Isolation and culture of human macrophages

All patients donating blood gave informed consent to the Sheffield blood Transfusion Service and all procedures have been approved by the University of Sheffield Ethics Committee. Mononuclear cells were isolated from platelet-depleted buffy coats (Blood Transfusion Service, Sheffield, UK) using Ficoll-Paque Plus (Amersham Pharmacia, St Albans, UK).

In brief, 50 million monocytes were plated into T75 tissue culture flasks (NUNC, UK) and after 2 h non-adherent cells were removed. The remaining adherent cells were cultured over 7 days in IMDM (Lonza, UK) supplemented with 2 mmol l−1 L-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2% human Ab serum (Lonza).

Endothelial cell cultures

Human umbilical vein endothelial cells were obtained from Promocell, (Heidelberg, Germany) and used in the experiments up to passage 8. Cells (150,000) were seeded for 24 h onto collagen-coated (0.1 mg ml−1, human type IV) membranes containing a 5-μM pore polyethylene terephthalate membrane (Neuroprobe).

Human multi-cellular tumour spheroids

Human prostate cancer cell line, LNCaP (ATCC CRL-1740), was seeded (5 × 103) in 100 μl medium into each well of a 2% agarose (Sigma, Dorset, UK)-coated 96-well tissue culture plate. After 7–10 days, each well contained a tumour spheroid with an average diameter of 700–800 μm.

Infection of primary macrophages

Day 3 MDMs were infected with a replication-deficient adenovirus (CMV-AdV5-GFP) at multiplicity of infection (MOI) 100. The E1A/B-deleted adenovirus, CMV-AdV5-GFP (driven by a CMV promoter), was isolated from a single plaque, expanded in 293 human embryonic kidney cells. All the viruses were purified by double caesium-gradient centrifugation, and titred by plaque assay on 293 cells with the titre expressed as plaque-forming units per cell. The MOI used in this study was previously optimized in macrophages and are described in ref. 17.

Cellular uptake of magnetic nanoparticles by macrophages

MDMs (infected with Ad-CMV-GFP) were cultured overnight with 100 μg ml−1 SPIOs (25 nm) (Sigma-Aldrich, Poole, UK). SPIO accumulation in cells was previously assessed by flow cytometry and confirmed by attraction of the cells towards a magnet placed at the side of the culture dish as observed by light microscopy (Leica Microsystems UK Ltd). Cell viability following SPIO uptake by macrophages was also measured by flow cytometry and compared with cells that were not incubated with SPIOs using the DNA dye propidium iodide (Sigma). Comparisons made using an unpaired Student’s t-test revealed no statistically significant difference between the two groups P=0.4 (Fig. 1b) N=3.

In vitro transendothelial flow assay

The TEM chamber was assembled as shown in Fig. 1a. SPIO-loaded MDM (1.5 × 105 cells per ml in PBS+2% FCS) was flowed over the human umbilical vein endothelial cell monolayer at typical venous flow rates (1.1885, ml min−1) at a sheer stress of 1.4 dyn cm−2, this is equivalent to blood flow through post-capillary venules. The TEM chamber was positioned directly in the isocentre at ∼5-mm distal of a 7 T magnet (Bruker BioSpecAVANCEII, 310-mm bore, MRI system B/C 70/30). The flow in the chamber was in the –z direction (in and out of the magnet bore). We used pulsed gradients 2-ms on and 7-ms off as described by Reigler et al.4. To steer SPIOs into the chamber containing tumour spheroids, we applied a pulsed –y gradient at 50% strength to avoid over-heating (∼300 mT m−1) for 30 min. Post MRT, a 1H volume resonator (Bruker, 300 MHz, 1 kW max, outer diameter 118 mm per inner diameter 72 mm) allowed the capture of Fast low angle shot (FLASH) and RARE MRI images.

Spheroid infiltration by MDMs was then assessed using a fluorescent microscope to detect the GFP-positive cells and flow cytometry using enzymatically dispersed spheroids. To determine the iron content within SPIO-loaded macrophages, cell pellets were solubilized in 70% nitric acid for 7–14 days before analysis. Iron concentrations were quantified against a calibration standard iron solution (Fischer Scientific) by atomic emission spectroscopy using Varian Vista-M PX.

Flow cytometric analysis

Single-cell suspensions were obtained by trypsinizing MDMs (including co-transduced MDMs). Cells were then incubated with for 30 min at 4 °C with mouse anti-CD14, 1:100 in PBS containing 1% bovine serum albumin (Sigma) to prevent nonspecific antibody binding. Alternatively, spheroids were digested using 0.25% trypsin/EDTA to separate the tumour cells and infiltrated macrophages and cell death was analysed by flow cytometry by adding propidium iodide (Sigma) to the cells immediately before running on the flow cytometer.

Orthotopic prostate xenograft model

All mouse procedures were conducted in accordance with the UK Home Office Regulations under the Animals (Scientific Procedures) Act 1986 and the awarded project licence number under which these protocols were performed is PPL:40/3424. In addition, the University of Sheffield Animal Welfare and Ethical Review Body approved all the in vivo experiments used in this study. Male CD1 athymic mice (aged 7–8 weeks, stock number 000711) were used in these studies (Charles Rivers, UK). Animals were randomized before beginning the treatment schedule and were kept in ventilated cages with food and water provided ad libitum. Animal group sizes were calculated by power analysis. In general, a maximum of five animals per group were used unless otherwise stated. One million LNCaP:LUC:GFP cells (a gift from Professor Magnus Essand, Uppsala Sweden) were mixed 1:1 in Matrigel and injected into the dorsolateral prostate. Tumour size was determined by administering luciferin (Molecular Probes) followed by bioluminescent IVIS imaging and measuring the daily weights of the mice. Tumour uptake was monitored by bioluminescence imaging using the IVIS Lumina II imaging system (Caliper Life Sciences). This detects live luciferase-labelled tumour cells, enabling real-time monitoring of tumour growth and spread in the mice. The mice were injected intraperitoneally with 90 mg kg−1 D-luciferin (Caliper Life Sciences) dissolved in sterile water and anaesthetized using 2.5% isoflurane (Abbott Scandinavia AB) in 100% oxygen at 3.5 l min−1 (for induction) in the anaesthesia chamber of the imaging system. Mice were transferred to the dark box and isoflurane was lowered to 1.5%. Images were taken every 3 min as a sequence of 10 images for every group of mice, once a week. Automatic contour regions of interest were created, and the tumour sizes (or tumour radiance) were quantified as photons per second per square centimetre per steradian. Progression and spread of tumours were evaluated by calculating the tumour radiance values from inoculated mice in each group. Tumour-bearing mice were used in experiments ∼14 days following implantation or 21 days in the metastases model when the pulmonary tumours develop following orthotopic implantation of the tumour cells into the prostate17. Mice not developing tumours were excluded from the experiments (<5%). All mice were closely monitored and any displaying signs of rapid weight loss, excessively large tumours (>1010 photons per second, or 15 mm in diameter) or any pain/suffering/distress sufficient to impede natural behaviour were culled.

Use of the MRI scanner to direct cell movement

Three million MDMs with or without SPIOs were administered via tail vein in 100 μl volume of PBS (n=5), control groups received 100 μl PBS (n=5) or 100 μl PBS containing 3 × 106 macrophages without SPIOs (n=5). Immediately after MDM administration, mice were anaesthetized with gaseous isoflurane (Abbott, UK) and then secured within a magnet-compatible holding capsule and MRT was carried out immediately using a 7 T small bore magnet with a 660 mT m−1 gradient insert (Bruker BGA 12-S).

Mice were split into two groups of n=5. Group 1 was a time-matched control without MRT and group 2 underwent 1 h of MRT with gradients pulsed 2-ms on, 7-ms off at 50% total strength (300 mT m−1); and applied direction selected for coarse steering to the tumour site for the prostate (−z and −y) (Fig. 2a). For steering to the lungs (+z and −y gradients), the absence of an x gradient should ensure even distribution of magnetic particles in each lung.

The force on magnetically labelled cells is dependent on whether the SPIOs have become magnetically saturated. When unsaturated, the force is dependent on the magnetic susceptibility of the SPIOs, the magnetic field and also the magnetic field gradient31. However, once the SPIOs reach saturation, the force is no longer dependent on the magnetic susceptibility of the particle but the saturation magnetization and as such only the magnetic field gradient will affect the force applied to the cells7. SPIOs typically reach magnetic saturation well below 1 T, for example, in Riegler et al.6, where the SPIOs become saturated at around 300 mT, therefore, MRT is feasible on clinical MRI systems provided the same magnetic field gradient is used ∼300 mT m−1.

Following MRI steering, high-resolution RARE (retention time (TR)=4.2 s, TE=12 ms, RARE factor 8, 512 × 192, no averaging, 9 slices 1-mm thick) and gated FLASH (TR=8.9 ms, TE=1.2 ms, 24 reps, 128 × 128, flip angle (FA) 15°) images of the tumour (prostate only) were captured using a 1H volume resonator (Bruker, 300 MHz, 1 kW max, outer diameter 118 mm/inner diameter 72 mm). Once complete, relaxometry using multi-slice multi-echo (TE 10 ms, echo spacing 10 ms, 16 echoes, TR 2 s, matrix size 256 × 256) and multiple gradient echo (TE 2.5 ms, echo spacing 3.7 ms, 12 echoes, TR 10 s, matrix size 128 × 128, FA 90°) was performed to assess the transverse relaxation rates. After treatment, animals were killed and tissues, including tumours, kidney, liver, lungs and spleen, were either paraffin wax embedded and fixed for immunohistochemistry or analysed by flow cytometry to determine macrophage uptake (see Supplementary Files for details).

Vascular permeability

A further study was performed to assess vascular permeability in mice. Mice were administered with 3 million SPIO-loaded MDM one group underwent MRT and the other remained in the scanner as described above. Immediately after targeting mice were injected i.v. with a 100 μl mixture of FITC-conjugated Lycopersicon esculentum (tomato) lectin (1 mg ml−1; Vector Laboratories) and Ricinus communis agglutinin I (2.5 mg ml−1; Vector Laboratories). Perfusion-fixation with 4% paraformaldehyde was performed 10 min following lectin administration. Harvested tissue was post-fixed in 4% paraformaldehyde, processed through graded sucrose and embedded in OCT medium (Tissue-Tek). Sections at ∼40 μm were counterstained with 4′,6-diamidino-2-phenylindole (0.05 mg ml−1, Invitrogen) and confocal image stacks were acquired by confocal microscopy (Nikon). Measurement of vascular volumes was performed on images from tumour-bearing mice with and without MRT targeting (n=3 mice per group and 5–10 fields of view).

In addition, vasculature leakage was also assessed using the contrast agent Gd-DPTA31,32. Mice receiving SPIO-loaded MDM with and without MRT were removed from the scanner and injected (via tail vein) with a 0.1 mmol Kg−1 dose of Gd-DPTA (Magnevist) (N=3 mice per group). Mice were then returned to the scanner and T1-weighted imaging (TR=100 ms, TE=3.7 ms, FA=30 degs, matrix size=256 × 256) was performed for 15 min (50 repetitions) post injection. Data were used to assess pooling of the contrast agent over time as an indicator of a leaky, damaged vasculature. Experiments were repeated with Gd-DPTA alone (no iron-labelled macrophages or MRT). Uptake of Gd-DTPA was monitored in tumour tissue over the 15-min period; any pooling would result in increased signal over this time period. We also monitored GD-DTPA uptake in the muscle surrounding the spinal cord (vertically away from the –y gradient targeted tumour region) as a control region where we expect no vascular disruption due to MRT. Direct comparisons in changes in signal intensity were made between these two regions to investigate any vascular damage in the targeted region. Finally, if there is no vascular damage, Gd-DTPA should enter the renal system; to confirm this in our groups, we imaged the kidneys.

Therapeutic studies

HSV1716 in vitro studies are described in the accompanying Supplementary Files. In vivo studies were performed as follows. Tumour-bearing mice received tail-vein injections of either 3 million MDM alone or armed with Seprehvir at MOI 50, 1 × 107 Seprehvir only or PBS (n=5 mice per group). Of note, three groups of mice were administered with MDM+OV, one group underwent MRT for 1 h, one sat in the MRI scanner but had no MRT (MDM+OV no MRT) and another group did not enter the MRI scanner (MDM+OV). Tumour size was monitored by IVIS Lumina II imaging (IVIS, Caliper Life Sciences). Animals were killed once tumours reached the maximum volume (>1010 photons per second) permitted by UK Home Office Regulations. Excised tissues including tumours, kidney, liver, lungs and spleen were embedded in OCT or paraffin wax for immunocytochemical/histological labelling studies.

Tissue analysis

Tissues were divided into two; one part was formalin fixed for immunohistological analysis and the other was dissected free of adherent fibrous and fatty tissue and treated with collagenase. Flow cytometry. Cell viability was determined using LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). All FACS data were analysed on an LSR II flow cytometer (BD Biosciences) using FlowJo software (Tree Star). Histology. Five-micron sections of all organs were incubated with specific antibodies for target antigens; for the vasculature we used CD31 (1:100; AbD Serotec) and for macrophages human CD68 (Dako, Ely, UK) at 1:100. A biotinylated secondary antibody system was used in conjunction with a streptavidin-conjugated horseradish peroxidase. Peroxidase activity was localized with diaminobenzidine (Vectastain Elite ABC kit, Vector Labs). To detect iron in the tumours (where cell densitites were high), sections were stained with Perls Prussian blue and counterstained with eosin for improved contrast. To detect cancer cells in the lungs, all lung sections were stained with epithelial cell adhesion molecule or haematoxylin and eosin. All immune-localization experiments were repeated on multiple tissue sections and included isotype-matched controls for determination of background staining. To assess necrosis, the area of necrosis within the whole-tumour section was determined visually, and the proportion of necrotic nonviable tumour areas over the whole section was calculated using ImageJ software (National Institute of Health). For each group, the mean percentage of necrosis and standard error were calculated. The results are presented as the mean tumour necrosis (%) for all tumours (five slices per each tumour) in each treatment group. Immunofluorescence. harvested tissue was post-fixed in 4% paraformaldehyde, processed through graded sucrose and embedded in OCT medium (Tissue-Tek) and stored at −80 °C. Frozen sections were dried for 10 min at room temperature and blocked in 5% horse serum+0.5% saponin in PBS for 30 min. Sections at ∼40 μm were stained with anti-GFP 1:200 (ab290 abcam, UK) and CD68 (1:100) for 1 h and then secondary antibodies donkey anti-rabbit alexa fluor 488 and goat anti-mouse 540 at 1:100 dilution (Invitrogen, Paisley, UK) and finally counterstained with prolong gold-antifade mountant with 4′,6-diamidino-2-phenylindole (0.05 mg ml−1, Invitrogen). Images of macrophage infiltration into primary and pulmonary LNCaP tumours were captured using a spinning disc confocal microscope (Olympus IX81, PerkinElmer, UK). Confocal image Z-stacks of tumours were captured at 1-μm increments at × 20 magnification.

Statistical analysis

Data are means±s.e.m. (Prism 5; GraphPad Software). Two-tailed Student’s t-test was used to analyse the statistical significance of the data unless otherwise stated. Differences were termed significant with a P value of <0.05.