Tóm tắt Luận án Anticancer effects of vaccine strain measles virus in combination with nimotuzumab in treatment of laryngeal cancer in vitro and in vivo

tyrosine kinase region is then phosphorylated. This phosphorylation leads to the activation of specific tyrosins and EGFR receptor-dependent intracellular signaling proteins subsequently leads to the transcription of target genes that promote cell proliferation, survival (apoptosis), invasion and metastases. EGFR is highly important in the pathogenesis of HNSCC and its expression was found in 92% of the HNSCC tumors. Moreover, the expression of EGFR is high in tumors in the advanced stage or in less differentiated tumors. 1.2.2. Nimotuzumab in treatment of Head and neck cancer Nimotuzumab is a monoclonal antibody that specifically binds to EGFR and blocks the activation of this receptor. Nimotuzumab recognizes the EGFR extracellular domain and competes for the binding site of EGF, prevents EGF to bind to its receptor, therefore, prevents the activation of EGFR, inhibits tyrosine kinase activity, consequently inhibiting the growth of tumor cells. In order to respond to EGFR blockaded by Nimotuzumab, tumor cells reduce the secretion of vascular proliferation factors, such as vascular endothelial growth factor (VEGF), which leads to reduced formation of vascular and increase the number of apoptotic cells. Nimotuzumab (Cimaher) has been shown to be effective in the treatment of advanced HNSCC and Nimotuzumab has been shown to be safe and has less serious complications. 1.3. Measles vaccine virus (MeV) in virus-based cancer therapy 1.3.1. Measles virus Measles virus is a single-stranded RNA (-) virus with a diameter of 100-300 nm, and belongs to the genus Morbillivirus, family Paramyxoviruses, surrounded by a helix capsid. The MeV envelope glycoproteins are the hemagglutinin (H) and fusion (F) proteins that mediate viral binding and integration with the host cells. In current OLV therapy, the use of Edmonston vaccine strains includes a lab strains, which is closely related to a clinical strain isolated from the throat of a baby named David Edmonston (1954) and was transplanted into different cells to create a less virulent and non-pathogenic MeV strain. 1.3.2. Receptors of MeV MeV uses three receptors, CD46, CD150 and nectin-4, to enter the target cells, the most important receptor is the CD46, which is a type 1 transmembrane glycoprotein that is common in all cells. The CD46 receptor is found to be highly expressed in cancer cells. In normal cells with low CD46 expression, MeV is likely to be infectious but the syncytial formation is negligible. In cancer cells with high CD46 expression, MeV infection leads to a strong synaptic formation: MeV binds to the receptor to enter the cells, replicate and cause the cells to form the symplasm and consequently kill the cells through a CD46 mediated mechanism. 1.3.3. Safety of attenuated Measles vaccine MeV meets the standards of an ideal OLV, which must have a high selection of tumors, non-pathogenicity, genetic stability and no disease transmission to the community. 1.3.4. The mechanism of cancer cell lysis 1.3.4.1. MeV directly kills tumor cells through syncytial formation The fusion between the infected cells and the adjacent normal cells forms syncytia. A virus-infected cell can merge 50-100 neighbouring cells to form a syncytium. This is a mechanism for spreading viruses without releasing viral particles from the host cells. The process of cell consolidation reduces the exposure of viruses to neutralizing antibodies of the host, that avoids the control and neutralization of the immune system. 1.3.4.2. Lysis of tumor cells mediated by stimulating specific anti-tumor immunity MeV produces two types of danger signals including damage-associated molecular pattern molecules (DAMP) and pathogen-associated molecular patterns (PAMP), which trigger specific immune responses that contribute to tumor cell lysis such as IFN, cytokines, activation of NK cells, macrophages, DCs, and T lymphocytes. 1.4. Combination of measles vaccine virus and Nimotuzumab monoclonal antibody in the treatment of cancer There were 2 clinical trials using MeV targeting EGFR to invade and lyze neuroblastoma cells and one trial with HNSCC cells. All three trials showed that MeV has a strong ability to inhibit and kill cancer cells by targeting EGFR both in vitro and in vivo. Nimotuzumab is a monoclonal antibody targeting EGFR and has been shown to be effective in the treatment of HNSCC. From the above evidence, we conducted a study using MeV in combination with monoclonal antibody Nimotuzumab in order to treat HNSCC in vitro and in vivo in order to improve the anticancer effects. CHAPTER 2: MATERIALS AND METHODS 2.1. Subjects and Materials 2.1.1. Animals: Nude mice BALB/c strain, 6-8 weeks old, weighing 18-22g, eligible for the experiment. 2.1.2. Measles virus Vaccine (MeV): Measles vaccine virus strains Edmonston. 2.1.3. Cell lines: head and head squamous cell carcinoma cells Hep2, monkey kidney cell (Vero cells). 2.1.4. Monoclonal antibody Nimotuzumab: Product CIMAher 2.1.5. Equipment used for the study 2.1.5.1. Equipment: NSK 150mm callipers, electronic scales TE3102S Sartorius, clean room, cell culture room, centrifuges, optical density reader, realtime PCR machine, flow cytometry, pipettes and etc. 2.1.5.2. Consumables: 6 and 96 well plates, tips, culture plates, bacterial filter, falcon tube, Eppendorf and etc.... 2.1.5.3. Chemicals and Reagents: M199, EMEM cell culture media, kits for MTT, Annexin V/PI Fluorescein isothiocyanate; RNA, cDNA synthesis kit, primers, master Mix, alcohol 70, 90 and etc.... 2.2. Methods: The study was conducted according to the standard experimental, prospective methods, compared and evaluated before, during and after treatment. 2.2.1. Evaluated the ability to inhibit cancer cells and apoptosis of MeV in combination with Nimotuzumab on Hep2 head and neck cancer cell line 2.2.1.1. Evaluation criteria - Determined viral concentration by CCID50 method. - Evaluated inhibition of Hep2 cells by MTT. - Evaluated apoptosis and necrosis by flow cytometry method. - Evaluated apoptosis through the expression of STAT3 and ISG15 genes by realtime PCR technique. 2.2.1.2. Techniques a, Cell Culture Hep2 and Vero cells were taken from freezers (-800C), thawed quickly (<1 minute). Added the cells into a culture dish together with an EMEM medium. The cells were then separated by using trypsin-EDTA 1X. b, Methods of proliferation MeV vaccine origin Infected Vero cells with MeV, after 9-10 days of infection, the viruses were collected, determined viral concentration by CCID50 titration method. Added Vero cells into a 96-well plate with a concentration of 104/ 200µl/well. MeV infection with a diluted concentration of stock virus from 10-2-10-7. After 5 days of MeV infection, methylene blue was used as a colorant to calculate CCID50. c, Evaluation of cell inhibition by MTT Using MeV and Nimotuzumab to treat Hep2 cells on 96-well plate with diluted concentrations of 10-2-10-8. Divided into 4 groups (control, MeV, Nimotuzumab, MeV+Nimotuzumab combination). At 72 and 96 hours after treatment, added MTT solution into the wells, measure the absorbance (OD) at 570 nm and calculate the result. d, Flow cytometry method for evaluation of cell apoptosis and necrosis Enriched Hep2 cells and added to 6 6-well plates. Used MeV and Nimotuzumab to treat Hep2 cells on 6-well plates. Divided into 4 groups (control, MeV, Nimotuzumab, combination of MeV and Nimotuzumab). Collect cells at 24, 48, 72 and 96 hours to assess apoptosis and necrosis. e, Evaluation of cell apoptosis through STAT3 and ISG15 expression by Realtime PCR Enriched Hep2 cells and introduced 6 6-well plates. Use MeV and Nimotuzumab to treat Hep2 cells on 6-well plates. Divided into 4 groups (control, MeV, Nimotuzumab, combination of MeV and Nimotuzumab). Collected cells at 48, 72 hours, RNA isolation, cDNA synthesis and conducting realtime PCR to evaluate STAT3 and ISG15 mRNA expression using GAPDH as a reference gene. 2.2.2. Evaluation of anti-cancer effects of MeV and Nimotuzumab on mouse model carrying head and neck tumors 2.2.2.1. Evaluation criteria Monitored tumor growing Monitored response to treatment in tumors of mice: Comparison of tumor size in groups by treatment time Monitored the mouse survival rate Apoptosis analysis of tumor cells by flow cytometry Analysed histopathological images of tumor cells Analysed cell superstructure 2.2.2.2. Techniques a, Experimental mouse care Nude mice were kept in cleanrooms according to the standard procedures. b, The method of creating head and neck cancer in naked mice The mouse was fixed and injected with 0.1 ml (106 cells) under the skin of the right thigh. Calculated the tumor volume by the formula: V = (D x R2) x 0.5 In which: V: tumor volume (mm3); D and R are the length and width of the tumor (mm). c, Evaluation method of treatment response When the tumor grew up to 7-10 mm in diameter, mice were randomly assigned to 4 groups of 10 mice each: - Control group: 0.1 mL tail injection of PBS / rat, single dose. - MeV: injecting MeV directly into the tumor 6 times, 2 times/week, at 107pfu/time/mouse. - Group of Nimotuzumab: intravenous tail with 0.1 ml of Nimotuzumab solution single dose of 100μg/mouse. - The MeV+Nimotuzumab group: MeV was injected into the tumor 6 times, 2 times/week, the dose was 107pfu/time/animal and was administered intravenously from mouse’s tail of 0.1 ml of Nimotuzumab solution with a single dose of 100μg/mouse. - Comparison of tumor volume in research groups over time - Monitored the survival rate d, Analysed cell apoptosis by flow cytometry: Separated cells from tissues of 4 groups and used Annexin V / PI kit to analyze apoptosis. e, Methods of tumor histopathological analysis: Tumors were separated from mouse thighs, paraffin cast and HE stained and read under a microscope. f, Methods of analysis of cell superstructure Scanned and read the results on the electron microscope transmitted through JEM 1400, JEOL, Japan (Institute 69, Command of the Ho Chi Minh Mausoleum). 2.3. Data analyses - Data were analyzed by using SPSS 20.0 and GraphPad Prism 6 software. CHAPTER 3: RESULTS 3.1. Anticancer effect of measles vaccine virus in combination with Nimotuzumab in vitro 3.1.1. Propagation of measles viruses and cell lines Figure 3.1: After 24 hours, the cells adhere to the culture plate, after 24 hours the number of Hep2 cell line was double, the cell grew well and reached about 80-90% of the culture surface after 6-7 days. After 2 weeks of culture, sufficient numbers Hep2 cells were collected for experiments. Figure 3.2. On day 6 after infection, the Vero cells were death and peeled off the plate surface. The cells were harvested, centrifuged to collect the supernatant, filtered the supernatant with a 0.45 µm filter to get the MeV particles. 3.1.2. Virus titration by CCID50 Figure 3.3. Results of virus titration CCID50 of MeV = 105+0,5/0.2 ml = 5x105,5/ml 3.1.3. Hep2 cells were treated with MeV to form syncytium in vitro Figure 3.4. Infected Hep2 cells forming syncytium 3.1.4. Evaluation of cell death by MTT 3.1.4.1. Results of cell death (Figures 3.6, 3.8) 3.1.4.2. The result of cell death at different time points after treatment with MeV by MTT Figure 3.9. The percentage of viable cells at different timepoints of viral therapy in the MeV group and MeV and Nimotuzumab combination group. Figure 3.10. The percentage of viable cells at different timepoints of group treated with Nimotuzumab. 3.1.5. Evaluation of apoptotic cell rates Figure 3.11. Morphology of apoptotic cells under a normal microscope: 24 hours after treatment with viruses, the cells were shown to shrink. 48, 72 hours, Hep2 cells showed clear viral infections (shrinking, membrane fusion cells to form syncytia). After 96 hours, cells peeled off, there were many necrotic cells and float on the surface of the culture medium. Figure 3.12. At the time points 48, 72 and 96 hours, the proportions dead cells in the control group was lower in the treated groups, and the death cell rate in the MeV+Nimotuzumab combination group was higher compared to the single treatment group (Pcombined group-control: <0.05). Figure 3.13-15. After 48 hours, the proportions of total apoptotic, early apoptotic, late apoptotic cells and necrotic cells were higher in the treatment group compared to the control group and were higher in the combination treatment group compared to the single treatment group (p < 0.05). Figure 3.15. Flow cytometry results of Hep2 cells at different time points after treated with MeV and Nimotuzumab with 2 MOI Q1 is the area with early apoptotic cells **:p < 0,01; ***:p < 0,0001 Figure 3.16. After 72 hours, the proportions of total apoptotic, early apoptotic, late apoptotic cells and necrotic cells was higher in the treatment group compared to in the control group and was higher in the combination treatment group compared to the single treatment group (p < 0.05). Figure 3.17-18. After 96 hours, the rate of necrotic cells in the MeV treatment groups (MeV and combination groups) were significantly higher (p < 0.05) compared to controls (p (MeV)-Control = 0.006; p (MeV + Nimotuzumab -Control = 0.001). Figure 3.19. The percentage of dead cells was increased gradually with the time of treatment with MeV and Nimotuzumab and was highest at 96 hours and lowest at 48 hours (p < 0.05 in MeV group and combined treatment group). Figure 3.20. The proportion of apoptotic cells was highest at 72 hours, this difference was statistically significant (p < 0.05 in MeV and Nimotuzumab). 3.1.5.3. Results of evaluating apoptotic cells by realtime PCR 3.2. Results of anti-cancer effects of MeV and Nimotuzumab on mouse model of immune deficiency carrying human head and neck cancer 3.2.1. Hep2 cell tumor results in naked mice Table 3.5. Three days after transplanting 106 Hep2 cells/mouse under the skin of the right thigh, 13/40 (32.5%) mice appeared to have tumors at the injection site, on the day 11 after transplantation, 40/40 mice (100%) had tumors. At the injection site, mice showed no manifestation of ulcers, necrosis, bleeding. Figure 3.26. Results of Hep2 transplantation under the skin C2 (A) day 3; (B) day 5; (C) day 7; (D) day 11 3.2.2. Results of anticancer effects of MeV and Nimotuzumab 3.2.2.1. Body condition of mice during the experiment Table 3.6. After Hep2 cell transplantation, mice still ate, exercised, responded to normal stimulation, dry anus, did not go loose. 3.2.2.2. Bodyweight in study mice Table 3.7. At most time points, the mean weight of mice in different groups was not statistically significant (p > 0.05). 3.2.2.3 Tumor volume in study mice Table 3.8. At day 0 - the first day of the treated group, the average tumor volume in all 4 groups of mice was similar, the difference was not statistically significant (p > 0.05). Figure 3.28-29. At all time points, the average tumor volume in the treated groups was lower compared to the control group. The average tumor volume in the MeV+Nimotuzumab combination group was lower compared to the single treatment groups with MeV or Nimotuzumab (Figure 3.29). Hình 3.29. Mean volume at different time points (mm3) *: p < 0,05; **:p < 0,01; ***:p < 0,001 3.2.2.4. Results of survival time, mortality rate of nude mice after treatment with MeV and Nimotuzumab Figure 3.30. After 60 days of follow-up, the mean survival time of mice in the treated group was longer compared to control group (the combination treatment group was 58.1 ± 4.33 days; the MeV group was 49.1 ± 16, 50 days; Nimotuzumab group was 45.4 ± 18.66 days, and the control group was 38.6 ± 18.76 days). However, this significant difference was only observed when compared between the control and the combination treatment group (p = 0.009). 3.2.2.5. Cumulative survival rates of mice between control and treated groups After 60 days of follow up, the number of dead mice and the cumulative survival rate was higher in the treated groups compared to control group. The number of dead mice and the cumulative survival rate in the combination treatment group was higher in the single treatment group: - Control group: 8 mice died, the cumulative survival rate was 0.2. - MeV group: 4 mice died, the cumulative survival rate was 0.6. - Nimo group: 6 mice died, the cumulative survival rate was 0.4. - MeV and Nimotuzumab combination group had 2 mice died: the cumulative survival rate was 0.8. 3.2.3. Results of apoptotic cells by flow cytometry on cells isolated from tumor tissue 3.2.4.Histopathological image of tumor on nude mice with Hep2 transplantation Figure 3.33. The histopathological image of Hep2 on nude mice showed less differentiated epithelial carcinoma cells. 3.2.5. Evaluation of Hep2 structure after treated with MeV and Nimotuzumab under transmission electron microscope Hình 3.35. Evaluation of Hep2 structure after treated with MeV and Nimotuzumab under transmission electron microscope (sample KH4, KH5) (A): Normal Hep2 tumor cells; C,D: Hep2 cells forming syncytium when treated with MeV (E) Chromatine condensation in post-treated Hep2tumor cells; (C), (F) Chrommosome fragmentation in post-treated Hep2tumor cells; (G) Many vacuoles in post-treated Hep2tumor cells; (H) necrotic Hep2 tumor cells. CHAPTER 4: DISCUSSION 4.1. Determinativo of virus concentration by CCID50 titration CCID50 titrations are based on the observations of cellular pathological effects under microscopy, so subjective errors can be encountered such as the misidentification of cell pathological effects in wells in the same concentration of substrates between the wells of different concentrations. In this study, we used MeV titration by CCID50 test using methylene blue as a colorant to evaluate the viral pathological effects of the virus to make an objective and accurate evaluation, and allow simultaneous visualization of pathological effects of cells in wells with the same virus concentration and in virus wells with different concentrations, from which CCID50 is calculated, we obtain the titration result of MeV. 4.2. Lysis Effects of MeV and Nimotuzumab on Hep2 cells in vitro 4.4.1. MeV and Nimotuzumab directly lyzed Hep2 cells by forming syncytium in vitro In this study, both in vitro (Hep2 cells under normal microscopes) and in vivo (superstructural structure of tissue under transmission electron microscopy) showed images of Hep2 cells forming syncytium when treated with MeV. This is consistent with the important mechanism of MeV, which cause the infected cells to form syncytia, that has been demonstrated previously. The syncytium allows the viruses to spread from one cell to another very quickly without releasing from the cells. This mechanism helps viruses minimize the effect of neutralizing antibodies. 4.4.2. Evaluation of cell suppression by MTT test In MTT essay, to evaluate the anticancer effect of MeV, other authors chose MeV concentrations (MOI) from 0.1-10 and Nimotuzumab from 7.8125 to 2.000 µg/ml after 12 to 120-hour (commonly at 24, 48 and 72 hours). In this study, we used the dilution concentration of viruses from 10-2 to 10-8 and Nimotuzumab concentrations from 25- 400 µg/ml at 72 hours and 96 hours to evaluate the effect of MeV on Hep2 cells in vitro in combination with Nimotuzumab. At 72 hours and 96 hours, the percentage of viable cells in the three treated groups was lower compared to the control group, the percentage of viable cells in the combined treatment group was lower compared to the single treatment group at all concentrations of MeV from 10-2 to 10-8 (p < 0.001). The percentage of viable cells was increased at all diluted concentrations of MeV from 10-2 to 10-8 (p < 0.05). However, even at a diluted concentration of MeV 10-8, the survival rate in the group treated with MeV and the combination group was still lower compared to the control group (p < 0.001). This results confirmed the lysis effect of MeV and Nimotuzumab on cancer cells. 4.4.3. MeV and Nimotuzumab lyzed Hep2 cells via apoptosis in vitro Our study has shown that MeV and Nimotuzumab have great ability to lyze Hep2 in vitro through activating apoptosis at all time points (from 48 hours, highest at 72 hours and decreased at 96 hours). The proportion of apoptotic cells in the control group was lower in comparison to the 3 treatment groups at all 3 time points (p < 0.05). The rate of early apoptotic cells in the combination group (MeV + Nimo) was highest at 72 hours, decreased to the lowest after 96 hours. The rate of early apoptotic cells was highest in MeV group at 48 hours, decreased to the lowest at 72 hours. In the Nimo group, the rate of early apoptotic cells was increased gradually over time, was lowest at 48 hours, then increased gradually at 72 hours and 96 hours. The proportion of late apoptosis cells in the groups treated with MeV and Nimotuzumab was highest at 72 hours (p < 0.05). The necrotic cell rate in the groups treated with MeV and Nimotuzumab was lowest at 72 hours and highest at 96 hours (p < 0.05). Many studies also chose the time points at days 2, 3, 4 and have proven the killing effect of MeV on many different human cancer cell lines such as cancer ovary, and colorectal cancer. 4.4.4. MeV and Nimotuzumab are effective in inhibiting cell proliferation through activation of STAT3 and ISG15 Realtime PCR results of our study showed that at both 48 hours and 72 hours, the mRNA expression of STAT3 and ISG15 in the treatment groups was higher compared to the control group. In the combination treatment group, there was a statistically significant higher compared to the single treatment groups (p < 0.001). These results indicate that MeV and Nimotuzumab inhibit tumor growth through apoptosis and the combination of MeV and Nimotuzumab has enhanced antitumor effect compared to single treatment with MeV or Nimotuzumab. The groups treated with viruses stimulate intracellular ISG15 expression, thereby stimulating immune cells, increasing IFN gamma production thereby limiting tumor cell growth, increasing apoptosis, and promoting cell necrosis. In the group treated with Nimotuzumab, it inhibits the binding of EGF to its receptor (EGRF) leading to the failure of activation intracellular signalling, inhibiting proliferation of tumor cell growth, vascular proliferation and increased apoptosis. 4.5. Therapeutic efficacy of MeV in combination with Nimotuzumab in nude mouse model with head and neck squamous cell carcinoma 4.5.1. MeV in combination with Nimotuzumab does not cause toxicity in nude mice with head and neck tumors The results of this study showed that MeV and Nimotuzumab did not cause toxicity in nude mice carrying Hep2 cell tumors. After injection of endothelial MeV (6 times, 2 times/week) and Nimotuzumab intravenous tail (single dose), the mice were normal, did not bleed, the injection sites were not contaminated, without ulcer and diarrhoea. The weight of the mice was increased gradually corresponding to the increased tumor volume. There was no difference in weight between the MeV and Nimotuzumab treatment groups and the control group. MeV and Nimotuzumab have also been shown to be effective in the treatment of several human cancers, safe and with less serious complications. 4.5.2. MeV and Nimotuzumab have an effect on limiting the development of head and neck squamous cell carcinoma Studies on nude models carrying human cancers or clinical trials (prostate, kidney, ovarian, neurological and esophageal cancers, etc.) confirmed the anti-cancer effects of MeV. Our results show that the combination treatment of MeV and Nimotuzumab enhances antitumor effect. At all time points, the tumor volume in the treatment group was lower compared to the control group. The tumor size in the combination treatment group was lower compared to the single treatment group (p combination group-control < 0,05). 4.5.3. The combination treatment with MeV and Nimotuzumab prolongs survival time of mice carrying head and neck squamous cell carcinoma. Our results are similar to those of other previous studies proving that MeV prolongs the survival time of nude mice with different cancer cell lines. After 60 days of follow-up, the average survival time in the MeV and Nimotuzumab group was 58.1 days, the MeV was 49.1 days; Nimotuzumab group was 45.4 days; Control group was 38.6 days. At the end of the experiment, the number of live mice in the control group was 2/10 (20%), the MeV group was 6/10 (60%, the Nimotuzumab group was 4/10 (40%), and the MeV + Nimotuzumab group was 8/10 (80%). Difference between the control group and MeV and Nimotuzumab combination was also observed (p < 0.05). The cumulative survival rate after 60 days of treatment with MeV and Nimotuzumab combination was 0.80, which is higher compared to MeV group (0.60), Nimotuzumab (0.40) and control