Rho kinase pathway

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Tumour necrosis factor-alpha increases extravasation of virus particles into tumour tissue by activating the Rho A/Rho kinase pathway Takahiro Seki a, Fionnadh Carroll b, Sam Illingworth c, Nicky Green b, c, Ryan Cawood a, Houria Bachtarzi a, Vladimir Šubr d, Kerry D. Fisher a, c, Leonard W. Seymour a,⁎ a

Department of Oncology, ORCRB, University of Oxford, Old Road Campus, Headington, Oxford, OX3 7DQ, UK Clinical Biomanufacturing Facility, Department of Medicine, University of Oxford, OX3 7JT, UK PsiOxus Therapeutics Ltd, Cherwell Innovation Centre, 77 Heyford Park, Upper Heyford, Oxfordshire, OX25 5HD, UK d Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq, Prague 6 16206, Czech Republic b c

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Article history: Received 12 May 2011 Accepted 14 August 2011 Available online 22 August 2011 Keywords: Drug delivery Adenovirus Vascular permeability Tumour necrosis factor-α Rho A/Rho kinase inhibitor EPR-effect

a b s t r a c t Tumour Necrosis Factor alpha (TNF) is a pleiotropic pro-inflammatory cytokine with known vascular permeabilising activity. It is employed during isolated limb perfusion to enhance delivery of chemotherapeutic drugs into tumour tissue. The use of conditionally-replicating lytic viruses, so called ‘oncolytic virotherapy’, provides a new approach to cancer treatment that is currently limited by the low efficiency of extravasation of viral particles into tumours. We report here evidence that TNF significantly enhances the delivery of virus particles through the endothelial layer to allow access to tumour cells both in vitro and in vivo. Intravenous administration of TNF resulted in a 3- to 6-fold increase in EL4 tumour uptake of Evans Blue/Albumin, adenovirus and long-circulating polymer coated adenovirus. Interestingly, endothelial permeabilisation could be suppressed in vitro and in vivo by Y-27632, a Rho kinase inhibitor, without inhibiting viral infection. These data indicate that TNF can enhance the delivery of virus particles into tumours through a Rho A/Rho kinase dependent mechanism and may be a valuable strategy for increasing the delivery of oncolytic viruses and other therapeutic agents. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Inefficient delivery of therapeutic agents to tumour deposits is a major limitation to successful cancer therapy and provides a particular challenge when agents are given intravenously to treat disseminated disease. The poorly structured tumour neo-vasculature, a consequence of rapid angiogenic growth, is often relatively leaky and allows extravasation of fluid into the tumour that leads to raised interstitial fluid pressure (IFP) within the tumour microenvironment. This is usually associated with poor drainage of tissue fluid via tumour lymphatics which limits interstitial fluid movement in deeper regions of the tumour. In perivascular regions, however, there is a high level of fluid extravasation, providing good delivery of drugs and nutrients to nearby tumour cells. The majority of this fluid returns to the bloodstream via the post-capillary venules, forming a kind of ‘perivascular fluid shunt’. The well-documented accumulation of macromolecules (such as albumin and macromolecular drugs) by the ‘Enhanced Permeability and Retention’ (EPR) effect [1] is thought to reflect this high level of perivascular fluid translocation, with macromolecules being deposited predominantly at the vascular edge of ⁎ Corresponding author at: Dept Oncology, University of Oxford, Old Road Campus, Headington, Oxford, OX3 7DQ, UK. Tel.: + 44 1865 617021; fax: + 44 1865 617022. E-mail address: [email protected] (L.W. Seymour). 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.08.022

the tumour where they can become incorporated into the expanding tumour stroma [2]. There have been various attempts to exploit these modified fluid translocation properties of tumours to improve the efficiency of delivery of anti-cancer drugs. Macromolecular drug conjugates and liposomes have been designed to try and exploit the EPR effect, and this phenomenon may contribute to the mechanism of action of agents such as the liposome Doxil, as well as increasing tumour uptake of many drugs that are tightly bound to plasma proteins. Attempts to further enhance the delivery of therapeutics to tumours have involved the study of vaso-modulating drugs and proteins aiming to permeabilise the tumour endothelial layer still further to allow increased uptake of therapeutic agents. One candidate protein, tumour necrosis factor alpha (TNF), has been extensively studied for its endothelial cell regulatory properties [3]. TNF is a pleiotropic proinflammatory cytokine that showed promising anticancer activity in murine models [4], apparently mediating a combination of permeabilising effects and direct toxicity on tumour-associated vasculature. Following these interesting preclinical findings, TNF was studied in a series of single agent intravenous phase I clinical trials in the 1980s, however body wide vascular leakage gave serious side effects and its systemic use was largely abandoned [5]. Interestingly TNF is now routinely used in the intra-arterial isolated limb perfusion treatment of sarcoma and melanoma, often in conjunction with melphalan [6,7]. In

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these procedures a 95% response rate can be observed, typically with a 26% complete response rate [8]. In this setting TNF appears to exert a selective permeabilisation effect on tumour-associated vasculature, allowing increased extravasation of therapeutic agents [7]. Accordingly we set out to explore whether TNF might be capable of increasing the uptake of particulate drugs across endothelial layers, particularly to improve delivery to disseminated tumours. Oncolytic adenoviruses have been studied extensively for virotherapy of cancer, where they replicate selectively within tumour cells and lyse them before spreading to infect adjacent cells. Despite great selectivity and potency, this approach is currently limited by inefficient delivery of virus particles to disseminated tumour deposits, particularly following intravenous injection [9]. We therefore wanted to investigate whether treatment with TNF could enhance the delivery of adenoviral particles through endothelial cell layers, and particularly whether it could enable increased delivery of virus particles into tumours in vivo leading to better therapeutic outcome.

TNF (concentrations up to 10 ng/ml) for 0.5 h, 2 h or 20 h which was added to both top compartments. After TNF treatment, the medium was replaced with fresh medium without phenol red. FITClabelled dextran (2 mg/ml, 4 kDa and 70 kDa; Sigma-Aldrich) was then added to the top compartment. Samples (50 μl) were taken from the bottom compartment at defined time points, and fluorescence levels were determined using a Wallac Victor Platereader with λex 480, λem 518 nm. HUVEC cells were treated as above. After TNF treatment, endothelial cells on the insert membrane were transferred to other plates in which A549 cells were seeded on the lower layer (24-well plate, 10 5 A549 cells per well), and 100 μl of Adluc was added into the top compartment (1000 vp/A549 cell) for 3 h. At 24 h post addition of the virus, media were removed and cells in both compartments were washed with PBS. Cells were then lysed and tested for luciferase levels using the luciferase reporter system (Promega) and a luminometer (LB9507, Berthold Technologies, Bad Wildbad, Germany). Protein levels were measured using the BCA assay (Sigma-Aldrich).

2. Materials and methods Recombinant human TNF (TNF) and mouse TNF (mTNF) were obtained from R&D Systems (Minneapolis, MN). Evans blue, formamide, Y-27632 and FITC-Dextran were purchased from Sigma-Aldrich (Poole, UK). Rhodamine-phalloidin was purchased from Cytoskeleton (Denver, CO). Dulbecco's Modified Eagle Medium (DMEM) and EGM2 were obtained from Lonza (Basel, Switzerland). 2.1. Adenovirus preparations and cell culture Human umbilical vein endothelial cells (HUVEC) were maintained in EGM-2 medium (Lonza) containing 2% foetal bovine serum (FBS). Mouse brain endothelioma bEnd.3 (a kind gift from Prof Adrian Harris), mouse ascites lymphoma lymphoblast EL4 (ECACC No. 85023105), and human lung carcinoma A549 (ECACC No. 86012804) were each maintained at 37 °C in a 5% CO2 humidified environment in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Batches of E1, E3-deleted type 5 adenovirus expressing luciferase under transcriptional control of the cytomegalovirus immediate early promoter (Adluc) were purchased from Hybrid BioSystems (Oxford, United Kingdom). For studies using tropism-ablated polymer coated virus, Adluc was coated with a multivalent polymer (20 mg/ml) based on poly(N-[2hydroxypropy]methacrylamide) (HPMA) bearing pendant side chains terminated in amino-reactive thiazolidone (TT) groups (as previously described [10], EC193, Mw average 87,300 Da; TT content, 10.37 mol%,). 2.2. Actin cytoskeleton immuno-fluorescence microscopy HUVEC and bEnd.3 cells were cultured on Lab-Tek chamber slides coated with gelatin. The cells were treated with TNF (0–10 ng/ml for either 0.5, 2 or 20 h). To visualise the actin cytoskeleton, cells were fixed in 4% formaldehyde in PBS, permeabilized with Triton X-100 (0.01% v/v) and then incubated with Rhodamine-phalloidin (70 nM) and mounted in Vectashield with DAPI (Vector Laboratories, Peterborough, U.K.). Fluorescence images were taken with a Nikon Eclipse 80i microscope and Hamamatsu ORC-ER CCD camera. 2.3. Assessment of endothelial monolayer permeability using FITCDextran as a tracer HUVEC or bEnd.3 cells were grown on Transwell filters (Costar; pore size, 0.4 μm) for 1 day after becoming confluent. These were judged to be the optimum conditions to allow reproducible culture of tightly packed cells for 24–48 h without significant cell loss or overgrowth. The endothelial cells were exposed to serial dilutions of

2.4. Assessment of endothelial monolayer permeability using polymer coated Adluc as a tracer HUVEC cells on Transwell filter membranes were treated with TNF as described above. The insert bearing the endothelial cells was then transferred to another plate seeded with A549 cells (10 5/well) containing fresh DMEM with 2% FBS. Polymer coated Adluc was added to the top compartment for 3 h at a concentration of 1000 vp/A549 cell. The number of virus particles transferring through the endothelial cell-coated membrane into the bottom compartment was quantified after 3 h with quantitative PCR (qPCR) [11]. DNA was extracted from samples of medium using the Genelute mammalian DNA extraction kit according to the manufacturer's instructions (G1N-350, Sigma-Aldrich). QPCR was performed on 5 μl of extracted sample using the primers, probe and equipment described by Green et al. [11]. 2.5. Assessment of TNF induced Evans blue dye extravasation in vivo Mouse ascites lymphoma lymphoblast EL4 (1 × 10 5 cells/mouse) cells were implanted subcutaneously (s.c.) in the dorsal skin of C57 Black 6 mice (5 wks, female, Biomedical Services, University of Oxford). When the EL4 tumour diameters reached 5–8 mm, TNF (1 μg/mouse) was injected intravenously. After 0.5 h, Evans blue dye (10 mg/kg in 0.1 ml PBS) was also injected intravenously. After 6 h, mice were euthanized and the tumours were removed and weighed. Evans blue dye in the tumour was extracted with formamide, and was quantified using a UVmini 1240 UV–vis spectrometer (Shimadzu, Kyoto, Japan) at 620 nm against known concentrations of Evans blue dye. To investigate whether Rho kinase plays a role in the permeability of tumour vasculature, some animals were injected with Rho kinase inhibitor Y-27632 0.5 h before TNF. 2.6. Assessment of TNF induced extravasation of Adluc and pcAdluc in vivo Mice bearing established EL4 tumours were treated with Y-27632 and TNF as described above, 0.5 h before intravenous injection of Adluc and pcAdluc (10 10 copies per mouse) in PBS. To assess the pharmacokinetics of virus particles, blood samples (20 μl) were taken at 0.5, 2 and 6 h after virus injection. Animals were put down after 6 h and tumours, blood and livers were taken and extracted using the Genelute mammalian DNA extraction kit. The number of the virus genomes in each tissue was determined using QPCR as previously described. Luciferase levels in organs were measured using the luciferase reporter system. All animal procedures were carried out in accordance with the United Kingdom Animals (Scientific

Procedures) Act 1986 and Oxford University local medical sciences ethical committee approval, under the terms of Home Office project licence 30/2333.

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2.7. Statistical analysis All data are expressed as means ± SEM. Throughout, p values were calculated by Student's T test and exact two-sided Wilcoxon tests using Microsoft Excel software. Significance was determined as a p value of b0.05. 3. Results 3.1. TNF induced endothelial monolayer permeability to FITC-Dextran in vitro Human umbilical vein endothelial cells have a partially activated, proliferating phenotype that may make them useful in vitro models for tumour-associated angiogenic vasculature. We hypothesised that TNF treatment might be able to increase the permeability of contiguous monolayers of HUVEC in vitro and this might serve as a reductionist model for the in vivo permeabilising activity. Accordingly HUVECs were seeded on trans-well filters and allowed to reach confluence and their use for permeability studies was investigated. Initial studies assessed the influence of TNF pre-treatment time (0.5 h, 2 h or 20 h) on the diffusion of FITC-labelled Dextran through the endothelial cell monolayer. FITC-Dextran molecules of either 4 kDa or 70 kDa average molecular weight were added to the top chamber of a trans-well assay and the movement of molecules into the bottom chamber was measured by quantifying FITC fluorescence. In the absence of an endothelial cell monolayer approximately 30–40% of the FITC-Dextran added to the top compartment migrated through the filter membrane after 3 h, with no difference in transfer between the 4 kDa and the 70 kDa Dextran molecules (Fig. 1A and B). In the presence of the endothelial cell monolayer the baseline transfer of both FITC labelled dextrans was reduced to below 15% but again molecular weight showed little to no effect. To determine whether TNF could increase the transfer of Dextran, HUVEC in the top compartment were incubated with TNF (10 ng/ml) for 0.5, 2 or 20 h before the addition of the FITC-Dextran. This treatment increased the quantity of FITC-Dextran passing through the cell monolayer and again this increase was independent of the dextran molecular weight with both the 4 kDa and the 70 kDa molecules passing through with equal efficiency (Fig. 1A and B). Transfer of FITC-Dextran increased with the length of pre-exposure to TNF, suggesting a time dependent increase in endothelial cell permeability, reaching a maximum after 20 h pre-exposure. TNF treatment (10 ng/ml) was confirmed to be non-toxic to endothelial cells after either 2 h or 20 h exposure (Fig. S1A and B), suggesting that the increased transfer of dextran observed was not due to endothelial cell death removing the physical barrier to transfer. To determine whether the TNF mediated increase in monolayer permeability we had observed in our trans-well assay might relate to shape changes resulting from the formation of actin stress fibres within endothelial cells we visualised actin polymerization in HUVECs using rhodamine labelled phalloidin, which binds directly to F-actin. Whilst there were very few actin stress fibres detectable in resting cells, fibres were clearly visible across the cell body of the endothelial cells after exposure to TNF for either 2 h or 20 h (Fig. 1A). However, this response was only clearly visible at doses of 10 ng/ml TNF or above, with lower doses showing little to no effect. Fig. 1. TNF increases stress fibre formation in HUVEC and induces an increase in endothelial monolayer permeability to FITC-Dextran. Confluent HUVEC monolayers grown on 0.4 μm membranes were treated with vehicle control (■), 10 ng/ml of TNF for 0.5 h (□), TNF for 2 h (▲), or with TNF for 20 h (●). No HUVEC on the membrane (◆) is the positive control. Medium was replaced and either 4 kDa (A) or 70 kDa (B) FITC-Dextran was added to the top compartment for varying lengths of time before determination of fluorescence levels at 24 h as a measure of membrane passthrough. (C) HUVEC were treated with TNF at defined doses for 2 h or 20 h, respectively (n = 6), followed by staining with Rhodamine-phalloidin to determine stress fibre formation by Fluorescence microscopy.

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The greatest actin stress fibre formation was observed after 20 h exposure to 10 ng/ml TNF, which is consistent with the increased transfer of FITC-Dextran through the trans-well filter (see earlier). These results indicate that TNF can significantly increase the formation of actin stress fibres in endothelial cells and can mediate enhanced

permeability of a human endothelial monolayer in vitro to macromolecules such as FITC-Dextran. 3.2. TNF induces endothelial monolayer permeability to virus migration in vitro The equal transfer of the 4 kDa and 70 kDa species of FITC-Dextran through the endothelial monolayer suggested activation of a paracellular or intercellular transfer mechanism with no significant size-related restrictions for soluble macromolecules in the region of 5–10 nm diameter. To assess whether TNF treatment could also facilitate the transfer of nanoparticles, the transfer of a reporter adenovirus (particles 110 nm diameter approx.) encoding luciferase (Adluc) through the endothelial cell monolayer was determined. For these studies HUVEC were plated as usual on the semipermeable membrane of the transwell assay and A549 lung carcinoma cells were seeded in the bottom compartment. Luciferase transgene expression in the A549 cells provided a sensitive indicator of virus transfer through the membrane. HUVEC monolayers were exposed to TNF for either 2 or 20 h followed by a 3 h incubation with Adluc at 1000 vp/cell (Fig. 2A). Luciferase levels were determined 24 h post initial infection. TNF pre-treatment of the HUVEC mediated a dose-dependent increase in the quantity of luciferase expression in the A549 cells, for example 2 h pre-exposure of HUVEC to TNF (25 ng/ml) increased luciferase expression in the A549 cells from 7.3 × 10 2 RLU/mg to 3.3 × 10 3 RLU/mg (Fig. 2B). Pre-exposure of HUVECs for 20 h mediated a similar increase in luciferase expression in the A549 cells, increasing from 3.2 × 10 2 RLU/mg to 2.4 × 10 3 RLU/mg (Fig. 2C). Whilst the level of infection of the HUVECs was unchanged following 2 h pre-exposure to TNF (Fig. 2D), 20 h pre-exposure to even low concentrations of TNF (down to 0.1 ng/ml) suppressed Adluc infection by 1.5–3 fold (Fig. 2E). This suggested that the anti-viral effect of TNF in primary endothelial cells occurs at far lower concentrations but longer time frame than the permeabilisation effect. To investigate whether virus transfer through the endothelial monolayer was saturable, transfer of a 10-fold higher virus concentration was investigated (10,000 vp/cell). After 20 h pre-treatment with TNF the permeability (measured as the proportion of virus particles transferred) was not significantly different from the 10-fold lower dose of Adluc (Fig. S2). This indicated that the trans-endothelial virus transfer process was not saturable over this concentration range, and is compatible with transfer in the fluid phase although high-capacity receptor-mediated interactions cannot be discounted. Together these data confirm that TNF can significantly increase the permeability of endothelial cell monolayers in vitro, allowing improved transfer of virus particles with an external diameter above 100 nm. 3.3. The involvement of actin cytoskeleton and Rho A/Rho kinase on TNFinduced virus permeability of endothelial cell monolayers Treatment with TNF causes rapid formation of actin stress fibres within endothelial cells that may play a role in the monolayer-permeabilising effects of TNF [12]. Actin polymerization is generally dependent on activation of the 19 kDa G protein Rho A, hence treatment with the Rho A/Rho kinase inhibitor Y-27632 [13,14] was used to assess involvement Fig. 2. TNF increases human endothelial monolayer permeability to Adluc in vitro. (A) Diagrammatical representation of the permeability assay performed with Adluc. Confluent HUVEC monolayers were grown on a 0.4 μm membrane and were treated with 0.1 to 25 ng/ml of TNF for 2 h (B and C) or 20 h (D and E). After the treatment for 2 h or 20 h the HUVEC monolayer was transferred to a plate in which A549 cells were seeded on the lower layer. 1000 vp/cell Adluc were added to the top compartment for either 3 h or 1.5 h. Luciferase levels in the A549 cells in the bottom compartment (B and D) and HUVEC on the top compartment (C and E) were measured using the luciferase reporter system and corrected for protein levels using the BCA assay.

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of actin polymerization in monolayer permeabilisation. Treatment with Y-27632 (100 μM for 20 h) showed no cytotoxicity to HUVECs (data not shown) although addition of Y-27632 0.5 h prior to TNF completely inhibited TNF induced actin polymerization (Fig. 3A). It also significantly suppressed the transfer of adenovirus particles through the HUVEC monolayer if cells were pre-exposed to Y-27632 for 0.5 h before either 2 h or 20 h exposure to TNF (Fig. 3B). No significant changes in the level of infection of HUVECs were observed using Y-27632 (Fig. 3C). 3.4. TNFα increases the permeability of mouse endothelial cell monolayers These data show that recombinant human TNF could increase the permeability of a human endothelial cell monolayer, however, prior to investigating the effect of human TNF in an animal model we sought to confirm the inter-species effect of human TNF on murine endothelial cell monolayers. A mouse endothelial cell line, bEnd.3, was pre-treated with recombinant human TNF at 10 ng/ml for either 2 h or 20 h. As shown in Fig. 3D, TNF significantly increased the penetration of adenoviral particles through the mouse endothelial cell monolayer into the bottom compartment. After 20 h pre-exposure to TNF the luciferase expression in the A549 cells increased from 2.5 × 103 RLU/mg to 7.7 × 103 RLU/mg (Fig. 3D). As with the HUVEC monolayers, this effect was significantly suppressed by 0.5 h treatment with Y-27632 before exposure to TNF (Fig. 3D). The addition of TNF to bEnd.3 cells increased the formation of actin stress fibres within the endothelial cells, with significant stress fibre formation mediated by doses at or above 10 ng/ml (Fig. S3-A). No TNF related cytotoxicity was observed in bEnd.3 cells at doses well above 10 ng/ml (Fig. S3-B). 3.5. Ability of TNF to increase the extravasation of Evans blue into tumours in vivo Evans blue is a dye which binds to plasma albumin and can be used to measure protein extravasation. To demonstrate whether TNF could increase the permeability of tumours to plasma albumin in vivo, EL4 tumour bearing mice were treated by intravenous injection of 1 μg/mouse TNF 30 min before i.v. injection of Evans blue (10 mg/kg). After 6 h mice were euthanized and the amount of Evans blue extravasating into the tumours was assessed visually and quantified after formamide extraction. EL-4 tumours taken from mice treated with TNF were obviously more blue than control tumours (Fig. 4A), suggesting significant enhanced leakage of albumin from tumour vasculature. The amount of Evans blue extravasating into the tumour tissue was significantly increased from 15.4 μg/g tumour to 27.9 μg/g tumour (Fig. 4B). This confirmed that TNF administration could significantly increase the permeability of tumours to macromolecules. To determine whether this effect was mediated by the activation of the Rho A/Rho kinase pathway, Y-27632 was injected intravenously 0.5 h before administration of TNF and Evans blue, as above. Treatment with Y-27632

Fig. 3. Rho kinase inhibitor prevents TNF induced permeability to Adluc. (A) HUVEC were pre-treated with 20 μM Y-27632 0.5 h before 10 ng/ml of TNF treatment. Cells were then stained with Rhodamine-phalloidin, as described in material and methods. Confluent HUVEC monolayers grown on 0.4 μm membranes and were pre-treated with 20 μM Y-27632 0.5 h before adding 10 ng/ml of TNF. The HUVEC monolayer was then transferred a plate seeded with A549 cell in the lower compartment. 1000 vp/cells of Adluc was added to the HUVEC (upper compartment) for 3 h. Luciferase levels in the A549 in the bottom compartment (B) and HUVEC on the top compartment (C) were measured using the luciferase reporter system and corrected for protein levels using the BCA assay. Confluent bEnd.3 monolayers grown on 0.4 μm transmebrane were pre-treated with 20 μM Y-27632 0.5 h before 10 ng/ml of TNF treatment, and then transferred to a plate in which A549 cells had been seeded in the lower compartment. 1000 vp/cells of Adluc was then added to the bEnd.3 containing top compartment for 3 h. Luciferase levels in the A549 cell in the bottom compartment (D) and bEnd.3 in the top compartment (E) were measured using the luciferase reporter system and corrected for protein content using the BCA assay.

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significantly decreased the extravasation of Evans blue into EL4 tumours when compared to TNF treatment alone (Fig. 4B), giving levels similar to control mice without TNF treatment (11.3 μg/g tumour compared to 15.4 μg/g tumour, respectively). 3.6. TNF induces endothelial permeabilisation and increased extravasation of adenovirus particles in vivo We then tested whether the vascular permeabilisation effect mediated by TNF in vivo would allow increased extravasation of Adluc particles into EL4 tumours. Intravenously administered Adluc particles (1× 1010 viral particles) showed blood stream kinetics with an alpha half-life of less than 2 min [9] and this remained unaffected by treatment with TNF (10 mg/kg, 30 min before Adluc) (Fig. S4-A). However, TNF treatment was able to increase the amount of Adluc accumulating in the EL4 tumour by N3-fold and also increased the luciferase activity in the tumour by N7-fold (Fig. 4C and D). Pre-treatment with Y27632 30 min before TNF significantly suppressed the tumour extravasation of Adluc particles when compared to injection of TNF alone (Fig. 4C). In the liver the number of Adluc genomes was also significantly increased by TNF treatment and this effect was decreased by Y-27632 treatment (Fig. 4E), however TNF significantly decreased the luciferase activity in the liver despite the increase in virus particle uptake (Fig. 4F). 3.7. TNF increases tumour permeability to polymer coated Adluc both in vitro and in vivo Coating of adenovirus with amino-reactive multivalent HPMAbased polymers ablates infectious tropism and provides a platform for retargeting infection towards specific receptors as well as allowing improved blood circulation kinetics in vivo that increases virus accumulation within the tumour stroma [15,16]. Consistent with previous experiments it was found that the infection of endothelial cells in vitro by Adluc coated with HPMA polymer (20 mg/ml EC-193, pcAdluc) (Fig. 5A) was decreased compared to the non polymer-coated virus (Fig. S5A) [15]. To investigate whether TNF treatment could increase the transfer of polymer coated adenovirus through endothelial monolayers in vitro, HUVECs were treated with TNF for either 2 h or 20 h at 10 ng/ml in the transwell assay before the addition of polymer coated Adluc at 1000 vp/A549 cell. The number of coated virus particles transferring through to the lower compartment was determined by QPCR. As shown in Fig. 5B, the amount of viral particles moving to the lower compartment was significantly enhanced by TNF treatment, approximately 10-fold compared to untreated cells. As with previous studies, 2 h pre-treatment with Y-27632 significantly decreased the number of virus particles accumulating in the bottom compartment compared to treatment with TNF alone (Fig. 5B). Finally, we assessed whether TNF could enhance the permeability of EL4 tumours in vivo to polymer coated adenovirus. Blood circulation kinetics showed a 10-fold longer clearance half-life of pcAdluc compared to non-polymer coated Adluc (Fig. S5-B compared to Fig. S4-A), however, no significant difference in blood Fig. 4. TNF increases EL4 tumour permeability to Evans blue and virus particles. EL4 cells (1 × 105 cells/mouse) were implanted subcutaneously (s.c.) in both dorsal flanks of C57BL6 mice. When the EL4 tumours were 5–10 mm in diameter, 1 μg/mouse of TNF was injected i.v. In the Rho kinase study, EL4-tumour bearing mice were pretreated with 1 mg/kg of Y-27632 for 0.5 h before TNF treatment. 0.5 h after TNF, 10 mg/kg Evans blue in 0.1 ml of PBS was injected i.v. 6 h post injection mice were killed, the tumours were removed and photographed (A). The dye in the tumour was extracted with formamide and quantified (B). To determine the effect of TNF on extravasation of Adluc particles into the tumour in vivo, EL4 tumour bearing mice (5 to 10 mm in diameter) were treated with and without Y-27632 for 0.5 h before TNF treatment. After a further 0.5 h Adluc (1010 genomes per mouse) was injected i.v. and 6 h later tumours and livers were harvested and the DNA was extracted. The number of virus genomes in tumour (C) and liver (E) was determined using qPCR, and luciferase levels in tumour (D) and liver (F) were measured using the luciferase reporter system.

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circulation kinetics of pcAdluc was observed following treatment with either TNF and/or Y-27632 (Fig. S5-B). Injection of TNF at 10 mg/kg 30 min before i.v. injection of 1 × 10 10 vp of Adluc allowed a N5-fold increase in pcAdluc extravasation into EL4 tumours compared to virus administration alone (Fig. 5C). The luciferase activity in the tumour was also increased by N3-fold by treatment with TNF compared to control tumours (Fig. 5D). In agreement with previous studies the increase in extravasation of polymer coated virus mediated by TNF was significantly suppressed by injection of Y-27632 30 min before TNF treatment (Fig. 5C). Whilst these results were similar to those observed with the non-polymer coated Adluc, no significant difference in number of virus particles in liver was observed with pcAdluc following TNF treatment (Fig. 5E) and only a 2-fold decrease in hepatic luciferase expression was observed (Fig. 5F). These data show that administration of TNF in conjunction with pcAdluc allows increased virus particle extravasation which leads to an increase in tumour associated gene expression compared to injection of pcAdluc alone. 4. Discussion Increasing the quantity of therapeutic agents entering tumours from the bloodstream is desirable to improve the performance of most systemic treatment strategies, and this is especially challenging for particulate therapeutics such as oncolytic virotherapy where size constraints are particularly restrictive [17,18]. Here we have shown in vitro that human TNF can significantly increase the permeability of both human and murine endothelial cell monolayers to FITC-labelled Dextran up to 70 kDa, and also to Adluc particles and polymer coated Adluc. In vivo, TNF can also significantly increase the extravasation of albumin (visualised using Evans blue dye), Adluc particles and polymer coated Adluc particles into syngeneic murine EL4 tumours without inhibiting virus infection. Transgene expression achieved by Adluc and pcAdluc within the tumour was up to 7-fold higher in the presence of TNF than in mice receiving equivalent treatments without TNF. This contrasts with its activity in normal liver where hepatic transgene expression was not increased despite elevated levels of extravasation. TNF is known to have an antiviral activity [19], although it appears to be more effective at inhibiting virus infection in normal liver than in tumour tissue. This may well reflect deficiencies in immune defenses that are often associated with tumours, as well as possible resistance to TNF [20]. TNF is a pleiotropic pro-inflammatory cytokine, known to have vaso-permeabilising activities as well as multiple roles in immunity and inflammation. It is produced as a membrane bound precursor which is cleaved to become a homo-trimer soluble molecule found in plasma. It binds to two receptors, p55 (TNFR1) and p75 (TNFR2) and can mediate a range of effects depending on the conditions and the target cell [7]. In angiogenic endothelial cells TNF appears to influence endothelial cell morphology and actin polymerization, predominantly by signalling through TNFR1 [21], leading to endothelial cell contraction and expansion of the intercellular junctions in the endothelial cell

Fig. 5. TNF increases the permeability of EL4 tumours to polymer coated Adluc and a Rho kinase inhibitor suppresses this permeabilisation effect both in vitro and in vivo. (A) Chemical structure of EC-193 polymer. Adluc was coated with EC-193 at 20 mg/ml, as described in material and methods. (B) Confluent HUVEC monolayers grown on 0.4 μm membranes were pre-treated with 20 μM Y-27632 0.5 h before 10 ng/ml of TNF treatment, and then transferred to new medium containing plate without A549, followed by adding 1000 vp/cells of pcAdluc for 3 h. Number of virus genomes in the bottom compartment was quantified by qPCR. To demonstrate whether TNF increases pcAdluc permeability into tumours in vivo, EL4 tumour bearing mice (5 to 10 mm in diameter) were treated with and without Y-27632 30 min before TNF treatment. After a further 30 min pcAdluc (1010 copies per mouse) was injected i.v. and tumours and livers were harvested 6 h later and the DNA was extracted. The number of virus genomes in each tissue was determined using by qPCR. Luciferase levels in each tissue were measured using the luciferase reporter system. (C) The number of virus and (D) the luciferase activity in tumour. (E) The number of virus and (F) the luciferase activity in liver.

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layer lining blood vessels. This allows increased fluid exchange between the blood stream and the tissue or tumour parenchyma and would be expected to promote more efficient delivery of therapeutic agents to tumours, as observed here. However at high doses, TNF exerts a direct antivascular (and anticancer) activity leading to endothelial cell death and vascular collapse [7]. Most clinical trials of TNF have used high doses to explore its direct antivascular effects and have encountered serious side effects. For example in a phase 1 clinical trial involving dual treatment with TNF and human IFN-γ in patients with advanced gastrointestinal cancer, the maximal tolerated dose (MTD) for each agent was 150 μg/m2/day administered on 5 consecutive days [22]. The main toxicity observed was vasoplegia and hyperbilirubinemia suggesting significant vascular damage. It was reported that the addition of IFN-γ synergistically enhanced the TNF-induced endothelial cell death and increased the antineoplastic response seen in this study [23–25]. Following observations such as these, systemic delivery of TNF was largely abandoned and local administration was explored as a means to avoid systemic toxicities. Lejeune et al. have subsequently demonstrated that TNF can be used clinically with dramatic results in a treatment regimen involving administration of TNF, IFN-γ, and melphalan through isolated limb arterial perfusion to treat sarcoma and metastatic melanoma of the limbs [7,8,26,27]. The success of the approach is thought to reflect a combination of direct anti-endothelial toxicity of TNF and induced endothelial leakage which allows enhanced delivery of the co-delivered alkylating agent melphalan to tumour cells [24,28]. Although the doses of TNF used in these isolated limb studies (1–3 mg/person) are higher than the maximum tolerated dose of TNF that can be given systemically to humans, it may be possible to employ lower doses of TNF to modulate vascular permeability and improve delivery without exerting a direct endothelial toxicity [29]. This possibility does not appear to have been fully explored in humans so far and an investigational study is probably warranted to explore use of low dose TNF to improve tumour delivery of anticancer agents (including imaging agents) given intravenously. The use of adenovirus systemically is limited by rapid inactivation by the immune system, poor particle kinetics and inefficient extravasation into tumour tissues. We have previously shown that polymer coating of adenovirus allows some of these limitations to be overcome and can increase the quantity of the virus accumulating in the tumour by extending its circulation kinetics [30]. In this study we have demonstrated that application of TNF did not alter the extended blood circulation kinetics of polymer-coated virus (pcAdluc) but that the TNF-mediated increase in vascular leakage allowed increased quantities of pcAdluc to accumulate in the EL4 murine tumours. The combination of these approaches therefore offers to maximise delivery of therapeutic viral particles to disseminated tumour nodules. We have shown previously that polymer coating of adenovirus particles can mask receptor-binding epitopes and inhibit infection in vitro, although infection is to some extent restored following accumulation within tumours in vivo [31]. The efficiency of infection in vivo is nevertheless low compared to unmodified virus, and the addition of tumour-selective ligands is a useful approach to maximise transgene expression at the target site [32]. It is presently unclear whether the doses of TNF needed to increase tumour vascular permeability (and uptake of systemic therapeutics) in human patients would be significantly lower than the MTD, and this should be determined in a clinical study. In any event, tumour vasculature-targeted forms of TNF are now well developed [33] that may allow selective delivery of TNF to its intended site of action, avoiding unwanted systemic toxicities. Regarding the molecular mechanism of TNF-enhanced uptake into tumours, it is already known that Rho kinase is crucially important for TNF induced vascular leakage and paracellular permeability in pulmonary microvascular endothelial cells [34,35]. Similarly van Nieuw Amerongen et al. reported that simvastatin, a Rho signalling inhibitor, suppressed vascular leakage of Evans blue into the thoracic and

abdominal part of the aorta in an atherosclerosis-induced Watanabe heritable hyperlipidemic rabbit model [36]. Accordingly the observation here that TNF-increased extravasation of both dextrans and virus particles in vitro and in vivo could be prevented by pre-exposure to the Rho A/Rho kinase inhibitor, Y-27632, confirms the central importance of this pathway. Interestingly, the basal level of extravasation of all dextrans and virus particles was unaffected by Y-27632 treatment, showing that the pathway that leads to TNF induced permeability is not the same as that involved in baseline tumour-associated vascular permeability. Although the possibility of receptor-mediated transport pathways cannot be formally excluded, the lack of saturation over the concentration range studied, coupled with similar TNF-mediated augmentation of extravasation for virus particles and also for dextrans, suggests a fluid phase transfer, compatible with increased leakiness due to actin-induced shape changes in the endothelial cells. Virotherapy is a promising strategy for the treatment of cancer, however, improving the efficiency of delivery to the target site and avoiding unwanted toxicities in non-target tissues is needed to maximise treatment and safety. The strategy reported here of exploiting TNF to induce vascular leakage to enhance viral delivery may have important clinical implications in the treatment of cancer. Supplementary materials related to this article can be found online at doi:10.1016/j.jconrel.2011.08.022. Acknowledgements This work was supported by Cancer Research UK (FC, RC, KDF). References [1] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [2] H. Maeda, L.W. Seymour, Y. Miyamoto, Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vivo, Bioconjug. Chem. 3 (1992) 351–362. [3] S.E. Goldblum, X. Ding, J. Campbell-Washington, TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction, Am. J. Physiol. 264 (1993) C894–C905. [4] T.L. Hagen, A.M. Eggermont, Tumor vascular therapy with TNF: critical review on animal models, Methods Mol. Med. 98 (2004) 227–246. [5] J.H. Schiller, B.E. Storer, P.L. Witt, D. Alberti, M.B. Tombes, R. Arzoomanian, R.A. Proctor, D. McCarthy, R.R. Brown, S.D. Voss, et al., Biological and clinical effects of intravenous tumor necrosis factor-alpha administered three times weekly, Cancer Res. 51 (1991) 1651–1658. [6] A.M. Eggermont, H. Schraffordt Koops, J.M. Klausner, B.B. Kroon, P.M. Schlag, D. Lienard, A.N. van Geel, H.J. Hoekstra, I. Meller, O.E. Nieweg, C. Kettelhack, G. Ben-Ari, J.C. Pector, F.J. Lejeune, Isolated limb perfusion with tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft tissue extremity sarcomas. The cumulative multicenter European experience, Ann. Surg. 224 (1996) 756–764 discussion 764–755. [7] F.J. Lejeune, D. Lienard, M. Matter, C. Ruegg, Efficiency of recombinant human TNF in human cancer therapy, Cancer Immun. 6 (2006) 6. [8] D.J. Grunhagen, F. Brunstein, W.J. Graveland, A.N. van Geel, J.H. de Wilt, A.M. Eggermont, One hundred consecutive isolated limb perfusions with TNF-alpha and melphalan in melanoma patients with multiple in-transit metastases, Ann. Surg. 240 (2004) 939–947 discussion 947–938. [9] R.C. Carlisle, Y. Di, A.M. Cerny, A.F. Sonnen, R.B. Sim, N.K. Green, V. Subr, K. Ulbrich, R.J. Gilbert, K.D. Fisher, R.W. Finberg, L.W. Seymour, Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1, Blood 113 (2009) 1909–1918. [10] V. Subr, L. Kostka, T. Selby-Milic, K. Fisher, K. Ulbrich, L.W. Seymour, R.C. Carlisle, Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components, J. Control. Release 135 (2009) 152–158. [11] N.K. Green, C.W. Herbert, S.J. Hale, A.B. Hale, V. Mautner, R. Harkins, T. Hermiston, K. Ulbrich, K.D. Fisher, L.W. Seymour, Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus, Gene Ther. 11 (2004) 1256–1263. [12] M. Koss, G.R. Pfeiffer II, Y. Wang, S.T. Thomas, M. Yerukhimovich, W.A. Gaarde, C.M. Doerschuk, Q. Wang, Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells, J. Immunol. 176 (2006) 1218–1227. [13] M. Amano, M. Ito, K. Kimura, Y. Fukata, K. Chihara, T. Nakano, Y. Matsuura, K. Kaibuchi, Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase), J. Biol. Chem. 271 (1996) 20246–20249. [14] K. Kimura, M. Ito, M. Amano, K. Chihara, Y. Fukata, M. Nakafuku, B. Yamamori, J. Feng, T. Nakano, K. Okawa, A. Iwamatsu, K. Kaibuchi, Regulation of myosin

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