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Journal of Breast Cancer logoLink to Journal of Breast Cancer
. 2023 Feb 17;26(2):186–200. doi: 10.4048/jbc.2023.26.e9

Inhibition of Tumor Growth and Metastasis by Newcastle Disease Virus Strain P05 in a Breast Cancer Mouse Model

Oscar Antonio Ortega-Rivera 1,2, Pamela Gallegos-Alcalá 1, Mariela Jiménez 1, J Luis Quintanar 3,✉, Flor Torres-Juarez 4, Bruno Rivas-Santiago 4, Susana del Toro-Arreola 5,6, Eva Salinas 1,✉
PMCID: PMC10139849  PMID: 37051644

Abstract

Purpose

Conventional therapies and surgery remain the standard treatment for breast cancer. However, combating the eventual development of metastasis is still a challenge. Newcastle disease virus (NDV) is one of the various species of viruses under clinical evaluation as a vector for oncolytic, gene-, and immune-stimulating therapies. The purpose of this study was to evaluate the antitumor activity of a recombinant NDV (rNDV-P05) in a breast cancer murine model.

Methods

Tumors were induced by injecting the cellular suspension (4T1 cell line) subcutaneously. The virus strain P05 was applied three times at intervals of seven days, starting seven days after tumor induction, and was completed 21 days later. Determination of tumor weight, spleen index, and lung metastasis were done after sacrificing the mice. Serum levels of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, and TNF-related apoptosis-inducing ligand (TRAIL) were quantified by enzyme-linked immunosorbent assay. CD8+ infiltrated cells were analyzed by immunofluorescence.

Results

rNDV-P05 showed a route-of-administration-dependent effect, demonstrating that the systemic administration of the virus significantly reduces the tumor mass and volume, spleen index, and abundance of metastatic clonogenic colonies in lung tissue, and increases the inhibition rate of the tumor. The intratumoral administration of rNDV-P05 was ineffective for all the parameters evaluated. Antitumor and antimetastatic capability of rNDV-P05 is mediated, at least partially, through its immune-stimulatory effect on the upregulation of TNF-α, TRAIL, IFN-α, and IFN-γ, and its ability to recruit CD8+ T cells into tumor tissue.

Conclusion

Systemic treatment with rNDV-P05 decreases the tumoral parameters in the breast cancer murine model.

Keywords: Immunotherapy, Neoplasm Metastasis, Neoplasms, Newcastle Disease Virus

INTRODUCTION

According to the World Health Organization, cancer is the second leading cause of death globally, and was responsible for 9.6 million deaths, or one out of six deaths in 2018 [1]. Of all tumors, breast cancer is the most common among women worldwide. Global cancer statistics estimated that approximately 2.1 million new breast cancer patients would have been diagnosed in 2018, representing almost 25% of cancer cases among women [2]. Among the breast cancer subtypes, triple-negative breast cancer is frequently characterized by relapse, reduced survival, and a heterogeneous nature that is negative for the expression of estrogen and progesterone receptors and the human epidermal growth factor receptor 2 [3]. Conventional chemotherapy, radiotherapy, and surgery are still the gold standard treatment for breast cancer. However, the eventual development of metastasis, even after an effective remission of primary tumors, makes triple-negative breast cancer more aggressive, and it has a poorer prognosis than the other breast cancer subtypes [4]. Thus, the study of potential alternative therapies to treat triple-negative breast cancer patients has generated growing interest among researchers [5].

Newcastle disease virus (NDV) is one of the various species of viruses that are under clinical evaluation as vectors for oncolytic, gene-, and immune-stimulating therapies [6]. NDV is a member of the Orthoavulavirus genus in the Paramyxoviridae family [7] and possesses a 15,186 nucleotide-negative single-stranded RNA genome that contains six genes, including those that encode the nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase, and RNA-dependent RNA polymerase [8]. NDV is classified into three pathotypes, depending on the severity of the disease it causes in birds: lentogenic (avirulent), mesogenic (intermediate), or velogenic (virulent) [8]. NDV has been demonstrated to mediate its oncolytic effect by activating both the intrinsic and extrinsic caspase-dependent pathways of cell death [9]. NDV-induced apoptosis is dependent on the upregulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and caspase activation [10]. In addition, NDV can also develop additional immune-stimulatory mechanisms by releasing danger signals during virus replication and stimulating immune cells to produce cytokines, such as interferons (IFNs) or TNF, which in turn lead to the activation of natural killer (NK) cells and sensitized T cells, among others [11,12,13,14,15,16,17,18,19,20]. Recent studies have demonstrated that different strains of the NDV can be used to treat breast cancer in mouse models [21,22,23], through either direct oncolysis or cytokine-mediated apoptosis. Our previous studies have demonstrated that recombinant NDV from a velogenic strain obtained from an outbreak in Puebla (Mexico) in 2005 (rNDV-P05) exhibited antitumor activity against a panel of different tumor cell lines, including one double-negative breast cancer (HCC1954 breast carcinoma) [24]. rNDV-P05 has already been demonstrated as an effective immune-stimulatory agent when cocultured with human peripheral blood mononuclear cells (PBMCs) from healthy volunteers, stimulating the release of antitumor cytokines such as TRAIL, IFNs, and TNF [24].

In the present study, we demonstrated that rNDV-P05 exhibits antitumor and immune-stimulatory activities in a mouse model with orthotopic and syngeneic breast cancer.

METHODS

Culture of the 4T1 cell line for the breast cancer model

The 4T1 cell line was acquired from the American Type Culture Collection (ATCC® CRL-2539™). Cells (1 × 106) were cultured in modified RPMI-1640 medium (2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4,500 mg/L glucose, and 1,500 mg/L sodium bicarbonate; ATCC® 30-2001™) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, USA), 50 IU/mL penicillin, and 50 μg/mL streptomycin (Sigma-Aldrich, St. Louis, USA). Cultures were maintained under high humidity conditions at 37°C with 5% CO2 and 95% air. The cells were sub-cultured 2–3 times per week and the confluence was maintained at less than 70%. For tumor induction in mice, cells were harvested in 0.5 mM EDTA and concentrated to 1 × 106 cells/mL in sterile phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) at pH 7.4 and kept on ice no longer than 1 hour before injection into animals.

Animals used for tumor induction and growth monitoring

Female mice (Mus musculus; strain BALB/cAnNHsd), 7–8 weeks old, were used for the model. A total of 32 mice were used and divided into 4 groups (n = 8 per group). Healthy mice were purchased from Envigo RMS S.A. de C.V. (Mexico City, Mexico) and were kept in the animal facility of the university under controlled light and temperature conditions. Both food (2018 Teklad Global 18% Protein Rodent Diet; Harlan, Lyon, France) and water were provided ad libitum. Animals were weighed using a digital scale (A&D Co., Tokyo, Japan), and the weight of the animals was recorded twice a week throughout the experiment. The tumor was induced as described in an earlier study [25]. Briefly, 50 μL of the cellular suspension at 1 × 106/mL (5 × 104 cells in total) was injected subcutaneously in the left fourth inguinal mammary fat pad. Seven days after the tumor was induced and reached a volume of approximately 19 ± 2.5 mm3, the mice were randomly assigned to the four groups, and the corresponding treatment was started. The primary tumor was measured twice a week, recording the length and width using a digital Vernier (Milomex Ltd., Pulloxhill, UK). The tumor volume was reported in mm3 and was calculated as follows [26]: (Length × Width2)/2. The cutoff tumor volume to euthanize the mice was set at 1,500 mm3.

In vivo treatment with rNDV-P05

The rNDV-P05 virus was obtained from a commercial attenuated live vaccine (Investigación Aplicada, S.A. de C.V., Tehuacán, Mexico) and was purified and characterized as described in an earlier study [24]. Two treatment approaches were assessed: systemic (via tail vein) and intratumoral administration. Sterile PBS (pH 7.4) was used as control for each treatment model. For the virus-treated groups, 1,000 hemagglutination units (HU) of rNDV-P05 in 50 μL of PBS were injected. This dose has been reported to be effective and safe for other lentogenic and velogenic strains [27]. A total of three interventions at intervals of seven days, starting seven days after tumor induction, were carried out. On the 28th day post-tumor induction (DPTI), the experiment was completed and the animals were euthanized.

Determination of tumor weight, spleen index, and lung metastasis

After the mice were euthanized, the tumor and spleen were excised and weighed. The percentage of tumor inhibition was determined as follows: [(Mean Weight of the Tumor in the Control Group − Mean Weight of the Tumor in the Treated Group)/Mean Weight of the Tumor in the Control Group] × 100. To calculate the spleen index of each animal, the following formula was used: (Spleen Weight/Body Weight) × 100. To examine the presence of metastasis, the whole lung was harvested and processed following a previously established protocol [25]. The lung samples were finely minced, digested with 1 mg/mL collagenase (from Clostridium histolyticum; Sigma-Aldrich) for 1 hour at 4°C, filtered through a 70 µm nylon cell strainer and washed three times with PBS. The obtained cellular suspension was cultured in six-well plates with RPMI-1640 medium (as detailed above) supplemented with 60 μM 6-thioguanine (Sigma-Aldrich) as a metastatic cell selective agent, and kept under the aforementioned conditions for seven days. Metastatic clonogenic colonies were identified by staining with 0.1% violet crystal dye, quantified, and represented as numbers per lung.

Enzyme-linked immunosorbent assay (ELISA) of serum samples

Blood was obtained at the end of the experiment and centrifuged at 2,000 × g for 10 minutes at room temperature (RT), and the obtained sera were kept at −80°C until utilization. The serum levels of IFN-α, IFN-γ, TNF-α, and TRAIL were quantified using commercial ELISA kits following the manufacturers’ instructions (mouse IFN-α platinum [eBioscience, Vienna, Austria]; mouse IFN-γ [Thermo Fisher Scientific, Inc., Waltham, USA]; mouse TNF-α [Life Technologies, Merelbeke, Belgium]; mouse TRAIL [CD253; Abcam, Cambridge, UK]). Absorbance was read using an iMark™ microplate reader (Bio-Rad Laboratories, Tokyo, Japan), and the concentration of each cytokine was determined by interpolation in a curve set with the standards supplied by the kit.

Immunofluorescence

Thin paraffin-embedded tumor sections were subjected to antigen retrieval using a Diva Decloaker kit (Biocare Medical, Pacheco, USA), following the manufacturer’s instructions. Subsequently, samples were blocked with bovine serum albumin 3%, and the primary antibody was added (polyclonal rabbit anti-mouse natural cytotoxicity triggering receptor 1 [NCR1] or polyclonal rabbit anti-mouse CD8, both at 1:25 dilution; AB214468 and AB203035, respectively; Abcam). The antibodies were incubated at RT overnight. Thereafter, the slides were washed twice and incubated with the secondary antibody (goat anti-rabbit IgG-Alexa594; AB150080 at 1:2,000 dilution; Abcam) for 2 hours at RT. Finally, after washing, 3 µM DRAQ7 (DR70250; BioStatus, Leicestershire, UK) was added for 20 minutes at RT, and the slides were mounted using Fluoroshield (Sigma-Aldrich). Images were acquired under a confocal microscope (DM2500; Leica, Wetzlar, Germany) and analyzed with LASX software (Leica). Image analysis was carried out by scanning samples in different directions using 40 randomly selected images per sample.

Statistical analysis

Data on tumor volume, body weight, cytokine levels, and NCR1+ and CD8+ infiltrated cells were represented as the mean ± standard error of the mean (SEM) of at least three independent trials. Statistical analyses were performed using the two-tailed unpaired Student’s t-test with a 95% confidence interval (CI). Data on tumor weight, tumor inhibition rate, spleen index, and metastatic clonogenic colonies were compared using the two-tailed nonparametric Mann-Whitney test with a 95% CI and represented as the median and interquartile range (IQR). A p-value < 0.05 was considered significant. All analyses were performed with GraphPad Prism 6.0 software (GraphPad Software, San Diego, USA).

Ethical approval

All animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee of the Autonomous University of Aguascalientes (approval date: 10/03/2013) and complied with the Animal Welfare Mexican Law (NOM-062-ZOO-1999) and National Institute of Health Guide for the Care and Use of Laboratory Animals.

RESULTS

rNDV-P05 reduces tumor volume and weight

To determine whether the rNDV-P05 virus possesses antineoplastic capacity in vivo, a model of syngeneic orthotopic breast cancer was established in female BALB/c mice, and two delivery methods were used, systemic and intratumoral (Figure 1A and B, respectively). Tumor growth kinetics for systemic delivery showed that at the beginning of treatment (DPTI 7), the control and rNDV-P05 groups showed similar tumor volumes and remained without a significant difference throughout DPTI 11 and DPTI 14 (Figure 1C). However, on DPTI 17 (after the second dose of rNDV-P05), a significant reduction in tumor volume was observed in treated animals compared to the control group (p < 0.01), and this was maintained on DPTI 21 (p < 0.01). On DPTI 24 and DPTI 28 (after the third virus dose), the tumor volume in treated animals was reduced by 68% and 53% in comparison to the control group (p < 0.001). On the other hand, when the virus was administered via an intratumoral injection, the tumor volume in both the groups was similar, with no significant reduction in the rNDV-P05 group compared to that in the control group on any of the days evaluated (Figure 1D). Individual kinetic curves from systemic (Figure 1E) vs. intratumoral (Figure 1F) treatment show that systemic treatment is more homogeneous than is intratumoral treatment. This is also reflected in the survival curve where systemic treatment shows a significant difference vs control (Figure 1G, p = 0.046). No significant difference was observed with intratumoral treatment (Figure 1H; p = 0.072). Therefore, further analysis was performed only with mice undergoing systemic therapy.

Figure 1. Effect of recombinant Newcastle disease virus (rNDV-P05) on 4T1-induced tumor volume and body weight. Schedule of tumor induction (4T1 cells) and the rNDV-P05 administration method, (A) systemic (IV injection) or (B) intratumoral (IT injection). Tumor growth kinetics for (C) systemic (IV injection) or (D) intratumoral (IT injection). Arrows indicate days in which rNDV-P05 or phosphate-buffered saline (control) was administered. Individual tumor growth kinetics for each administration method, (E) systemic or (F) intratumoral. Survival plots (Kaplan-Meier) for each administration method, (G) systemic or (H) intratumoral. The data are represented as mean ± standard error of the mean (n = 8). The unpaired t-test (two-tailed, 95% confidence value) was used to compare between groups.

Figure 1

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IV = intravenous; IT = intratumoral; DPTI = day post-tumor induction.

*p < 0.01; †p < 0.001 vs. control.

In addition to analyzing the tumor volume, the tumors were removed from mice (Supplementary Figures 1, 2, 3, 4) and weighed (Figure 2A). The systemic rNDV-P05 group showed a significant reduction in tumor weight than did the control group (1.15 g [IQR 0.62] vs. 0.86 g [IQR 0.28], p = 0.0256; Figure 2A). The percentage of tumor inhibition rate was calculated, and it was observed that systemic rNDV-P05 administration significantly inhibited the mass (in grams) of the tumor by 31% (Figure 2B; p = 0.0218). Representative images of tumor sections from the control group and the intravenous NDV-P05 treated group stained with hematoxylin and eosin are shown in Figure 2C and D, respectively.

Figure 2. Effect of the systemic administration of recombinant Newcastle disease virus (rNDV-P05) on the 4T1-induced tumor, spleen, and lungs. (A) Tumors were collected from mice and weighed, and a representative image from each group is displayed. The bar = 1 cm. (B) The tumor inhibition rate is presented per group. Images (10×) from tumor sections stained with hematoxylin and eosin from the (C) control group and (D) NDV-P05 treated group (E) Analysis of the number of metastatic clonogenic colonies from the lungs of each mouse. Representative images of metastatic clonogenic colonies grown in vitro from the lungs are shown. Blue-violet dots indicate the presence of a 4T1 metastatic clonogenic colony. (F) The spleen index is presented as the percentage per group, and a representative image from each group is displayed. The bar = 1 cm. Data are presented as median and interquartile range (n = 8). Mann-Whitney test (95% confidence value) was used to compare between groups. The p < 0.05 was considered to be statistically significant vs. control.

Figure 2

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rNDV-P05 diminishes metastasis in the lungs

Another key feature to evaluate was the capability of rNDV-P05 to protect mice from the metastasis of 4T1-induced primary tumors, especially in the lungs. Primary lung tissue culture was performed with a selective agent for 4T1 tumor cells, as described in the Methods section. After seven days of growth under normal culture conditions, the presence or absence of 4T1 metastatic clonogenic colonies was determined visually (Figure 2E). In the case of systemic rNDV-P05, a marked and significant reduction in the abundance of metastatic clonogenic colonies was observed vs. the control group (91 colonies [IQR 48.3] vs. 7 colonies [IQR 18], p = 0.0062; Figure 2E). An increased spleen index in 4T1 tumor-bearing mice is reportedly associated with secondary cancer development [28,29]. Macroscopically, spleens from mice receiving systemic rNDV-P05 were smaller than those of the control mice (Figure 2F), and when the spleen index was calculated, it was 24% lower than that in the controls (p = 0.0031, Figure 2F). These data demonstrate that intravenous treatment with rNDV-P05 reduces both 4T1 cell metastasis in the lung and the spleen index.

Systemic administration of rNDV-P05 increases serum antitumor cytokine levels and CD8+ cell infiltration in primary tumors

After demonstrating that the systemic delivery of rNDV-P05 resulted in a considerable reduction in the primary tumor volume and weight as well as metastasis in the lung, we sought to determine the underlying mechanism. As TNF-α, IFN-α, IFN-β, and TRAIL are cytokines closely related to antitumor immunity [30,31,32,33], we investigated their levels in the sera of mice receiving systemic rNDV-P05. We found that the levels of TNF-α, IFN-α, TRAIL, and IFN-γ were significantly elevated in the rNDV-P05 group compared to that in the control group on DPTI 28 (76.8 ± 12.3 pg/mL vs < 46 pg/mL [not detected], p < 0.0001; 192.1 ± 23.5 pg/mL vs. 90.6 ± 3.9 pg/mL, p = 0.0012; 39.8 ± 10.6 pg/mL vs. 6.6 ± 3.2 pg/mL, p = 0.0102; 102.1 ± 9.1 pg/mL vs. 70.4 ± 6.5 pg/mL, p = 0.0093; Figure 3).

Figure 3. Cytokine levels in the serum of mice systemically administered with recombinant Newcastle disease virus (rNDV-P05). The concentrations of (A) TNF, (B) IFN-α, (C) TRAIL, and (D) IFN-γ in the serum of animals from the systemic administration (intravenous injection) groups were measured by enzyme-linked immunosorbent assay at day 28 post-tumor induction. The sera from four mice of each group were tested in duplicate (n = 8). Data are represented as the mean ± standard error of the mean. The p < 0.05 was considered statistically significant vs. control.

Figure 3

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TNF = tumor necrosis factor; IFN = interferon; TRAIL = tumor necrosis factor-related apoptosis-inducing ligand; ND = not detected.

Additionally, we evaluated the infiltration of NCR1+ and CD8+ cells in primary tumors by employing immunofluorescence (Figure 4A). NCR1 and CD8 are considered accurate surface markers for NK and cytotoxic T cell identification, respectively, and both cell populations are associated with good prognosis for breast cancer [34,35]. Our results showed a significantly higher infiltration of CD8+ cells in tumors from mice treated with systemic rNDV-P05 vs control (p < 0.0027; Figure 4B). The number of NCR1+ cells infiltrated in tumors from rNDV-P05 mice was lower than that in the control group (p = 0.0039; Figure 4C).

Figure 4. NCR1+ and CD8+ cells infiltrated 4T1-induced tumors in mice systemically administered with recombinant Newcastle disease virus (rNDV-P05). (A) Immunofluorescence staining of NCR1 and CD8-positive cells (green) localized inside primary tumors. A background control was evaluated using only the secondary antibody. The number of (B) CD8+ and (C) NCR1+ cells/sample. Primary tumors from four mice of each group were evaluated. Data are represented as mean ± standard error of the mean. The p < 0.05 was considered to be statistically significant vs. control.

Figure 4

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NCR1 = natural cytotoxicity triggering receptor 1.

DISCUSSION

The 4T1 mouse mammary carcinoma cell line is a triple-negative breast cancer cell line that is well documented to be very aggressive and highly metastatic [25,36]. Primary breast tumors established in BALB/c mice using the 4T1 cell line are a well-recognized model of simulating metastatic breast cancer or phase IV breast cancer, which typically metastasizes to the lung, lymph nodes, and liver [37,38,39]. Till date, the development of different therapies for the treatment of breast cancer using the 4T1 model has been documented [38,40], including the use of oncolytic viruses [41,42], for which the efficacy in terms of the reduction in the size of the primary tumor or the inhibition of a metastatic process is variable. There are a few studies with this model of breast cancer using NDV as a therapeutic agent, but none have used the strain rNDV-P05 [21,22,23]. The results of our study demonstrated, for the first time, that the systemic administration of rNDV-P05 to mice strongly impacts 4T1 mammary primary tumor development by decreasing tumor volume and weight. In addition, the systemic administration of rNDV-P05 prevents the metastasis of 4T1-induced tumors to the lungs and the increase in the spleen index of animals. None of these effects were observed when rNDV-P05 was administered intratumorally. Our study also provides additional evidence for the major antitumor properties of systemically delivered rNDV-P05 in this triple-negative breast cancer model, showing increased serum levels of TNF-α, IFN-α, IFN-γ and TRAIL and tumor infiltration of CD8+ cells and decreased NCR1+ cells in the mammary tumor.

The intratumoral administration of the NDV strains AF2440 or Anhinga has been shown to reduce the weight and volume of 4T1-induced tumors [21,22,23]. We did not observe any significant decrease in the tumor volume or mass when 1,000 HU of rNDV-P05 were administered intratumorally every seven days. In previous studies, a maximum dose of 64 HU daily [21], 27 HU/kg twice a week [22], or 1 × 108 plaque-forming unit daily [23] have been used, indicating that the amount and frequency of the dose, as well as the virus strain, have a relevant effect on 4T1 tumors when injected directly into the tumor. Conversely, we found that the growth of the primary tumor was significantly inhibited when rNDV-P05 was injected systemically, which indicates that for the present experimental conditions, the best route of administration is systemic. Our results are in agreement with a clinical trial on systemic therapy with the NDV strain PV701 that shows a promising result as an important therapy for patients with advanced solid tumors [43]. In this clinical study, the authors characterized the safety profile of intravenously administered NDV PV701 and made significant improvements in patient tolerability. The side effects following NDV PV701 dosing appeared to be manageable and can be categorized into three classes: a) flu-like symptoms; b) tumor-site specific adverse events; c) infusion reactions [43]. However, due to the variability and virus strain-dependent response of the NDV, similar approaches as described by the authors must be followed when different strains are planned to be tested in humans. The antitumor efficacy of other NDV strains has already been confirmed using different routes of administration with models of colon cancer and lymphoma in immune-compromised mice [27]. In that study, two strains, one velogenic (73T) and the other lentogenic (Ulster), and multiple doses of 1,000 HU were used. The growth of the colon tumor was inhibited only by the intratumoral administration of the Ulster strain, while no antitumor effect was observed after the systemic administration of the 73T or Ulster strain. The treatment provided intravenously to combat lymphoma did not have a favorable effect on the animals treated with either virus strain. Altogether, these results indicate that for each cancer type and viral therapy, the optimum administration route must be determined, as previously evidenced [42].

It is important to evaluate the anti-metastatic activity of the rNDV-P05 strain to better predict the protective effect that might occur in patients. Therefore, we decided to examine the presence of metastatic colonies in the lung, one of the main organs affected by metastasis, both in a 4T1 mammary carcinoma mouse model and in human breast cancer [39]. The high metastatic ability of the 4T1 cells is also reflected by an increase in spleen mass, which is usually determined as an increase in the spleen index [28,38,39]. These variables have already been reported as important indicators when declaring the efficacy of any therapy in a model of breast cancer induced with the 4T1 cell line [25,28,38,40,41,42]. We have found that treatment with rNDV-P05 virus injected intravenously reduces the presence of 4T1 metastatic clonogenic colonies in the lungs, and that there were animals in whom the presence of these colonies was not detected. Systemic rNDV-P05 administration was found to reduce spleen enlargement. These results are of enormous importance, since the poor prognosis of patients with stage IV breast cancer is largely due to the presence of metastases in organs other than the mammary gland [3,4]. Even though breast cancer metastasis to the spleen in humans is rare, the reported cases are increasing due to improved imaging modalities and more attentive patient follow-up [44]. However, as explained above, for the 4T1 model, the spleen index is an important indicator for determining the success of therapy [25,28,38,40,41,42]. Further spleen histological analysis might provide more information about the pathological status of this organ in experimental metastatic breast cancer. Notably, previous evaluation of NDV in a 4T1 cancer model [21,22,23] did not present evidence or results of the reduction in metastasis in the lungs or other organs.

The mechanism by which the virus inhibits both the size of the primary tumor and the process of metastasis in the lung has partially been explored by the present study. Since intravenous (systemic) therapy showed antitumor effects and our previous in vitro results have shown increased cytokine secretion by PBMCs in response to rNDV-P05 [24], we speculated that the effectiveness of the therapy is likely due to an immune-stimulatory process. We observed that treatment with rNDV-P05 increased the TNF-α, IFN-α, IFN-γ, and TRAIL serum levels, the same proinflammatory and antitumoral cytokines that are secreted in vitro by human PBMCs stimulated by rNDV-P05 [24]. Furthermore, these findings are in accordance with those found with other NDV strains administered either intratumorally or systemically [10,11,12,13,14]. NDV-induced apoptosis is dependent on the upregulation of TRAIL and caspase activation, which cause opening of mitochondrial permeability transition pores, loss of mitochondrial membrane potential, and subsequent activation of apoptosis [10]. Besides, NDV can also develop additional immune-stimulatory mechanisms. NDV provokes the release of danger signals into the tumor cell cytoplasm during virus replication [11,12,13] and stimulates the immune cells to produce cytokines, such as IFNs or TNF [14]. In turn, these inflammatory cytokines lead to the activation of NK cells, monocytes, macrophages, and sensitized T cells, stimulating both innate and adaptive immune responses [14,15,16,17,18,19,20]. Nevertheless, a direct oncolytic capacity should not be discarded; we previously demonstrated that rNDV-P05 exhibits antitumor activity against a panel of different tumor cell lines, including HCC1954 breast carcinoma, a tumor cell line of double-negative breast cancer [24]. In addition, it has already been confirmed that the induction of antitumor cytokines by NDV is capable of recruiting or activating cells involved in immunosurveillance, such as CD4+ Th1 cells, CD8+ cytotoxic T cells, and NK cells, ultimately reflecting the inhibition of malignant cell proliferation [45,46]. We observed a significant increase in tumor-infiltrating CD8+ cells in mice systemically treated with rNDV-P05, likely explaining the reduction in the size and weight of tumors in this group. However, it was surprising that the presence of NK cells was lower in that group than that in the control group, even though it has been demonstrated that NDV activates and recruits NK cells to tumor sites [45,46]. As seen with other immune responses [47,48], an NDV strain-dependent factor is probably involved in these different responses. It will be interesting to assess whether the antitumor effect can be increased when combined with other types of therapies, whether conventional, such as chemotherapy, or alternatives, such as immunotherapy. To date, NDV oncolytic virotherapy has demonstrated biosafety in preclinical studies in human-like animal models or in clinical trials, revealing the absence of NDV-related lesions and hematologic or biochemical abnormalities [5,49].

The evaluation of rNDV-P05 in a syngeneic orthotopic model of 4T1 cell-induced breast cancer in BALB/c mice shows that the systemic administration of the virus significantly reduces the tumor mass and volume, spleen index, and metastatic clonogenic colonies in lung tissue, and increases the inhibition rate of the tumor. The intratumoral administration model with rNDV-P05 was ineffective for all the parameters evaluated. The antitumor and antimetastatic capability of rNDV-P05 is due to, at least partially, its immune-stimulatory properties and its ability to recruit CD8+ T cells into tumor tissue. Previous results, together with our findings, clearly exemplify how the antitumor activity of NDV has three key variables: i) the strain used; ii) the type of cancer to be treated; and iii) the route of administration of the virus.

ACKNOWLEDGMENTS

Oscar Antonio Ortega-Rivera has a doctoral fellowship from the National Council of Science and Technology of Mexico.

Footnotes

Funding: This work was supported by the National Council of Science and Technology of Mexico (Grant number 207977).

Conflict of Interest: The authors declare that they have no competing interests.

Author Contributions:
  • Conceptualization: Ortega-Rivera OA, Rivas-Santiago B, Del Toro-Arreola S, Salinas E, Quintanar JL.
  • Investigation: Ortega-Rivera OA, Gallegos-Alcalá P, Jiménez M, Torres Juarez F.
  • Methodology: Ortega-Rivera OA, Gallegos-Alcalá P, Jiménez M, Torres Juarez F.
  • Project administration: Salinas E, Quintanar JL.
  • Resources: Quintanar JL.
  • Supervision: Rivas-Santiago B, Del Toro-Arreola S, Salinas E, Quintanar JL.
  • Writing - original draft: Ortega-Rivera OA, Salinas E.
  • Writing - review & editing: Salinas E, Quintanar JL.

SUPPLEMENTARY MATERIALS

Supplementary Figure 1

Tumors collected at the end of the study from the group treated with recombinant Newcastle disease virus (rNDV-P05) systemically.

jbc-26-186-s001.ppt (343KB, ppt)
Supplementary Figure 2

Tumors collected at the end of the study from the control group treated with phosphate-buffered saline systemically.

jbc-26-186-s002.ppt (276.5KB, ppt)
Supplementary Figure 3

Tumors collected at the end of the study from the group treated with recombinant Newcastle disease virus (rNDV-P05) via intratumoral injection.

jbc-26-186-s003.ppt (349KB, ppt)
Supplementary Figure 4

Tumors collected at the end of the study from the control group treated with phosphate-buffered saline via intratumoral injection.

jbc-26-186-s004.ppt (316KB, ppt)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1

Tumors collected at the end of the study from the group treated with recombinant Newcastle disease virus (rNDV-P05) systemically.

jbc-26-186-s001.ppt (343KB, ppt)
Supplementary Figure 2

Tumors collected at the end of the study from the control group treated with phosphate-buffered saline systemically.

jbc-26-186-s002.ppt (276.5KB, ppt)
Supplementary Figure 3

Tumors collected at the end of the study from the group treated with recombinant Newcastle disease virus (rNDV-P05) via intratumoral injection.

jbc-26-186-s003.ppt (349KB, ppt)
Supplementary Figure 4

Tumors collected at the end of the study from the control group treated with phosphate-buffered saline via intratumoral injection.

jbc-26-186-s004.ppt (316KB, ppt)

Articles from Journal of Breast Cancer are provided here courtesy of Korean Breast Cancer Society

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