The cloning vectors used in this study, expressing bacterial nitroreductase gene (NTR2), mutant HSV1-thymidine kinase (HSV1-sr39TK), HSV1-sr39TK-NTR-fusion, and Fluc-EGFP fusion constructs were from our plasmid bank (Cellular Pathway Imaging Laboratory, Stanford). Plasmid extraction, DNA gel extraction, and genomic DNA extraction kits were purchased from Qiagen (Valencia, CA, USA).
Cell culture reagents, including culture media, fetal bovine serum (FBS), antibiotics (streptomycin and penicillin), and Lipofectamine 2000 transfection reagent were purchased from Invitrogen (Carlsbad, CA, USA). MDA-MB-231 cell line (ATCC HTB-26) was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were tested for pathogens by VSC diagnostic lab, Stanford University. MDA-MB-231 cell line was cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and 1% penicillin-streptomycin.
Stable cell lines
To make MDA-MB-231 stable cell lines, modified pcDNA3.1 (PURO) vectors expressing HSV1-sr39TK, NTR, or HSV1-sr39TK-NTR fusion proteins and lentivirus expressing Fluc-EGFP fusion proteins were used. Initially we created MDA-MB-231 cells stably expressing HSV1-sr39TK, NTR, or HSV1-sr39TK-NTR fusion by puromycin antibiotic selection (100 ng/ml). Clones of cells expressing near equal level of each enzyme (assessed by RT-PCR) were used for further transduction with lentivirus expressing Fluc-EGFP to co-express both therapeutic gene and an imaging reporter gene. To control the level of Fluc-EGFP at near equal expression, cells were sorted by FACS in a similar window after transduction. MDA-MB-231 stable cells were maintained in puromycin stress throughout the study. Single colonies of stable cells were expanded and evaluated for the functionality of NTR enzyme by incubating with a CytoCy5S (red-shifted NTR substrate; GE Healthcare, Piscataway, NJ, USA) for the detection of emitted fluorescent signal and 3H-PCV uptake for HSV1-sr39TK. CytoCy5S (excitation 628 nm/emission 638 nm) was dissolved in DMSO to a stock concentration of 2 mg/ml. The substrate was used for all in vitro and in vivo experiments by diluting either in PBS or in cell culture medium. Cells containing luciferase and EGFP fusion constructs were screened by FACS. Clones expressing equivalent levels of NTR and HSV1-sr39TK (as measured by immunoblot analysis) were selected.
To determine the expression levels of TK in cells transfected with HSV1-sr39TK and HSV1-sr39TK-NTR, immunoblot analysis using anti-TK-antibody was performed. Cells were lysed in 100 µl of RIPA lysis buffer (Pierce Biotechnology, Rockford, IL, USA), and 30 µg of total protein from stable MDA-MB-231 cells expressing HSV1-sr39TK, NTR, and HSV1-sr39TK-NTR, in 1X Lamelli loading buffer with β-mercaptoethanol (Life Technologies, Grand island, NY, USA), were heated at 95ºC for 5min, resolved using a 4-12% SDS-polyacrylamide gel electrophoresis gradient gel (Invitrogen, Carlsbad, CA, USA), and electroblotted onto a 0.2 µm pore size nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T, pH 7.6) for 1h and probed overnight at 4ºC on a rotating platform with the rabbit anti-human TK polyclonal antibody followed by incubation with peroxidase conjugated goat-anti rabbit IgG antibody (1:10,000 dilution, Rockland Immunochemicals, Gilbersville, PA, USA). The membranes were then re-probed with a mouse monoclonal antibody against human GAPDH (1:3,000, Novus Biologicals, Littleton, CO) to control for protein loading. Immunoblots were developed using the LumiGlo enhanced chemiluminescence method (Cell Signaling, Boston, MA, USA), following manufacturer's instructions using IVIS-Lumina imaging system (Caliper Life Sciences, MA, USA).
3H-Penciclovir (PCV) uptake assay
Quantitative evaluation of the functional effect of TK in cells stably transfected with HSV1-sr39TK, and HSV1-sr39TK-NTR fusions was performed by 3
H-Penciclovir uptake assay as described in Sekar et al., 2012 20
. Briefly, MDA-MB-231 cells stably expressing HSV1-sr39TK, NTR, and HSV1-sr39TK-NTR along with control cells were plated (2 X 105
cells per well) in 12-well plates. Twenty-four hours after plating, cells were treated with 0.5 µCi of 8-3
H-Penciclovir (specific activity 14.9 Ci mmol-1
; Moravek Biochemicals, La Brea, CA, USA). After 3 h of incubation at 37ºC with 5% CO2
, cells were washed twice with ice-cold PBS and lysed in 0.5 ml of 0.1N NaOH, analyzed by scintillation counter by adding 10 ml of cytoscint scintillation cocktail solution (Fisher Scientific, USA). The radioactivity readings were normalized to protein concentrations measured from a 20 µl aliquot of cell lysates.
Fluorescence-activated cell sorting (FACS) analysis for CytoCy5S uptake in cells expressing NTR
MDA-MB-231 cells were seeded in 10 cm culture plates to 60 - 70% confluency and transfected with plasmids expressing HSV1-TK, NTR, and HSV1-sr39TK-NTR after overnight incubation. After 24 h of further incubation, cells were treated with CytoCy5S (100 ng ml-1) dye for 1h and subjected to FACS analysis. Cells expressing NTR and HSV1-sr39TK-NTR were sorted using CytoCy5S reduction signal and maintained for further studies.
Fluorescence-activated cell sorter (FACS) for PI based apoptosis analysis
Stable MDA-MB-231 cells were seeded in 12-well culture plates and grown to 30-40% confluency, followed by 72 h treatment with various concentrations of GCV and CB1954 in various combinations (1 μg/ml GCV, 5 μM CB1954, 10 μM CB1954, 1 μg GCV+5 μM CB1954, 1 μg GCV+10 μM CB1954); untreated MDA-MB-231 cells were used as control. For a time course study, 1 μg of GCV+10 μM CB1954 was administered to cells that were observed for 5 days. Cells were trypsinized, washed, and analyzed for dead or apoptotic cells by staining with propidium iodide (15 nM) for 15 min, followed by flow cytometry (FACS Aria III, BD Biosciences, San Jose, CA, USA) at the Stanford FACS Facility. Data were analyzed by FlowJo FACS analysis software (Tree Star, Ashland, OR, USA). GCV and CB1954 were purchased from Sigma-Aldrich (St Louis, MO, USA).
Tumor xenograft studies and imaging
The Institute Animal Research Committee at Stanford approved all animal handling. All animals (CD1, nude) were purchased from Charles River Laboratories (Wilmington, MA, USA). The mice (N = 5 per treatment group) were intravenously injected with 1X105 MDA-MB-231 triple-negative breast cancer cells (200 µl in 2 min injections), stably co-expressing HSV1-TK-NTR and Fluc-EGFP fusion proteins, and allowed to establish metastatic tumors primarily in lungs. Animals were optically imaged by IVIS spectrum (IVIS-Spectrum Imaging System, Caliper Life Sciences, MA), followed by D-luciferin substrate injection every alternative day until desirable metastatic tumor was detected. Mice were divided into three groups, and treated with (1) GCV (40 mg/kg body weight), (2) CB1954 (40 mg/kg body weight) and (3) CB1954 (40 mg/kg) and GCV (40 mg/kg body weight), with two doses of prodrugs administered spaced 5 days apart. Drugs were given intraperitoneally by mixing 10% of PEG400 in 250 µl volume with sterile physiological saline. All animals were imaged before drug treatment to obtain a baseline image by optical CCD camera. The animals were imaged by an optical imaging camera on a daily basis following treatment initiation, and both the imaging and treatment continued for 10 days before the metastatic tumor grew to a level lethal to animal.
Positron Emission Tomography imaging and image analysis
Positron Emission Tomography/Computed tomography (PET/CT) imaging was performed prior to, and 9 days post, initiation of therapy with [18
F]-FHBG, a PET reporter probe for imaging the PET reporter genes, HSV1-TK and its mutant HSV1-sr39tk, is currently undergoing clinical evaluation 21
. In each imaging session, mice were anesthetized with 2% isoflurane in oxygen at 2 L/min, and 7.4 - 8.9 MBq of [18
F]-FHBG in 200 μl was intravenously injected via tail vein. Five-minute static PET scans were performed 3 hours after injection by using a micro-PET/CT (1.4-mm resolution, Inveon; Siemens, Malvern, PA, USA) preceded by CT scans for anatomic reference. Volumes of interest (VOIs) were drawn using the image analysis software IRW (Siemens). VOIs were drawn for lung tumors based on the CT scan and centered on the activity profile peak. Data (%ID/g) was corrected for partial volume effect and spillover 22
using calibration factors obtained from scanning a cylinder containing phantoms of different sizes.
The tumor xenografts of MDA-MB-231 cells expressing various combinations of therapeutic reporter genes (NTR, HSV1-sr39TK HSV1-sr39TK-NTR), after treatment or co-treatment with GCV and CB1954, were used for assessing the prodrug-induced therapeutic effects by visualizing apoptotic cells. For this, a portion of the tumor was frozen in OCT (TissueTek) and sectioned to 10 µm by a Cryomicrotome (Leica CM1850, Wetzlar, Germany). A terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) assay was performed with a Trevigen TACS 2 TdT-DAB (diaminobenzidine) in situ Apoptosis Detection Kit (TREVIGEN, Gaithersburg, MD, USA) according to the manufacturer's instructions. After staining, the slides were scanned in a Nanozoomer 2.0RS (Hamamatsu, Japan) digital scanner and assessed for diaminobenzidine staining of apoptotic cells using Nanozoomer Digital Pathology software.
Tumor xenografts were frozen in OCT cryoprotective fixing medium, sliced at 5 µm thickness by Leica cryomicrotome. Sections were stained by undiluted hematoxylene (Sigma-Aldrich, USA) for two minutes, rinsed in running water, and differentiated in 1% HCl acid/alcohol for 30 sec. They were then washed and immersed into Bluing solution (Fisher, USA) for 1 min, washed in running water and rinsed in 10 dips of 95% alcohol. After this, slides were counterstained in eosin by dipping into 1:5 ethanol-diluted Eosin solution (Fisher) for a total of less than 30 sec, and dehydrated through 95% alcohol, absolute alcohol, and xylene for 5 minutes each. Slides were mounted with xylene based mounting medium (Permount, Sigma) and imaged by Nanozoomer (Hamamatsu, Japan) digital slide scanner.