Evaluation of the in vitro and in vivo effect of liposomal doxorubicin along with oncolytic Newcastle disease virus on 4T1 cell line: Animal preclinical research

Abstract Background Breast cancer is one of the most common malignancies in women, with one in 20 globally. Oncolytic viruses have recently been the first step in the biological treatment of cancer, either genetically engineered or naturally occurring. They increase specifically inside cancer cells and destroy them without damaging normal tissues or producing a host immune response against tumour cells or expressing transgenes. One of the most known members of this family is the Newcastle disease virus (NDV), a natural oncolytic virus that selectively induces apoptosis and DNA fragmentation in human cancer cells. Methods This study performed biochemical and molecular investigations with variable doses of NDV (32, 64, 128 HAU) and liposomal doxorubicin (9 mg/kg) on mouse triple‐negative mammary carcinoma cell line 4T1 and BALB/c models tumours for the first time. Results Real‐time quantitative PCR analysis in NDV‐treated animal tumours showed increased expression of P21, P27 and P53 genes and decreased expression of CD34, integrin Alpha 5, VEGF and VEGF‐R genes. Additional assessments in treated mouse models also showed that NDV increased ROS production, induced apoptosis, reduced tumour size and significantly improved prognosis, with no adverse effect on normal tissues. Conclusions These findings all together might indicate that NDV in combination with chemotherapy drugs could improve prognosis in cancer patients although many more conditions should be considered.

In 2012, approximately 1.7 million people were diagnosed with breast cancer worldwide, and about half a million people died from this cancer (Ferlay et al., 2015). Significant prognostic factors include visceral, brain, and multiple metastases to other organs, which deteriorate the prognosis. In contrast, many cancers, such as bone cancer, and youngsters have a better prognosis (Mariotto et al., 2017). Treatment with oncolytic viruses is one of the most robust and most advanced methods in recent years. Oncolytic viruses are either genetically engineered or naturally occurring. They proliferate specifically inside cancer cells and destroy them without damaging normal tissues. Induce and initiate systemic antitumour and enhance the antitumour effect of the primary drug (Fukuhara et al., 2016;Msaouel et al., 2018). These viruses can directly kill tumour cells through various mechanisms, but they are insufficient to destroy tumour tissue completely and must be combined with other exogenous or other therapies (Wei et al., 2018). Newcastle disease virus is a single-stranded, negative-sense RNA virus with an envelope belonging to the genus Avulav of the family Paramyxoviridae. According to Newcastle studies, they are classified into lentogenic, mesogenic and velogenic groups. The velogenic strain results in 100% poultry mortality (Samour, 2014;Alexander, 2000). Although Newcastle disease causes severe infections in birds, it is nonpathogenic to humans. It can only cause conjunctivitis in humans, and no human-tohuman transmission of the virus has been reported (Alexander, 2000).
The LaSota Newcastle strain is the lentogenic type of virus and a natural oncolytic virus that selectively infects human tumour cells, binds to the malignant cell membrane, then enters the cytoplasm through endocytosis, causing apoptosis and DNA fragmentation in cancer cells Czeglédi et al., 2006;Al-Shammari et al., 2016).
Anthracyclines, especially doxorubicin, which has anticancer activity and significant toxicity, have long been isolated from the bacterium Streptomyces peucetius var, and they are the mainstay of cancer treatment. The first liposomal encapsulated anticancer drug to receive clinical confirmation against a wide range of tumours, including solid tumours, transplantable leukaemia and lymphoma, was doxorubicin.
However, it has many side effects such as nausea, vomiting, alopecia, increased serum aminotransferase, acute liver injury and jaundice.
Recent studies have focused on developing new liposomal formulations, A polyethylene glycol coating can wrap around the liposome to prevent it from phagocytising to form the liposomal doxorubicin, which is only 100 nm in diameter, that reduce the side effects of the drug, and because it is less toxic, higher doses of the drug can be prescribed, consequently advancing treatment (National Center for Biotechnology Information 2021; Tardi et al., 1996;Gabizon, 2001). Chemotherapy drugs have improved cancer treatment, but these drugs have many side effects, and recurrence of the disease has been seen in many cancers. So many efforts have been made to reduce the dose of chemotherapy drugs. The outcome is using chemotherapy drugs alongside oncolytic viruses to increase their function (Allen & Chonn, 1987;Ottolino-Perry et al., 2010).
This study examines the effects of the Newcastle virus and liposomal doxorubicin drug together in the 4T1 cell line.

Animal tumour model
Female inbred BALB/c mouse models at the age of 3 weeks were housed at a temperature of 22−27 ο C with a 12-h light / dark cycle in a pathogen-free isolation room. They were kept in this situation to adapt to the environment for a week. The Animal Ethics Committee approved all in vivo experiments in this study.
The mice were divided randomly into groups (5 mouse models per group). A total 1×10 (Wei et al., 2018) 4T1 cells/100 μL in the logarithmic growth phase were subcutaneously (sub-Q) injected into the right flank region of the BALB/c mouse models. The size of tumour masses was determined every three days. Tumour volume was measured using the following equation (Faustino-Rocha et al., 2013;Vassileva et al., 2008;Zandi et al., 2019).

LaSota strain of Newcastle disease virus
LaSota Newcastle is a lentogenic strain of the virus and is relatively stable in the standard environment (Czeglédi et al., 2006). For the proliferation of NDV, the particles were injected into the allantoic cavity of embryonated chicken eggs aged between 7-9 days. After being incubated for five days at 37 • C, it was incubated at 4 • C for 24 h to kill the foetus. In the last step, the NDV was harvested from the allantoic fluid and filtered through a 0.22 μm filter, and centrifuged (3000 rpm, 30 min, 4 • C) have been used to purify it of debris for injected intraperitoneally in mouse models.
Haemagglutination assay (HA) test was performed to determine the proliferation of NDV (Santry et al., 2018). The haemagglutination units were shown per mL as HAU/mL.

Cell viability assay
The

Apoptosis and necrosis detection
The 4T1 cell line was cultured on a 6-well plate until it reached 70 to 80% of the total well capacity. Cells interacted with the IC50 dose of NDV in two groups (6 and 12 h). The treated cells were harvested by trypsin and centrifuged at room temperature for 5 min at 1200 rpm.
The cell pellet was resuspended in phosphate-buffered saline and analysed by a flow cytometer (BD FACSCalibur, USA) and FlowJo (v10.5.3) software. Each experiment was assayed five times.

RNA extraction and real-time PCR
Total RNA was extracted from tumour tissue using the TRIzol reagent (Tiangen, Cat. No. 15596-026, Beijing, China). 200 μL chloroform was added to the solution, shaken for 30 s, incubated at room temperature for 15 min, and centrifuged at 14,000 rpm at 4 • C for 15 min.
The supernatant was transferred to a new RNase-free microtube with an equal volume of cold isopropanol and stored at minus 80 • C for 30 min. The tubes were centrifuged at 14,000 rpm at 4 • C for 10 min and mixed with 70% ethanol. Finally, the sample was centrifuged at 7000 rpm at 4 • C for 10 min, then combined with Tris-EDTA and kept at minus 80 • C. The concentration of extracted RNA was measured using a UV spectrophotometer (NanoDrop One Microvolume UV-Vis spectrophotometer, Thermofisher, USA). cDNA was synthesised using reverse-transcribed RNA by a kit (QuantiNova SYBR Green PCR) following the manufacturer's procedure. The reverse transcription reaction was performed at 37 • C for 1 h, and the inactivation of ( Figure 1).

Evaluation of intracellular reactive oxygen species (ROS) generation by fluorescence microscopic methods
The DCFH diacetate form (DCFH-DA) was used to measure the net intracellular generation levels of ROS. The 4T1 cell line was cultured on a 48-well plate. After it reached 70%-80% of the total well capacity, it interacts cells with IC50 dose of NDV in two groups (6 and 12 h).
DCFH-DA was added to the well and incubated for 45 min. The wells were washed twice with PBS. Cells were imaged in the epifluorescence mode with a blue filter. All observations and control processes were perpetrated with a computer connected by IEEE1394.

Histological staining
Slides from tumours samples, spleen, liver, stomach, heart, brain, kidneys and lungs were collected for haematoxylin and eosin (H&E

Chemotherapy regimens
Two groups of five mouse models with 4T1 tumours (with and without IC50 dose of NDV injection) were randomly allocated to various treatment groups on day 14 of tumour inoculation, were used chemotherapy regimens. The chemotherapy groups were treated with liposomal doxorubicin (sinadoxosome, liposomal) via an intraperitoneal bolus injection of 9 mg/kg/week for three successive weeks.
Cardiotoxicity, skin toxicity, mucositis, weight loss, anaemia, aller-gic reactions, vomiting, alopecia and any other signs or symptoms of toxicity were evaluated (Ansari et al., 2017;Coleman et al., 2006). All mouse models were followed until relapse of tumours or death.

Data analysis and statistics
All data are described as standard deviation (SD). The normal allocation of each set was determined using a two-way analysis of variance (twoway ANOVA), and the datasets that did not have a normal distribution were normalised in SPSS software with a two-step approach. The level of significance was set to p < 0.05.
To evaluate the cytotoxicity, and the apoptotic effect of LaSota strain on 4T1 cell line, GraphPad Prism version 8.4.0 and SPSS version 22.0 were used for the statistical significance analyses. phase from 65% to 47% and an ascending course in phase S and Sub G1 ( Figure 2I).

In vitro effects of Newcastle disease virus LaSota strain on 4T1 cell line
The highest titre of NDV-LaSota strain for the MTT experiment to measure the effect of NDV on cell growth was 256 HAU. The IC50 titre was obtained 64 HAU by inducing 50% mortality of 4T1 cells.
The Annexin/PI test was used to evaluate the actual apoptosis rate in the 4T1 cell line after 6 and 12 h of exposure to NDV. The results of primary apoptosis depicted a downwards trend from 3.58% to 2.97% and 1.95% at 6 and 12 h, sequentially. The results of primary apoptosis depicted a downwards trend from 3.58% to 2.97% and 1.95% at 6 and 12 h, sequentially, as well as late apoptosis that decreased from 8.02% to 7.73% and 6.89% in the mentioned interactions. Live cell counts showed a reduction from 83% to 64% ( Figure 2II).
MTT test was used to show the effect of NDV on cell growth. 8HA was the highest titre of NDV-LaSota strain evaluated in the MTT test, and the IC50 titre was 64 HAU ( Figure 2III).

In vivo inhibition of mammary tumour growth by Newcastle disease virus LaSota strain in mouse models
A total of 1 × 10 6 /100 μL of 4T1 cancer cells (triple-negative breast cancer mouse model cell line) were subcutaneously injected into the female BALB/c mouse models' right flank and followed up until the tumour volume reached ∼1000 mm 3 to evaluate whether NDV inhibits malignant tumour growth in vivo. Afterwards, the mouse models were F I G U R E 3 (I) Comparison of tumour sizes in each group showed that the treated mouse models significantly reduced their tumour size. (II) Tumour size alterations up to 15 days after the first day of treatment indicate that the tumour size decreased with an increasing drug dose.
divided into seven groups. The NDV were administrated in three different doses, 32, 64 and 128 HAU, respectively. Another group was treated with NDV (64HAU) in conjunction with liposomal doxorubicin (3 mg/kg). The other group was only treated with liposomal doxorubicin (9 mg/kg). The last groups were considered as the placebo and control.
All injections were performed seven days apart.
Images taken from the treated mouse models showed an outstanding reduction in tumour size when the NDV and liposomal doxorubicin inject together. Tumour degradation in NDV-treated mouse models proved a special relationship between tumour degradation rate and dose of NDV. So that after 21 days, a significant reduction in tumour size was observed even in low doses. After approximately 21 days of continuous injection, the mouse models treated with doxorubicin became tumour-free ( Figure 3I). However, in mouse models treated with IC50 dose of NDV and doxorubicin together, the tumour size reduction rate was 160-170 mm (Ferlay et al., 2015) per day, and no tumours were detected after about 10 days ( Figure 3II). It was astonishing to figure out that the tumour size in mouse models treated with IC50 dose of NDV and doxorubicin together reduced by roughly a quarter in less than five days without affecting other organs such as the heart, skin, brain or kidneys. Examination of the body weight and organs of mouse models in the treated groups and control groups showed that the Newcastle virus did not cause a significant change in the weight of mouse models. Additional tests, such as complete blood count (CBC), serum electrolyte and chemistry, liver and kidney function test, amylase, and lipase, demonstrated no abnormalities in the typical mouse models treated by NDV compared to the control group ( Figure 4). The long-term effects of NDV were evaluated over 1 year.
No complications were seen in NDV-treated mouse models, and they became pregnant twice during this period. Also, the next generation had no complications and was able to reproduce.

Comparative histopathological examinations of treated and control tumourised mouse models
Highly condensed nuclei are called pyknotic nuclei (Elmore, 2007), which in the H&E image, it is used as a marker to detect cancer cells, and NDV activated internal apoptotic pathways of these cells.
Histopathological analysis of NDV with doxorubicin-treated mouse model tumours revealed a significant increase in apoptosis compared with the control and NDV or liposomal doxorubicin-treated groups.

F I G U R E 4
Comparison of blood characterisation and weight of mouse models and their organs in the groups compared to the control group demonstrated that blood factors and body weight in mouse models treated with NDV and liposomal doxorubicin were not significantly distinct from the control group.
Cytoplasmic degradation is predominant in Newcastle with liposomal doxorubicin-treated malignant tissues; as the tissue matrix of the exposed tumours was not affected, the chance of necrosis was excluded ( Figure 5I). To demonstrate selective apoptosis in the tumour mass used IHC staining with P53 and Ki67 as markers of apoptosis and proliferative, respectively (Ozer et al., 2012;Zandi et al., 2021) ( Figure 5II). Also, H&E investigations of areas directly exposed to NDV as well as the body's vital organs, including the liver, kidneys, spleen, stomach, lungs, brain, and heart, were not demonstrated any pathological effects or any alternation, which indicates that the Newcastle virus will not negatively affect them ( Figure 6I and II). Immunohistochemical staining revealed that NDV+liposomal doxorubicin-treated tumours expressed higher P53 due to increased apoptotic and necrotic tumour cells and lower Ki67 expression as proliferative markers ( Figure 5II). P21, P16 and P53 are components of the apoptosis pathway, which were significantly upregulated just in NDV-treated tumours in quantitative real-time polymerase chain reaction (RT-PCR) analysis. Furthermore, spreading and adhesion transcriptomes including integrin α−5, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGF-R), and CD34 genes were downregulated in treated cohorts ( Figure 7I).

survival analysis
All mouse models died within roughly 50 days in the control and placebo groups. The Kaplan-Meier survival curves for the NDV and NDV-doxorubicin group demonstrated significantly higher survival rates than the control group, and they survived after 180 days.
In addition, pathology analysis proved that tissues were treated with NDV-liposomal doxorubicin had the least ulcer and necrosis ( Figure 7II).

DISCUSSION
Oncolytic viruses are an essential therapeutic protocol against cancer.
Newcastle disease virus is a natural oncolytic virus that induces adaptive immunity against tumours by selectively affecting the cancerous cells without harming the normal or untransformed ones (Wei et al., 2018;Schwaiger et al., 2017;Schirrmacher, 2015). In the study designed by Keshelava et al. (2009) Krishnamurthy et al. (2006) determined that NDV can eliminate human fibrosarcoma cells quickly; also, in human skin fibroblasts as control cells, it had no effect. NDV replication is independent of DNA replication in the host cell, and it can inhibit cancer cells by cytolysis or viral replication, which induces apoptosis and breaks the therapy resistance of tumour cells (Schirrmacher, 2015;Elankumaran et al., 2006).
Newcastle virus induces apoptosis in cancer cells by activating the death receptor (external) and mitochondrial (intrinsic) pathways. The drug combination is the most extensive method in treating cancer, and the main ambition is to reduce the drug dose and its side effect (Chou, 2010). Rajmani's investigations reported that NDV HN protein induces apoptosis in the human cervical cancer cell line by activating the internal SAPK/JNK pathway (Shim, 2015). Also, Ghrici demonstrated that the NDV activates mitochondrial transport pores and caspase-8 and induces apoptosis in MCF-7 human breast cancer cells (Ghrici et al., 2013 F I G U R E 6 (I) Histological examination indicated that NDV eliminates tumour cells, in addition, it does not cause any damage to vital organs, including the liver, kidneys, spleen, lungs, stomach, brain, and heart. The scale bars are set to 5 μm. (II) Histopathological analysis of the sections directly exposed to NDV demonstrates no evidence of apoptosis or necrosis. Furthermore, it can be seen that cellular assembly and morphology stayed intact. The scale bars are set to 5 μm.

CONCLUSION
In outline, we found an innate ability of NDV to induce selective apoptosis in cancer cells and annihilate malignant tumours in ani-mal models with clinically validated outcomes. NDV was used to treat the tumours of female mouse models. The Newcastle virus in humans has very few side effects compared to chemotherapy.
These side effects are temporary and disappear in 1 to 2 days.
Histopathological analyses and cellular/molecular examinations demonstrated activation of apoptosis and suppression of proliferative markers in treated malignant cell lines and mouse model tumours.
Due to the many side effects of chemotherapy drugs, this method can be used as an adjunctive therapy to decrease side effects, increase the antitumour efficacy, and offer a novel insight into using oncolytic viruses, especially Newcastle disease virus, in cancer treatment.
F I G U R E 7 (I) Used real-time reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the expression level of P16, P21, P53, CD34, integrin α5, VEGF and VEGF-R on day 26. NDV induced apoptosis in malignant cells via the overexpression of P21, P27 and P53 and downregulation of CD33, integrin α5, VEGF, and VEGF-A in exposed groups. In this study, the GAPDH gene was used as a control. All the data are exhibited as means ± SD. (II) Kaplan-Meier survival curves of different mouse models in each group. There is no distinction between the control and placebo groups' survival curves (log-rank test). NDV-liposomal doxorubicin group had the most survival rate. Survival rates depended on the dose of NDV and decreased in the lower dose of NDV.