Abstract
Advanced melanoma cells, characterized by resistance to chemotherapy, have been shown to be highly sensitive to oncolysis by Newcastle disease virus (NDV). In the present study, we investigated the capacity of NDV to specifically infect and spread into solid tissues of human melanoma and lung carcinoma, in vivo and ex vivo. For this purpose a new model of SCID-beige mice implanted with human melanoma was developed. Surprisingly, the replication competent NDV-MTH and the attenuated, single-cycle replication NDV-HUJ strains, demonstrated a similar oncolytic activity in the melanoma-implanted mice. Further, ex vivo analysis, using organ cultures derived from the melanoma tissues indicated a limited spread of the two NDV strains in the tissue. Extracellular matrix (ECM) molecules, notably heparin sulfate and collagen, were found to limit viral spread in the tissue. This observation was validated with yet another solid tumour of human lung carcinoma. Taken together, the results indicate that the ECM acts as a barrier to virus spread within solid tumour tissues and that this restriction must be overcome to achieve effective oncolysis with NDV.
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A supplementary figure is available with the online version of this paper.
Introduction
For an effective oncolytic activity, a virus has to specifically infect tumour cells and be able to spread within the solid tissue to eliminate most tumour cells. While many studies have been aimed at genetic modification of viruses to selectively infect and replicate in tumour cells, little is known about the potential of these oncolytic viruses to effectively spread in a solid tumour following the initial infection (Smith et al., 2011). Newcastle disease virus (NDV) is an avian paramyxovirus with a selective oncolytic effect on tumour cells in culture and in animal models (Fábián et al., 2001, 2007; Lorence et al., 1988; Sinkovics & Horvath, 2000; Yaacov et al., 2008). Several clinical studies have already indicated the safety and potential efficacy of NDV treatment in a variety of cancers, such as glioblastoma (Freeman et al., 2006; Steiner et al., 2004), melanoma (Batliwalla et al., 1998), colorectal carcinoma (Ockert et al., 1996; Schlag et al., 1992), breast carcinoma (Ahlert et al., 1997) and head and neck squamous cell carcinoma (Karcher et al., 2004). The oncolytic activity of NDV has been studied mostly using mesogenic strains of NDV that productively replicate in mammalian cells. These NDV strains specifically infected human tumour cells in culture and completed a productive replication to produce infectious progeny (Nagai et al., 1980). The effective oncolytic activity of mesogenic viral strains in animal tumour models was therefore explained by selective infection followed by spread of progeny virus within the solid tumour, to bring about shrinkage of the tumour mass (Ravindra et al., 2009). We have previously reported the isolation of an attenuated (lentogenic) strain of NDV (HUJ) that selectively infected and induced apoptotic death of lung carcinoma cells in culture and furthermore displayed oncolytic activity in mouse models of lung and prostate cancers (Yaacov et al., 2008). Notwithstanding, the attenuated NDV-HUJ strain is a single-cycle virus that is unable to produce infectious progeny virus in mammalian cells. Thus, in contrast to NDV-MTH, the attenuated NDV-HUJ is not expected to spread efficiently in mammalian cells beyond the first cycle of infection. Using primary human melanoma cells we found that apoptosis induced by NDV-HUJ is dependent on the expression of livin, a member of the inhibitor of apoptosis proteins family (Lazar et al., 2010). Livin has a dual function, while the intact protein is anti-apoptotic; cleavage of a peptide at the amino-terminus converted it into a pro-apoptotic protein (Abd-Elrahman et al., 2009; Nachmias et al., 2003). Furthermore, infection of advanced melanoma cells with NDV-HUJ induced cleavage of livin and thus effective apoptosis (Lazar et al., 2010). In the present study, we compared the oncolytic activity of a single-cycle attenuated NDV-HUJ with the multi-cycle mesogenic strain NDV-MTH; the latter strain has been extensively applied in cancer preclinical and clinical studies (Apostolidis et al., 2007; Csatary et al., 1993; Wagner et al., 2006). Our initial working hypothesis suggested a superior anti-tumour activity of NDV-MTH as it potentially replicates and spreads within the tumour tissue. Surprisingly, the oncolytic activity of NDV-MTH was similar to that of the attenuated NDV-HUJ in primary human melanoma cells implanted in SCID-beige mice. Further analysis in vivo and ex vivo indicated a restriction to NDV-MTH progeny spread within the solid tumour tissue and thus the oncolytic activity of the attenuated NDV-HUJ and the replicative NDV-MTH were similar.
Results
Replicative and single-cycle NDV strains display similar oncolytic activity in vivo
Since the attenuated single-cycle NDV-HUJ displayed oncolytic activity in several experimental systems (Lazar et al., 2010; Yaacov et al., 2008), we set out to compare its anti-tumour activity with that of a replicative NDV-MTH strain that had been extensively used in preclinical models and in human trials (Kelly & Russell, 2007). To this end, a new model of human advanced melanoma tumour implanted in SCID-beige mice was established (see Methods). When the length of the subcutaneous tumours reached 4 mm, NDV was repeatedly injected intratumourally (IT) twice a week and the volume of tumours was measured. The results illustrated in Fig. 1 indicate that treatment with both strains, NDV-HUJ and NDV-MTH, reduced tumour volume compared with the control PBS-treated group of mice (P = 0.024 and P = 0.123, respectively). Taken together, the results indicate that NDV-HUJ, an attenuated virus strain, effectively reduced melanoma growth in vivo.
The oncolytic activity of two NDV strains on human melanoma in immunodeficient SCID-beige mice. Human advanced melanoma tumour model was established in SCID-beige mice essentially as described previously (Abd-Elrahman et al., 2009). Mice were injected subcutaneously at the flank with 2×106 cells (351 advanced melanoma). When tumour length reached 4 mm, mice were injected IT with NDV-HUJ (1×109 IU) or NDV-MTH (2×108 IU) or PBS, twice a week for 35 days (Methods). The statistical analysis was determined by a one-tail exact Mann–Whitney test adjusted for multiple comparisons using the Bonferroni correction with a k value of 3, to account for the number of times the same group of mice was used. Bars represent sem.
IT spread of NDV
Since both the replicative NDV-MTH and the single-cycle NDV-HUJ reduced tumour volume in the mice, we next analysed the capacity of these strains to spread in the tumour tissue in vivo, by in situ immunohistochemical staining of the treated tumours. Only limited areas in the solid tumour tissues were positive for NDV antigens, after repeated IT injection with both NDV strains (Fig. 2b–c). The immune-staining pattern of slices derived from the tumour tissues indicated a plaque-like infection around the virus-injected site. This observation suggests a restriction on viral spread in the solid tumour, even with the multi-cycle NDV-MTH strain.
Distribution of NDV-HUJ and NDV-MTH after in vivo infection. Two weeks after termination of the NDV injections IT (at 35 days) mice were injected once again with NDV, according to standard conditions and 24 h later mice were killed, tumours were removed and formalin-fixed. Tissues were cut into 4 µm sections and deparaffinized. Slides from each sample were immunostained with anti-NDV sera to detect NDV antigens (Methods).
To further evaluate NDV spread in solid tumour tissue, we compared NDV replication and spread in two experimental systems, in vivo and ex vivo. For the ex vivo model, melanoma tumour tissues were excised from euthanized control mice (PBS treated) and organ cultures were prepared, as described in Methods. The tumour tissues remain viable for the duration of the experiment of 48 h, as shown in previous work (Massler et al., 2011) (Fig. S1, available in JGV Online). Following infection of the ex vivo cultured tissues by NDV-HUJ and NDV-MTH progeny virus were collected at 24 and 48 h and titrated on QT6 cells (Fig. 3a). The results indicated that tumours infected ex vivo with the replicative NDV-MTH, but not with the single-cycle NDV-HUJ, produced infectious progeny virus. Addition of NDV-specific antibodies to the indicator QT6 cells, following the infection, reduced the titre of progeny NDV-MTH, but not of NDV-HUJ, indicating that the released progeny NDV-MTH virus was neutralized (Fig. 3a). In a parallel experiment (Fig. 3b), mice bearing tumours were IT-injected in vivo with NDV-HUJ or NDV-MTH and 24 h post-injection mice were euthanized and the infected tumour tissues were removed and prepared as organ culture. Forty-eight hours post-explantation of the in vivo-infected tumours, condition medium was collected from the organ cultures and titrated on QT6 cells, using the same controls as in the ex vivo experiment (Fig. 3a–b). Surprisingly, no progeny virus was released from the in vivo IT-infected tumour tissues (Fig. 3b).
Tissue spread of NDV-MTH virus following in vivo and ex vivo infection. (a) Ex vivo: primary human melanoma tumours were excised from mock-infected mice (PBS-treated group). Tissues were prepared for organ culture as described in Methods. Organ cultures were infected with NDV-HUJ (1×106 IU) or NDV-MTH. A control tissue, for zero time infection, was treated after 2 h with neutralizing anti-NDV serum (1 : 500) to prevent reinfection by the newly released progeny virus (marked as: +Ab). At 48 h post-infection, media of the organ cultures were collected and progeny NDV titrated on QT6 cells. Total cell RNA was isolated from the organ cultures (Yaacov et al., 2008). (b) In vivo: mice bearing primary human melanoma tumours were injected IT with NDV-HUJ (1×106 IU) or NDV-MTH. After 24 h infection mice were killed, infected tumours were removed and prepared for organ culture (Methods). After 24 h in organ culture, media were collected and NDV was titrated on QT6 cells. Total cell RNA was isolated from the infected tissues (Methods).
Taken together, these results indicate a major difference in the release of progeny virus from the in vivo- and ex vivo-infected human melanoma tissues. To further investigate whether NDV can, in fact, initiate a replication cycle following injection IT in vivo, total RNA was extracted from the in vivo-injected tumours, as well as from organ cultures infected ex vivo, and viral RNA replication was tested by RT-PCR (Fig. 4). Specific PCR primers, to detect NDV genomic (–) RNA strand or the (+) mRNA, were used. Results presented in Fig. 4 indicate synthesis of both the (+) and (–) RNA strands in tumours infected in vivo and ex vivo. Control RNA prepared from the organ culture tissues, after just 2 h of infection was negative for both the (+) and (−) RNA strands of NDV, indicating that the (+) mRNA, detected after 48 h of infection, demonstrated de novo RNA synthesis and not a residual parental virus adsorbed to the tissues. Thus, both NDV-HUJ and NDV-MTH initiate an infection cycle in the tumour tissue after ex vivo or in vivo infection. We concluded, therefore, that restriction to NDV-MTH spread in the tissue is post-viral transcription (Fig. 4) and probably post-protein synthesis (Fig. 2).
NDV RNA transcription in the tumour tissues. RT-PCR to detect NDV transcription was conducted with both the ex vivo- and in vivo-infected tissues. At the indicated times post-infection tissues were harvested, total RNA isolated and RT-PCR was preformed, to distinguish between the (−) and (+) RNAs of NDV (Methods). In parallel RT-PCR was conducted on cell actin mRNA to control for amounts of RNA.
The effect of extracellular matrix (ECM) molecules on NDV spread
Since the main barrier to NDV-MTH spread, following IT injection in vivo, appears to be post-transcription, we reasoned that ECM constituents might limit the spread of progeny virus within the solid tissue. To investigate this possibility, we treated melanoma tumour tissues in organ culture with the ECM-degrading enzymes, collagenase-II and heparinase-I, prior to infection ex vivo. The enzymic treatment with ECM-degrading enzymes did not change viability of the tissues, as measured by the glucose consumption assay (Fig. S1a). At 48 h post-infection, treated tissues were taken for immunohistochmical staining with anti-NDV sera (Fig. 5a) and for real-time PCR analysis to detect NDV genomic (−) RNA (Fig. 5b). The immune histochemical analysis indicated that infection of tumour tissue slices ex vivo with NDV-MTH was mostly limited to the borders of the solid tissue, which was exposed to the virus during the infection [Fig. 5a(i)], while the centre of the tissue was mainly virus free. Notwithstanding, treatment with heparinase or collagenase improved viral spread and distribution within the tissue, and viral antigens could be detected in most areas away from the tissue borders [Fig. 5a(iii)–(iv)]. To further study the effect of ECM components on viral infection and spread in the solid tissue, we analysed levels of viral RNA in the enzyme-treated tissues at 48 h post-infection. Real-time RT-PCR analysis indicated a significant increase of NDV RNA in tissues treated with both collagenase and heparinase (Fig. 5b). Taken together the results indicate a limited capacity of the replication competent NDV-MTH to penetrate and spread within the melanoma tissue beyond the surface of the tissue slice. Removal of the tissue collagen and heparan sulfate enabled the virus to further penetrate and replicate within the tissue.
Effect of ECM-degrading enzymes on NDV spread in human melanoma tumour tissues. Primary human melanoma tumour tissues were removed and prepared for organ culture (Methods). Tissues were pre-treated with collagenase-II (50 µg ml−1) or heparinase-I (1 U). Next, the tissues were infected for 48 h by NDV-MTH (1×105 IU). (a) Same tissue samples were formalin-fixed and prepared for immunohistochemistry with anti-NDV sera (Methods). Red arrows, NDV-infected cells. (b) Relative amount of NDV genomic RNA was measured using real-time PCR (Methods). Asterisk represents statistical significance (P<0.05) calculated using the t-test method.
Since the primary human melanoma cells were grown into a tumour tissue in SCID-beige mice, it is likely that some of the ECM components in the tumour are mouse derived and the tissue does not reflect an authentic human tumour tissue. To extend the generality of this phenomenon, we have studied yet another tumour tissue of human lung carcinoma. To this end, lung carcinoma tissues were obtained from the surgery room and immediately prepared for organ culture. Viability of the human lung tissues was maintained for at least 48 h, the duration of infection with the viruses (Massler et al., 2011). Pre-treatment of the lung carcinoma tissue with ECM-degrading enzymes did not change the viability of the tissues, as measured by the glucose consumption assay (Fig. S1b) and typical lung structures were observed by histology analysis of the control and pre-treated tissues [Fig. 6a(i)]. Viral infection and spread in the lung carcinoma tissues was followed by immunohistochemical staining for NDV antigens and by real-time RT- PCR to detect NDV (−) RNA in the infected tissues (Fig. 6a–b). The treatment with collagenase and heparinase before infection increased viral distribution in the lung tissue as observed in multiple histology slides of the tissues [Fig. 6a(iii)–(iv)]. Moreover, quantitative RT-PCR analysis indicated a significant elevation of viral (−) RNA synthesis (five- and sevenfold) in the collagenase and heparinase pre-treated lung tissues. The consequence of enzymic degradation of the lung ECM components on NDV RNA synthesis and spread was observed in carcinoma lung tissues obtained from three different patients.
Effect of ECM-degrading enzymes on NDV infection and spread in human lung carcinoma tissue. Non-small-cell lung carcinoma tissues were obtained from the surgery room together with the adjacent normal lung of the same patient (human tissues were obtained under Hadassah Hospital IRB approval no. 203-17.02.06). Tissues were prepared for organ cultures and pre-treated with collagenase-II (50 µg ml−1) or heparinase-I (1 U) for 1 h (Methods). Organ cultures were infected for 48 h with NDV-MTH (1×105 IU). (a) Infected tissues were prepared for immunohistochemical staining to detect NDV antigens (Methods). Green arrow indicates alveoli structure and red arrows indicate cells infected with NDV. (b) Relative amounts of NDV genomic RNA measured by real-time RT-PCR (Methods). Asterisk represents statistical significance (P<0.05) calculated using the t-test method.
Discussion
Targeting of viruses to tumour cells may be achieved through different mechanisms and the oncolytic potential of these viruses has been demonstrated in a variety of preclinical and some initial clinical trials (Cattaneo et al., 2008; Kelly & Russell, 2007). Yet, more work is clearly needed before oncolytic viruses join the mainstream of anti-cancer treatment protocols. Potentially, the main advantage of oncolytic viruses over other targeted drugs, i.e. chemotherapy and antibodies, is the ability of viruses to replicate and expand within the tissue, to selectively obliterate all tumour cells.
In the present work, we investigated the capacity of NDV to replicate and spread in two solid tumours of human origin, melanoma and lung carcinoma. To this end, a new in vivo model of human primary melanoma cells implanted in SCID-beige mice was developed to analyse the correlation between viral replication and spread in the tumour tissue and the subsequent oncolytic capacity. Two NDV strains were compared in the present work, NDV-HUJ and NDV-MTH. The latter strain is a mesogenic virus with high replication and oncolytic capacity in human cells (Csatary et al., 1993). To our surprise, tumour regression in SCID-beige mice implanted with human melanoma cells was observed after IT injection of both the single-cycle attenuated NDV-HUJ and the replicative strain NDV-MTH. How can one explain the significant reduction of tumour size when only a limited number of cells appear to be infected? SCID-beige mice are immune deficient and thus it is unlikely that immune response against viral antigens shown before (Schirrmacher et al., 1997) is responsible for the oncolytic activity. However, some innate immune mechanisms induced by macrophages or dendritic cells, as well as interferons that are stimulated in the melanoma cells by the virus (Lazar et al., 2010), may participate in the oncolytic effect through a bystander activity. Furthermore, analysis of viral RNA and protein synthesis following NDV injection into the tumour in vivo indicated that both NDV strains initiate a replication cycle in the tissue (Figs 2 and 4). Immunohistochemical analysis, with antibodies to NDV, shows that the virus is mostly restricted to the area of initial injection and does not spread to distant regions of the melanoma tissue (Fig. 2).
NDV may spread within a solid tissue either through direct cell-to-cell infection by cell membrane fusion, or by production of free extracellular progeny virus that reinfect and further spread within the tissue (Smith et al., 2011). Little is known regarding the contribution of each mechanism to viral spread within a solid tissue, a critical issue in the development and effective application of oncolytic viruses. To address this question, in the context of oncolytic NDV-MTH, we compared production of free progeny virus following infection of the tumour (IT) in vivo to infection of the same tissue but under ex vivo conditions, subsequent to the establishment of the tissue in organ culture. The human melanoma tissues grown in SCID-beige mice and the lung carcinomas obtained directly from the surgery room maintain the original cell composition and the extracellular components when cultured ex vivo. The organ culture system is thus amenable to investigate mechanisms of virus replication and spread in solid tissues and to validate animal experimental systems versus human tissues (Cohen et al., 2011; Kolodkin-Gal et al., 2008; Kunicher et al., 2008, 2011).
Surprisingly, no free progeny virus was released following IT in vivo injection of the virus and subsequent tissue explantation, while significant amounts of progeny virus were released to the medium after ex vivo infection of the melanoma tissues (Fig. 3). Previous studies related to the question of solid tissue permeability to viruses, such as herpes simplex virus 1 (HSV1) and adenovirus, have indicated that penetration and ex vivo infection are limited to two to three layers of cells at the periphery of the solid tissue (Kolodkin-Gal et al., 2008; Kuriyama et al., 2000; Maillard et al., 1998). Similarly, only the periphery of the melanoma tumour is infected when replicative NDV-MTH is added to organ cultures ex vivo to result in the production of free progeny virus. Why then was no progeny virus observed when the tumour tissue was infected IT in vivo and subsequently explanted as organ culture (Fig. 3)? The observation that NDV-MTH initiates the replication cycle following IT injection in vivo, based on viral RNA and protein synthesis, but fails to release free infectious virus to the medium is likely due to entrapment of progeny virus within the solid tissue around the site of injection (Figs 2 and 4). We suggest therefore, that differences in the outcome of infection between the two methods (in vivo and ex vivo) are due to the mode of viral application. It should be noted that following infection of the same melanoma cells, grown as monolayer cultures with NDV-MTH, the budding and secretion of progeny virus to the medium is very efficient and high titre of infectious virus is recovered in the medium supernatant, but not from the cell pellet (unpublished data). It is therefore, unlikely that virus maturation or release is blocked due to non-permissiveness of melanoma cells. What are the constituents of the solid tissue that so effectively restrict the release of free progeny NDV and consequently spread within the tumour? Previous investigations have shown that ECM constituents, such as collagen (McKee et al., 2006), hyaluronic acid (Guedan et al., 2010) and heparan sulfate (Watanabe et al., 2010) interfere with the oncolytic activity of adenovirus and HSV1. Based on these observations recombinant viral vectors that express ECM-degrading enzymes, such as matrix metalloproteinase-8 (Cheng et al., 2007) and heparanase (Watanabe et al., 2010), or proteins that regulate the ECM synthesis relaxin (Krishnamurthy et al., 2006) and decorin (Choi et al., 2010) were shown to enhance the oncolytic activity. Notwithstanding, the safety of such recombinant viruses may be problematic in a clinical setting as these proteins may also enhance the spread of metastatic tumour cells (Uno et al., 2001; Victor et al., 1999). Indeed, pre-treatment of human melanoma tissues with collagenase and heparinase effectively enhanced NDV-MTH infection and spread within the melanoma tissues (Fig. 5). To strengthen this observation, we have developed an organ culture system of human lung carcinoma tissue infected ex vivo with NDV. While the lung tissue is spongy and thus may be relatively accessible to viral particles, pre-treatment with ECM-degrading enzymes enhanced NDV expression and spread within the tissue (Fig. 6).
In summary, our results indicate that while NDV demonstrate oncolytic activity in a variety of tumours, the spread of virus within solid tumours is a major impediment for an effective treatment. Therefore, the choice of the tumour to be treated in clinical studies must take into consideration the tissue accessibility of oncolytic virus. Application of the organ culture system with human-derived tumour tissues as described in this work should facilitate the design of future clinical studies with oncolytic viruses.
Methods
Cells.
Advanced melanoma specimens were obtained by the surgeon in consultation with oncologists at the Hadassah Medical Center. The study was approved by the Institutional Review Board and informed consent was obtained from study participants. The method of primary melanoma cell culture was described previously (Lotem et al., 2002). Melanoma cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL), 10 % FCS (Gibco-BRL), HEPES (10 mM), penicillin–streptomycin (0.1 mg ml−1) and glutamine (2 mM), at 37 °C in CO2 incubator.
NDV strains.
NDV-HUJ was cloned from the parental lentogenic strain Hitchner B1 as described before (Freeman et al., 2006; Lazar et al., 2010; Yaacov et al., 2008) and propagated in specific-pathogen-free fertilized eggs. Extensive biological characterization and sequence analysis of the genomic RNA indicated a unique strain of NDV (Lazar et al., 2010; Yaacov et al., 2008). While NDV-HUJ replicates efficiently in fertilized eggs, infection of mammalian cells is aborted at a late stage and no infectious progeny are released from the infected cells. The mesogenic NDV-MTH-68 (Csatary et al., 1993) efficiently replicates in both avian and mammalian cells and was grown in fertilized eggs. NDV grown in eggs was purified on sucrose gradients (20–60 %, w/w) using standard methods (Csatary et al., 1993). For titration, IU, NDV viruses were plated on QT6 cells and viral titres analysed by FACS, after staining with anti-NDV serum (Yaacov et al., 2008). For titration of NDV-MTH anti-NDV serum was added to the infected culture to neutralize the newly released progeny virus.
NDV RNA synthesis.
Total RNA was isolated from tissues using the RNeasy RNA isolation kit (Qiagen). To distinguish between the (−) and (+) RNA strands of NDV, a two-step RT-PCR was applied, as described previously (Lazar et al., 2010; Yaacov et al., 2008). RT reaction to identify the (+) strand was carried out with a reverse (negative-sense) primer to the NDV leader (+) strand RNA sequence (nt 330) 5′-TGCCTGAGTGGTTTGTTGGC-3′ from the NDV NP gene and to identify the (−) strand RT reaction was carried out with a forward (positive-sense) primer, Leader Start (nt 1) 5′-ACCAAACAGAGAATCGGTGAG-3′ (nt 21). For PCR amplification of the NDV cDNA, we used the forward (positive-sense) primer, Leader Start (nt 1) 5′-ACCAAACAGAGAATCGGTGAG-3′ (nt 21) and the reverse primer, Leader End (nt 330) 5′-TGCCTGAGTGGTTTGTTGGC-3′ (nt 310), from the NDV NP gene to produce a dsDNA fragment of 330 bp, containing the Leader and part of the NP gene region. To control for a non-specific amplification of NDV RNA positive-strand due to secondary RNA structures, a control RT assay without primers was performed with each RNA sample. DNA products were collected after 25 PCR cycles and run on 1 % agarose gel (Yaacov et al., 2008). PCR of cell β-actin mRNA was carried out as a control, on all RNA samples, using primers of two different exons, to prevent amplification of cell DNA contamination.
Actin forward (5′-CCAACCGTGAAAAGATGACC-3′) and actin reverse (5′-GCTGTGGTGGTGAAGCTGTA-3′) primers were used to produce DNA of 270 bp.
Quantitative RT-PCR.
Total RNA (0.5 mg) was isolated using the RNeasy RNA isolation kit (Qiagen). In order to distinguish between the (−) and (+) RNA strands of NDV, a two-step RT-PCR was applied (Lazar et al., 2010; Yaacov et al., 2008). To identify the (−) strand, the genomic RNA, RT reaction was carried out with a forward (positive-sense) primer, Leader Start 5′-ACCAAACAGAGAATCGGTGAG-3′. To identify GAPDH gene expression cDNA was performed using reveres primer 5′-TGACGGTGCCATGGAATTTG-3′. Real-time PCR was performed on 2 ng cDNA using Quantimix Easy Syg kit (Biotools). SYBR green (Molecular Probes) was used to detect the PCR products. Primers used were GAPDH forward 5′-ATGGGGAAGGTGAAGGTCGG-3′ and reverse 5′-TGACGGTGCCATGGAATTTG-3′, and for NDV the forward Leader Start 5′-ACCAAACAGAGAATCGGTGAG-3′, and the reverse primer, Leader End 5′-TGCCTGAGTGGTTTGTTGGC-3′. PCR parameters consisted of 5 min Taq activation at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 70 °C for 35 s. The last step is fluorescence acquisition done at 95 °C for 15 s, 60 °C for 15 s and 95 °C for 15 s. Standard PCR curves were generated and the relative amount of mRNA was normalized to GAPDH mRNA. Specificity was verified by melt curve analysis and agarose gel electrophoresis.
Analysis of NDV surface proteins in the infected cells.
To determine NDV titres, infected QT6 cells were stained with chicken sera against NDV (1 : 500) for 1 h and with goat anti-chicken IgG-FITC or CY5-conjugated (1 : 500; Jackson) for 30 min. Cells were counter stained with propidium iodide (0.5 µg ml−1; Sigma), an indicator of cell viability. The relative levels of surface NDV antigens were assessed by FACS analysis, FACSort (Becton Dickinson). Data were analysed using FCS Express software (De Novo Software).
Human melanoma tumour in SCID-beige mice.
Briefly, mice experiments were done under ethics guidelines (Ethics Committee no. MD 09-12030-5). SCID-beige male mice, 6–7 weeks old, were inoculated subcutaneously in the flank with (2×106 IU/0.1 ml) human primary advanced melanoma cells (351 ADV) (Lazar et al., 2010). Tumour volume was measured and calculated on the basis of the formula V = (L W2)/2 (L, length; W, width) (Euhus et al., 1986). NDV was injected IT twice weekly for 35 days and mice were monitored for an additional 14 days to evaluate tumour growth kinetics in the absence of treatment. After an additional 14 days, mice were euthanized, tumour tissues removed and prepared for the organ culture experiments and for histological examination.
The organ culture ex vivo model
Fourteen days after the end of virus treatment (see above), the mice were injected again (IT) with 1×106 IU of NDV-HUJ or NDV-MTH and 24 h post-infection mice were killed and tumour tissue was excised for organ culture. Organ cultures (500 µm thick slices) were prepared as described previously (Cohen et al., 2011; Kolodkin-Gal et al., 2008; Kunicher et al., 2008, 2011) and grown for 48 h in DMEM (Gibco-BRL), 10 % FCS (Gibco-BRL), 10 mM HEPES, 0.1 mg ml−1 penicillin–streptomycin and 2 mM glutamine. Organ cultures were kept ex vivo at 37 °C in a 5 % CO2 incubator. In a parallel experiment, a control tumour tissue (from PBS IT-injected mice) was excised and prepared for organ culture. The tumour slices were incubated with NDV for 2 h adsorption time, washed twice with fresh medium and incubated for a further 48 h. Organ culture supernatants were collected and NDV was titrated on QT6 cells using FACS. The detection limit of the test is 103 IU (Yaacov et al., 2008). The tissues were taken for immunohistochemical staining.
Treatment of tumour tissues with extracellular-degrading enzymes and infection with NDV.
Two experimental systems were used to analyse the effect of extracellular-degrading enzymes on viral spread. (i) Primary human melanoma cells (351 ADV) were injected into SCID-beige mice. When tumour volume was about 1 cm, mice were euthanized and tumour tissue was removed and prepared for organ culture (see above). (ii) Human lung tumour tissues of non-small-cell lung carcinoma were prepared for organ cultures (tissue samples were obtained from the surgery room together with normal lung tissue from the same patient, under Hadassah Hospital IRB approval 203-17-0.2.06). The organ cultures were pre-treated for 1 h in medium-free serum with human collagenase-II (50 µg ml−1; Worthington) or flavobacterium heparinase-I (1 U; Sigma). Organ cultures were subsequently infected with NDV-MTH (1×105 IU) or NDV-HUJ (1×105 IU) for 48 h and the infected tissues were taken for immunohistochemical staining.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded tissue samples were cut (4 µm) and deparaffinized antigen retrieval was done at 115 °C for 3 min in 40 mM Tris/HCl pH 8.8, 1 mM EDTA buffer and incubated for 1 h with rabbit anti-NDV serum (1 : 1000) (Yaacov et al., 2008). Antigen was detected using HRP-conjugated secondary antibodies for 30 min (goat anti-rabbit Ig; DAKOCytomation) and DAB (DAKOCytomation). Contra-staining was done with haematoxylin (DAKOCytomation).
Acknowledgements
This work was supported by a European community grant, the Clinigene network of excellence and by a grant of the Israeli Academy of Sciences. Statistical analysis was kindly provided by Professor Norman Grover.