Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.


Introduction
Virotherapy is an alternative therapy against cancer, which takes advantage of the cytolytic activity of viruses during their infective cycle and the absence of response mechanisms of the tumor cells against viruses. Fusogenic oncolytic viruses (FOVs) show some advantages over nonfusogenic viruses when used against cancer cells, mainly because FOVs can induce tumor immunogenic cell death (ICD), producing cellular structures with strong immune-stimulatory effects [1]. The first virus used against cancer was a modified herpes simplex virus. It was aimed at obtaining efficient and safe therapy against unresectable stages of melanoma. This therapy was approved in 2015 by the FDA. Since then, other therapies based on oncolytic viruses alone or in combination with other treatments are being researched [2]. Moreover, some viruses have demonstrated promising results in clinical trials [3,4]. The mode of action of these therapies is associated with efficient malignant cell death, mediated by the direct viral cytotoxic effect and/or stimulation of the immune system [5,6].
Reovirus (RV) is a double-stranded RNA (dsRNA) virus without a membranous envelope expressing a nonstructural small fusion-associated membrane protein (FAST protein (Fusion-Associated Small Transmembrane protein)) in an active conformation in the cell membrane of infected cells [7]. This protein expressed at late stages for the viral cycle leads to syncytium formation, a mechanism involved in the horizontal propagation of the viral infection [8,9]. RV also displays tropism and efficiently replicates in tumor cells with the activated Ras pathway [10]. These characteristics allow the use of RV in oncological therapy, either alone or combined with the conventional and nonconventional treatments [11]. For instance, a combination of RV and cisplatin enhanced cytotoxicity in the human and murine melanoma cell lines in vitro and murine tumors in vivo synergistically [12]. Intratumor (i.t.) reovirus injection, together with intravenous (i.v.) anti-PD-1 antibody, significantly enhanced survival of mice with subcutaneous B16 melanoma. In both cases, the mechanism is dependent on the activation of antitumor immune responses [13]. Currently, RV is used in cancer therapeutics under the name REOLYSIN®. This product corresponds to a formulation of the human reovirus (HRV), tested at the preclinical stage and Phase I, Phase II, and Phase III clinical studies in a broad range of cancer indications [11]. For example, REOLYSIN® combined with carboplatin and paclitaxel is a safe and potentially efficacious therapy for patients with advanced malignant melanoma [14]. Evidence suggests that the antitumoral mechanism associated with RV involves the activation of the immune response related to fusogenic activity and ICD induction. These effects have only been reported for reovirus FAST expression. Le Boeuf and coworkers, using an interferon-sensitive vesicular stomatitis virus (mutant VSVΔM51) encoding the fusogenic p14 FAST protein, showed an increased anticancer effect in two different in vitro cancer systems (MCF-7 and 4T1). This strategy also produced positive results in vivo, extending the survival of animals in 4T1 and CT26 metastatic colon cancer, with a mechanism associated with the activation of antitu-mor immune responses [15]. In this study, we evaluated the effect of in situ transfection of the avian RV (ARV) FAST protein, named p10, on murine B16 melanoma tumor growth and induction of an immune response using chitosan nanoparticles (CH-NPs) as a vehicle to deliver DNA into cancer cells.

Materials and Methods
2.1. Nanoparticle Generation and Characterization. The gene coding the p10 protein of avian reovirus (ARV-p10) inserted into the vector pUC57 was subcloned into the commercial expression vector for eukaryotic cells pIRES2 (BD Biosciences Clontech, PT3267-5) using the same strategy that we described previously [16]. Complexes were generated by the coacervation method and characterized as we previously described using chitosan at an N/P 20 ratio, chosen due to its homogeneity and transfection efficiency.
Transfection efficiency was verified in B16 cells using a green fluorescence protein expression vector as a reporter (pcDNA3.1-GFP), determined by the percentage of GFPpositive cells (GFP+) relative to untreated cells 48 hours posttreatment. Fluorescence was monitored by flow cytometry using BD Accuri C6 equipment (BD Biosciences, USA). Lipofectamine 2000 (Invitrogen, 11668027) was used as a positive transfection control, and naked pcDNA3.1-GFP was used as a negative control.

ARV-p10 Protein Expression.
For ARV-p10 detection, B16 cells at a confluence of 40-60% were transfected with NPs of chitosan and pIRES-ARV (N/P 20) and Lipofectamine (Invitrogen, 11668027). At 48 hours posttransfection, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, washed again, and blocked with goat serum at 10% in PBS for 1 hour at room temperature. Subsequently, they were incubated with a rabbit anti-p10 polyclonal serum (gently donated by Dr. Roy Duncan, Dalhousie University, Nova Scotia) at a dilution of 1 : 500 in 10% goat serum at 4°C overnight with gentle agitation. Cells were washed for 10 min with PBS three times in gentle agitation B16 cell suspensions of 2 × 10 5 living cells were used to induce tumor development in C57BL/6J mice by subcutaneous (s.c.) injection into the lumbar region (challenge), as described previously [17]. Once a tumor reached a volume of 2.0 mm 3 , animals were separated into three groups: (i) without treatment, (ii) CH: treated with chitosan, and (iii) NP-ARV: treated with NPs of chitosan+pIRES-ARV. The treatment with NP-ARV consisted of an intratumor injection of an NP suspension synthesized at an N/P 20, composed by 122 μg of chitosan and 10 μg of plasmid pIRES-ARV, in 100 μl of PBS. The same amount of chitosan was used to treat the CH group of mice. The tumor growth was evaluated by measuring the tumor size using a caliper and calculating tumor volume according to the half-sphere formula (V = 2/πr 3 , where V corresponds to volume in mm 3 and r to tumor radius in mm). A maximum tumor volume (MTV) of 260 mm 3 was used as the endpoint criterion, at which time the animals were sacrificed by cervical dislocation and processed for subsequent analyses.
2.7. Splenocytes and Tumor-Infiltrating Lymphocytes. The spleen and tumor of each animal were removed. The spleen was disgregated in a 100-mesh metal grid and then treated with ACK buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , and 1 mM Na 2 EDTA, pH 7.3) for 5 min with gentle shaking to remove, by differential lysis, erythrocytes. After centrifugation for 7 min at 600 g, the splenocytes were resuspended in RPMI 1640 medium with 10% FBS (Biological, DW105804-127-1A).
In parallel, the tumor was removed and received on a plate with 5% FBS (Biological, DW105804-127-1A) in HBSS buffer (Gibco, 24020117) on ice and disintegrated using scissors. The homogenate was collected and treated with 1 mg/ml type IV collagenase (Sigma, C-5138) and 0.05 mg/ml DNAse (Promega, M6101) for 30 minutes at 37°C with gentle agitation. The digested extract was screened using a 100mesh, and the filtrate was washed with HBSS 5% FBS and centrifuged at 600 g for 7 minutes at 4°C. The cell pellet obtained was treated with ACK erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , and 1 mM Na 2 EDTA, pH 7.3) for 5 min at room temperature. The washing and spinning steps were repeated, the supernatant was removed, and the pellet was resuspended in 40% Percoll (GE Healthcare, 17-0891-01) in HBSS. The same volume of Percoll at 70% was added under the cells-40% Percoll suspension using a glass Pasteur pipette. It was centrifuged at 750 g for 20 min at room temperature with low acceleration and deceleration. Then, the T lymphocytes present between the two phases formed after centrifugation were removed and washed with HBSS 5% FBS at 600 g for 7 minutes at 4°C. Finally, tumor-infiltrating lymphocytes (TIL) were resuspended in RPMI 1640 medium with 10% FBS (Biological, DW105804-127-1A).

T Lymphocyte Activation.
For the detection of the intracellular IFNγ and IL-17A cytokines, specific of the Th1 and Th17 subpopulations, respectively, 2 × 10 6 splenocytes and TIL isolated were activated with 0.25 μM of 20 PMA (Sigma, P1585) and 1 μg/ml of Ionomycin (StemCell, 73722). Simultaneously, the vesicular transit was blocked to avoid the release of the cytokines to the extracellular medium, using 10 μg/ml of Brefeldin A (StemCell, 73012). Both treatments were performed for 4 hours at 37°C. with 5% CO 2 . As a negative activation control, cells treated only with Brefeldin A (Nuñez, Saez et al. 2013) were used.

Statistical Analysis.
Results were graphed as average value ± SEM and analyzed by the nonparametric Mann-Whitney test. The effect on tumor growth was analyzed by Fischer's exact test. A confidence value of 95% was used. All analyses were performed with the GraphPad Prism 5.01 computer program (GraphPad Software, Inc., USA).

Results
3.1. Nanoparticle Characterization. Chitosan/pIRES-ARV complexes were synthesized at an N/P ratio of 20 as described in Materials and Methods and as described previously [18]. Under these conditions, the formation of the complexes between pIRES-ARV and CH was confirmed by electrophoretic migration delay in comparison to pIRES-ARV alone, indicating a successful complexation interaction with chitosan (Figure 1(a)). These complexes showed a nanoparticle diameter average close to 100 nm (Figure 1(b)) and a zeta potential value of 1.79 mV (Figure 1(c)). This characterization indicates that complexes between CH and pIRES-ARV   (Figures 2(a) and 2(b)). These results indicate that expression vectors present in the NPs were incorporated by the B16 cells, which allows the expression of the arv-p10 gene and later the ARV-p10 protein synthesis.
The fusogenic activity of ARV-p10 protein was determined by evaluating the presence of syncytia 48 h posttransfection of B16 cells. We observed large multinucleated cells that indicate a syncytium formation process in Lipo-ARVand NP-ARV-transfected cells, but not in nontransfected cells (Figure 2(c)). The number of formed syncytia in Lipo-ARV and NP-ARV was significantly increased in comparison to nontransfected cells but with no difference between both transfection methods (Figure 2(d)). Moreover, in NP-ARVtransfected cells, the syncytium presence was associated with a 20% decrease in B16 cell viability at 120 hours posttransfection (Figure 2(e)). These results show that the transfection of B16 tumor cells using NP-ARV allows the expression of a fusogenically active ARV-p10 protein, which decreases cell viability because of syncytium formation.

Intratumoral Expression of ARV-p10 Protein with
Chitosan Nanoparticles. Our next aim was to determine      (Figure 3(a)). Animals were monitored daily, evaluating tumor growth until MTV was achieved (Figure 3(b)). The NP-ARV and CH treatments delayed tumor growth on 50% and 20% of mice, respectively, in comparison to control counterparts, but with a nonsignificant difference between both treatments (Figure 3(b)). Despite this tumor growth delay, the CD4 + and CD8 + lymphocytes in the tumor (Figures 4(a) and 4(b)) and spleen tissue (Figures 4(c) and 4(d)) did not show changes for both treated groups. However, a significant increase in splenic CD4 + IFNγ + (Th1 lymphocytes) cells was observed in both cases in comparison to the control group ( Figure 5(a)). Altogether, these results suggest that the tumor growth delay induced by the CH and NP-ARV treatments could be associated with a Th1 IFNγ type of immune response.

Discussion
Intratumoral expression of viral fusion proteins is a promising strategy as gene therapy against cancer. In this work, we evaluated the effect of in vitro and in vivo expression of avian reovirus p10 protein (ARV-p10), using chitosan nanoparticles (CH-NPs) as a transfection vehicle of murine B16 melanoma cells. We observed that ARV-p10 expression resulted in syncytium formation associated with a cytotoxic effect in vitro, and intratumor treatment with NP-ARV caused a mild delay in tumor development associated with an increase in splenic Th1 + IFNγ + lymphocytes.
The syncytium generation in B16 tumor cells 48 hours posttransfection with NP-ARV supports the high fusogenic activity described for ARV-p10 protein, classified as a promiscuous fusogen, which is sufficient to induce cell fusion and syncytium formation in various cell lines [7,19]. Altogether, our results resemble previous studies using a highly fusogenic variant of GALV-F protein, which induces unstable syncytia in human tumor cells, causing potent cytotoxicity in vitro [20,21] and inhibition of tumor growth in vivo [20,22].
Antitumoral reovirus-mediated effects have been associated with changes in immune responses by Prestwich and coworkers, using immunodeficient mice. These authors showed that modifying immune responses is critical for the antitumoral effect of reovirus [23]. Intravenously administered reovirus reduces B16 metastatic lymph nodes and increases antitumor immunity. Similarly, infected Mel888 cells induce DCs and activate autologous peripheral blood lymphocytes [24]; and human DCs loaded with reovirusinfected human melanoma Mel888 cells induce NK cell activation and tumor-specific cytotoxicity [25]. Errington and coworkers demonstrated that reovirus replicates in human melanoma cell lines and i.t. injection of reovirus induces tumor regression in a xenograft model of melanoma, with a mechanism involving modulation of inflammatory responses [26]. In mice, i.t. administration of reovirus into melanoma B16F10 or Lewis Lung Carcinoma (LLC) models prolongs survival and delays tumor growth [27]. In our case, NP-ARV treatment increased cell fusion, delayed tumor growth, and elevated levels of splenic Th1 + IFNγ + lymphocytes. However, similar results were observed with NP alone, suggesting a possible chitosan NP-mediated effect on Th1 cells. This fact would support previous works showing that chitosan activates dendritic cells [28,29] and macrophages [30,31] to release IL-1β and IL-12, both cytokines critical for the  BioMed Research International polarization of CD4 cells to a Th1 phenotype, which is known to participate in antitumor immunity [32].
In conclusion, we showed that the ARV-p10 protein expression using CH-NPs in murine melanoma cells induces efficient tumor cell fusion in vitro. This process was associated with a mild cytotoxic effect and an antitumor response in vivo. Altogether, these results provide a stepping stone towards future research on improving the ARV-p10 protein expression using CH-NPs as an expression vehicle.

Conclusion
In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. However, its use to treat melanoma tumors produces no difference in in vivo antitumoral impact in comparison to chitosan treatment. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.  Figure 5: Effect of NP-ARV treatment on splenic CD4 + subpopulations. Representative dot plots of splenic T CD4 + subpopulation (left) and graph bar of the percentage of each population (right) for the (a) CD4 + IFNγ + , (b) CD4 + IL-17A + , and (c) CD4 + Foxp3 + lymphocytes in the nontreated animals (control), chitosan alone (CH), and NP-ARV-treated group. Bars correspond to average ± standard error; individual experiments are also graphed; statistical analyses were performed using the Mann-Whitney test. 9 BioMed Research International