGC CCT GAG TC- 3′ and reverse: 5′-GCT GCC TCA ACA CCT CAA C-3′). PC14PE6-RFP cell lysates were used as positive control for human ß-actin. The PCR reaction was performed in a T-Gradient Thermoblock PCR machine (Biometra, Göttingen, Germany).
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 followed by dehydration in 10% and 30% sucrose/PBS (Carl Roth, Karlsruhe, Germany) and finally embedded in Tissue-Tek® O.C.T. (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands). Tumor samples were sectioned (15 μm) with a cryostat 2800 Frigocut (Leica Microsystems GmbH, Wetzlar, Germany) and stored at −80°C. Antibody-labeling was performed following fixation in ice-cold aceton. The primary antibody was incubated for 1 h. After washing with PBS, sections were labeled for 30 min with the secondary antibody and finally mounted in Mowiol 4–88.
Whole animal sectioning and H&E staining of tissue sections
Mice were euthanized, perfused with 4% paraformaldehyde, and whole mice were fixed in 4% formalin for 2 days followed by decalcification in 10% formic acid/4% formalin for up to 5 days. Decalcified specimens were dehydrated and embedded in paraffin. Tissue sections (5 μm) were stained by H&E staining.
The fluorescence-labeled preparations were examined using a MZ16 FA Stereo-Fluorescence microscope (Leica) equipped with a digital DC500 CCD camera and the Leica IM1000 4.0 software (1300 × 1030 pixel RGB-color images). Furthermore, a Leica TCS SP2 AOBS confocal laser microscope equipped with argon, helium-neon and UV lasers and the LCS 2.16 software (1024 × 1024 pixel RGB-color images) was used. Digital images were processed with Photoshop 7.0 (Adobe Systems, Mountain View, CA) and merged to generate overlay images.
Fluorescence intensity measurements and microvessel density
Fluorescence intensity of the CD31-, Ly-6G-, MHCII-labeling as well as the vascular density was measured in 15-μm-thick cryostat sections of control tumors and infected areas of GLV-1h68-colonized tumors on digital images as described earlier . For all experiments the mean value was calculated for nine images (three images each of three different control and GLV-1h68-infected tumors) and presented with standard deviation.
Antibodies, reagents and treatment of animals
Endothelial cells of blood vessels were labeled with hamster anti-mouse CD31 antibody (Chemicon, International, Temecula, CA). Platelets were labeled with rat anti-mouse CD41 antibody (GeneTex Inc., Irvine, CA). Immune cells were labeled using rat anti-mouse MHCII antibody (B, dendritic cells, monocytes, macrophages) and rat-anti Ly-6G antibody (neutrophils) (eBioscience, San Diego, CA). Vaccinia virus was labeled with rabbit anti-Vaccinia virus (abcam, Cambridge, UK).
The DyLight649-conjugated secondary antibodies (donkey) were obtained from Jackson ImmunoResearch (West Grove, PA).
For the labeling of functional blood vessels in tumors, mice were intravenously injected with 100 μg of biotinylated-Lycopersicum esculentum Lectin (Vector Laboratories, Burlingame, CA). Three minutes later, tumors were removed and prepared for histology. Tumor cross-sections (15 μm) were labeled with DyLight649-conjugated streptavidin (Sigma Aldrich, Taufkirchen, Germany) to visualize the Lectin-labeled functional tumor vasculature.
A two-tailed Student’s t test was used for statistical analysis. P values of < 0.05 were considered statistically significant (*p < 0.05, ** p < 0.01, *** p < 0.001).