Anti-Tumor Effects Of Combination Measles Vaccine Virus And Nimotuzumab Treatment On Nude Mice Bearing Human Larynx Cancer - Ngo Thu Hang

Journal of military pharmaco-medicine n o 5-2019 156 ANTI-TUMOR EFFECTS OF COMBINATION MEASLES VACCINE VIRUS AND NIMOTUZUMAB TREATMENT ON NUDE MICE BEARING HUMAN LARYNX CANCER Ngo Thu Hang1,2; Ho Anh Son1; Nguyen Linh Toan1 SUMMARY Objectives: To evaluate the antitumor effects of the combination treatment with measles vaccine virus and nimotuzumab in vivo. Materials and methods: We used human larynx cancer cell to form tumors on nude mice by xenograft model and to evaluate the anti-cancer effects of measles vaccine virus + nimotuzumab combination by assessing the survival time of mice and the rate of tumor cells induced apoptosis. Results: After 60 days of treatment by measles vaccine virus, nimotuzumab and measles vaccine virus + nimotuzumab combination, the medial tumor volumes were smaller and the survival time of mice lasted longer in nude mice treated with measles vaccine virus + nimotuzumab combination compared to mice treated with measles vaccine virus or nimotuzumab alone (p < 0.05). The apoptotic cell rate in the group treated with measles vaccine virus + nimotuzumab combination was significantly higher compared to the groups treated with measles vaccine virus or nimotuzumab alone (p < 0.05). Conclusions: Combination of measles vaccine virus and nimotuzumab treatment has a greater anti-tumor effect than single treatment with only measles vaccine virus or nimotuzumab on nude mice bearing human larynx cancer by xenograft model. * Keywords: Human larynx cancer; Nude mice; Measles vaccine virus; Nimotuzumab INTRODUCTION Head and neck cancers rank fourth among the most popular malignant diseases and are one of the most common cancers in men worldwide [1]. Among all cancers, laryngeal cancers are the second most common malignancy after nasopharyngeal cancers. According to statistics in various countries, laryngeal cancer accounts for 2% of all cancers. The disease is diagnosed in more than 10,000 men and 3,000 women in the United State each year. Most of them are at the age of 65. In Vietnam, laryngeal cancer ranks second after nasopharyngeal cancers among head and neck cancers and ranks tenth among all cancers, with the high incidence among men aged 40 - 60 [2]. Epidermal growth factor receptor (EGFR) has been observed to be overexpressed in the tumor tissues of head and neck cancer patients. EGFR has been shown to play an important role in cells growth, metastasis and proliferation of tumors development of head and neck cancers [3]. 1. Vietnam Military Medical University 2. Hadong Medical College Corresponding author: Nguyen Linh Toan (toannl@vmmu.edu.vn) Date received: 12/04/2019 Date accepted: 20/05/2019 Journal of military pharmaco-medicine n o 5-2019 157 Oncolytic virus therapy is based on the mechanism that the oncolytic viruses have the capability to selectively enter and replicate in infected tumor cells, subsequently, either directly kill the infected cells or stimulate immune responses against the tumor cells [4, 5]. Nimotuzumab, a monoclonal antibody, can bind specifically to EGFR and prevent the activation of the receptor [6]. In this study, we evaluate the anticancer effectiveness of measles viruses (Mev) against laryngeal cancer in combination with nimotuzumab in vivo. MATERIALS AND METHODS 1. Cell lines. Vero cell line (kidney, African green monkey) and human laryngeal squamous cell carcinoma Hep-2 cell lines (ATCC CCL-23, laryngeal SCC) were purchased from the American Type Cell Culture (ATCC, Manassas, VA, the United States). Vero cells were cultured in M199 medium (Biowest, France) supplemented with 10% of foetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (ATCC) to propagate vaccine strain MeV. Hep-2 cells were cultured in Eagle’s minimum essential medium (EMEM) (ATCC-formulated F-12K) (Catalog No.30-2004) medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin in a 75 cm2 culture flash. The cells were maintained in a condition of 37oC in a humidified incubator with 5% CO2. The cells were transferred when they reached 80% of culture flash area. The cells were harvested by using Trypsin EDTA, centrifuged to remove the culture medium. The density of the cancer cells was determined using Neubauer counters and optical microscopes. The standard density of 107 cells/mL was prepared for further experiments. 2. Propagation of measles viruses. MeV was plaque purified from Priorix (GlaxoSmithKline, the UK). The procedure to prepare MeV for further experiments has been described in detail in our previous study [7]. We also confirmed the presence of only MeV in each viral clone by using RT-PCR with specific primer pairs [7]. 3. Animal experiments. Six to eight-week-old male BALB/c nude mice were purchased from Charlie- River (the USA) and were kept under pathogen-free conditions in accordance with Animal Center Guidelines. The procedures were approved by Vietnam Military Medical University. To evaluate the effect of MeV, nimotuzumab or MeV + nimotuzumab combination on tumor growth and survival time in nude mice bearing Hep-2 tumors, the mice were inoculated with 106 Hep-2 cells in 100 µL FBS on the right rear flanks of male nude mice. One week after inoculation, the formation of tumor in mice was checked twice per week. When the tumor reached the size of 7 - 10 mm in diameter (approximately 6 - 7 days after inoculation), the mice were divided into 4 groups, 10 mice in each group. The mice were treated with PBS as control, MeV multiple doses (107 CFU/mouse/time, twice a week for 3 weeks) by intratumor injection and nimotuzumab was injected through the tail vein with single dose of 100 μg/mouse as well as treated with MeV + nimotuzumab Journal of military pharmaco-medicine n o 5-2019 158 combination. Tumor volume and survival time were observed and recorded. Tumor volume was calculated from calliper measurements of length and width of masses (volume (mm3) = length x 1/2 x width2). Relative tumor volume was calculated as the volume at a given time divided by volume on indicated time points after initiation of treatment. 4. Apoptosis assessment by flow cytometry. In order to access the programmed cell death of tumor cells, annexin V/PI kit was used to analyze sub-G1 DNA. Briefly, tumors were from nude mice, washed three times with PBS, transferred to a clean petry dish, divided into blocks with equal weight about 100 mg/block, then cut the blocks into the smaller pieces before put into the 1.5 mL tubes. Added 1.0 mL TE 1X solution (trypsin 0.25%), and incubated at 4°C for 12 hours. Removed the supernatant and the tumor cells were incubated 37°C for 20 minutes. Trypsin was removed by adding 500 µL of BPS into each tube, mixed by vortexing, centrifuged at 500 RCF for 1 minute and removed the supernatant. This step was repeated for three times to ensure trypsin was removed properly. Then, 700 µL BPS was added, mixed gently and checked cell death with trypan blue then cells were adjusted into a concentration of 106 cells/mL in 1X annexin-binding buffer solution. Subsequently, 100 µL of cell suspension were dyed with 5 µL of FITC annexin V and 1 µL of a PI solution, incubated at room temperature in dark for 15 minutes, then added 400 µL of 1X annexin-binding buffer, mix well and kept on ice. The cells were loaded on BD FacsLysis and fluorescence signals were measured at emission wavelengths of 530 nm (for FITC) and greater than 575 nm (for PI). * Statistical analysis: Data were analyzed with the GraphPad Prism 6.0 software (GraphPad Software, California, USA) and SPSS v.20 (SPSS Statistics, IBM, Armonk, NY, USA). Student’s t-test or Mann-Whitney U-test or Fisher’s exact test was applied to compare between groups wherever appropriate. The Kaplan- Meier method and the log-rank test were used to compare the survival time of nude mice between groups. Statistical significance was defined as p value ≤ 0.05. RESULTS 1. Laryngeal tumor formation in nude mice. Table 1: Result of laryngeal tumor formation in nude mice. Result Day after inoculation D0, n (%) D3, n (%) D7, n (%) D8, n (%) D1, n (%) Number of mice bearing tumor 0/40 13/40 26/40 36/40 40/40 Percentage 0% 32.5% 65% 90% 100% Journal of military pharmaco-medicine n o 5-2019 159 Figure 1: Laryngeal tumors were formed in nude mice. (A: day 3 after inoculated with Hep-2 cells; B: day 7; C: day 11; D: day 11) After inoculation of 106 Hep-2 cells suspended in 100 µL PBS into the right rear flanks of male nude mice, we regularly checked the formation and growth of laryngeal tumors by observing and measuring (figure 1). Three days after the inoculation, the tumors appeared at the injection site in 13/40 mice (32.5%), 7 days after the inoculation, 26/40 mice (65%) appeared tumors, and until day 11 all mice (40/40 = 100%) were observed bearing the tumors (table 1). Tumors appeared at the injection sites with no signs of ulceration, necrosis and bleeding. Mice weight remained unchanged and mice health in all groups was at good condition (figure 1). 2. Anti-tumor effects of MeV + nimotuzumab treatment in mouse model. D a y 0 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 2 0 0 3 0 0 D a y 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 D a y 1 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 5 0 0 1 0 0 0 1 5 0 0 D a y 4 3 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 4 7 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 0 4 0 0 0 6 0 0 0 *** D a y 5 4 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 0 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 2 0 0 3 0 0 D a y 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 D a y 1 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 5 0 0 1 0 0 0 1 5 0 0 D a y 4 3 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 4 7 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 0 4 0 0 0 6 0 0 0 *** D a y 5 4 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 0 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 2 0 0 3 0 0 D a y 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 D a y 1 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 5 0 0 1 0 0 0 1 5 0 0 D a y 4 3 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 4 7 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 0 4 0 0 0 6 0 0 0 *** D a y 5 4 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 0 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 2 0 0 3 0 0 D a y 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 D a y 1 5 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 5 0 0 1 0 0 0 1 5 0 0 D a y 4 3 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 *** D a y 4 7 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 2 0 0 0 4 0 0 0 6 0 0 0 *** D a y 5 4 T u m o r v o lu m e ( m m 3 ) C o n tr o l M e V N im o C o m b in a t i o n 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 * * ***** **** * ** *** ** ** *** Figure 2: Tumor volume of different treated groups. (*: p < 0.05; **: p < 0.01; ***: p < 0.001) Journal of military pharmaco-medicine n o 5-2019 160 We evaluated the tumor suppressive effect of MeV, nimotuzumab and MeV + nimotuzumab combination in human laryngeal cancer xenograft tumor model. Mice bearing laryngeal tumor were injected intratumorally with multiple doses of MeV, nimotuzumab and MeV + nimotuzumab combination (twice per week for 3 weeks). Tumor size and survival time were examined at different time points (on day 5, 15, 29, 40, 43, 47, 54 and day 60) after treatment. The results showed that the tumor volume was increasing after the laryngeal cancer cells were transplanted into the mouse. After treated with MeV, nimotuzumab and MeV + nimotuzumab combination, on day 43, 47 and 54, the tumor volume of treatment groups was significantly smaller compared to control (p < 0.0001), and tumor volume in the group treated with MeV + nimotuzumab combination was also significantly smaller compared to groups treated with MeV and nimotuzumab alone (figure 2). A S u rv iv a l ti m e ( d a y ) B Figure 3: Cumulative survival rate (A) and survival time (B) of nude mice bearing laryngeal tumor after 60 days. During follow up, eight mice in the control group died on day 12, 15, 33, 36, 40, 43, and day 57; four mice in the group treated with MeV died on day 22, 29, 47, and day 54; six mice in the group treated with nimotuzumab died on day 15, 22, 40, 47, and day 57; while only two mice in the group treated with MeV + nimotuzumab combination died on day 54 and day 57. After 60 days of follow-up, the survival rate in the treated group (18/30, 60%) was higher compared to the untreated group (2/10, 20%) (p < 0.05) (figure 3A). Similarly, the survival time of the combined Journal of military pharmaco-medicine n o 5-2019 161 treated group was higher compared to single treated groups and the control group (figure 3B). This result indicated that treatment with MeV and nimotuzumab increased the survival rate and time in the human laryngeal cancer xenograft tumor model. 3. Treatment with MeV and nimotuzumab induces apoptosis of tumor cells. p < 0 .0 0 1 A p o p t o ti c c e ll s ( % ) c o n tr o l M e V N im o C o m b in a t i o n 0 2 0 4 0 6 0 8 0 1 0 0 Figure 4: The proportion of apoptotic cells isolated from different groups. Apoptosis of tumor cells isolated from tumors was assessed by flow cytometry. The result showed that the proportion of apoptotic cells from treatment groups was higher than that of apoptotic cells from the untreated group. The results showed that the percentages of apoptotic cells isolated from the group treated with MeV + nimotuzumab combination were significantly higher compared to that of apoptotic cells isolated from the groups treated with MeV or nimotuzumab alone (p < 0.001). DISCUSSION In this study, we show that treatment with MeV in combination with nimotuzumab has a greater anti-cancer effect on a nude mouse model with head and neck cancer. The anti-cancer effect indicates as reduction of tumor size, increasing survival rate and time of mice bearing tumors. In order to evaluate the antitumor effect of MeV in combination with nimotuzumab in nude mice, we inoculated the laryngeal cancer cells Hep-2 under the skin of the mouse that is suitable for MeV delivery through intratumor injection and delivery of monoclonal antibodies through the tail vein. We injected the virus directly into the tumor to minimize viral inactivation by neutralizing antibodies in the blood, and to increase the exposure and penetration of the viruses into the tumor. EGFR plays a crucial role in the pathogenesis of head and neck squamous cell carcinoma. The overexpression of EGFR and its ligands TGF-α are frequently observed in head and neck cancer, and the presence of EGFR and TGF-α mRNA with a high level is also observed in tumor tissues as 92% and 87%, respectively [3]. Nimotuzumab is capable to bind specifically to EGFR and prevents the activation of the receptor [6]. Nimotuzumab recognizes the extracellular domain of EGFR, competently bind to EGFR that subsequently prevents the binding and activating effect of EGFR ligand. In order to respond to the blocked EGFR by nimotuzumab, the tumor cells reduce the secretion of vascular hypertrophy Journal of military pharmaco-medicine n o 5-2019 162 factors that results in decreasing in blood vessel formation and increasing the number of dead cells through apoptosis [8]. Moreover, it is demonstrated that nimotuzumab also plays a role in promoting other mechanisms of the immune response such as T cells, natural killer cells and contributes to cytotoxicity dependent effects. Oncolytic virus therapy is based on the mechanism that oncolytic viruses have the ability to selectively enter and replicate in tumor cells. Once oncolytic viruses infected tumor cells, its replication results in the lysis of tumor cells. Subsequently, the newly produced oncolytic viruses continuingly infect other cancer cells. Another mechanism is that the oncolytic viruses also stimulate the immune response against tumor cells [4, 5]. Some studies using measles viruses to treat bone marrow cancer [9] and neurological cancer [10], especially Mayo Clinic’s research have used measles viruses in treatment for 21 ovarian cancer patients who had previously treated with taxol and platinum. The results indicated that patients who were treated with measles viruses had longer survival time (12.5 months) as twice as that in the untreated group (6 months) and no side effects were observed [11]. This study has demonstrated that the use of live-attenuated MeV vaccine combined with the monoclonal antibody nimotuzumab to treat laryngeal cancer is effective. The important point of this study is that the combined use of oncolytic viruses and monoclonal antibodies increases the effectiveness of tumor therapy compared to single treatment with only MeV or nimotuzumab. However, the mechanism by which the anticancer effect of combined treatment has not known and require to be investigated further. 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