MINISTRY OF EDUCATION MINISTRY OF HEALTH AND TRAINING HANOI MEDICAL UNIVERSITY NGUYEN THI MY THANH ANTI CANCER EFFICACY OF VACCINE STRAIN MEASLES AGAINST HUMAN LUNG CANCER IN EXPERIMENT Speciality All[.]
MINISTRY OF EDUCATION MINISTRY OF HEALTH AND TRAINING HANOI MEDICAL UNIVERSITY NGUYEN THI MY THANH ANTI-CANCER EFFICACY OF VACCINE-STRAIN MEASLES AGAINST HUMAN LUNG CANCER IN EXPERIMENT Speciality: Allergy and Immunity Code: 62720109 (Bio-Medicine science Code: 9720101) DOCTORAL THESIS HANOI- 2023 THE THESIS WAS COMPLETED AT HANOI MEDICAL UNIVERSITY Supervisor: Prof Nguyen Linh Toan, MD PhD Dr Nguyen Van Do, MD PhD The 1st reviewer: A/Prof Dr Nguyen Thi Xuan The 2nd reviewer: A/Prof Vu Xuan Nghia, MD.PhD The 3rd reviewer: A/Prof Luong Thi Lan Anh, MD.PhD The thesis was defended in front of the thesis review committee at Hanoi Medical University The thesis can be founded in: - National library - Military Medical Academy’s library THE LIST OF PUBLISHCATION RESULTS OF THE THESIS Tạo khối ung thư phổi không tế bào nhỏ người thực nghiệm Tạp chí nghiên cứu Y học Số 2022 Trang (182188) https://doi.org/10.52852/tcncyh.v150i2.737 Hiệu kháng ung thư virus sởi mơ hình chuột thiếu hụt miễn dịch mang khối ung thư phổi người Tạp chí nghiên cứu Y học, Số tháng năm 2022 Trang (317 -323) ( https://doi.org/10.52852/tcncyh.v156i8.1083 INTRODUCTION Lung cancer is the second most common cancer worldwide, after breast cancer According to statistics of the World Health Organization (WHO), in 2020 there will be about 2.21 million of new lung cancer cases and 1.8 million lung cancer deaths in the world Lung cancer remains a challenge for clinicians The disease is often detected at a late stage, the tumor is large and metastatic, the 5-year survival rate is estimated at less than 18% for all stages of the disease More than 50% of cases of lung cancer diagnosis died in duration of year Oncolytic virus (OV) cancer therapy was introduced in the early 1990s and early 20th century Many phase I-III clinical trials have demonstrated good tolerability of OV therapy and a lot of promise Live, attenuated measles vaccine virus (MeV) has been shown anticancer effecacy in the treatment of several types of cancer: cutaneous T-cell lymphoma, lung cancer, etc with promising results In Vietnam, using MeV to treat cancer is a potential therapy that is of interest to research Recently, the Military Medical University has studied the effecacy of MeV used alone and in combination to treat many different lines of human blood cancer, the results showed a clear anti-cancer effectiveness To date, there have been no studies to evaluate the effectiveness of MeV against human lung cancer From there, we carried out the project: “Study on Anti-cancer efficacy of vaccine-strain measles viruses against human lung cancer in experiment” Objectives Assessment of cytotoxic efficacy and apoptosis of MeV in human lung cancer (H460 and A549 cell lines) in vitro Assessment of anti-tumor efficacy of MeV against human lung tumor in nude mice xenograft model bearing H460 cell tumor Essential and scientific meaning of the thesis Studying anti-cancer efficacy of measles against human lung cancer in vitro and in nude mice xenograft models, that can be a premise for subsequent researches as well as clinical trials to evaluate the safety and mechanisms of measles vaccine virus for lung cancer treatment The new main contributions of the thesis Completing the procedure of human lung cancer cell (H460 and A549 cell lines) culture in vitro, transplanting H460 cell tumor under thigh skin of nude mice Evaluation of the anti-tumor efficacy of MeV against human lung cancer H460 and A549 cell lines in vitro and H460 cell line tumor transplanted in nude mice xenograft model From there, it forms the basis for clinical trials of MeV for lung cancer treatmen Thesis layout: The thesis has 130 pages, including: Introduction (2 pages), Chapter 1: Overview (34 pages), Chapter 2: Subjects and methods (28 pages), Chapter 3: Results (32 pages), Chapter 4: Discussion (31 pages), Conclusion (2 pages), Recommendations (1 page) The thesis has 132 (English references: 130) CHAPTER 1: OVERVIEW 1.1 Lung cancer 1.1.1 Current status of lung cancer in the world In 2018, amount of Americans died from lung cancer was similar these of a total of the next three most common cancers (colon, breast, and prostate) In 2020, the overall incidence of lung cancer of men in Europe is about 100/100,000, more than double the rate of women (45/100,000) 1.1.2 Lung cancer status in Vietnam In Vietnam, lung cancer is the most commonly diagnosed cancer in men and the fourth most common cancer in women From 2013 to 2017, the age-standardized rates for Hanoi and Ho Chi Minh City were 32.03/100,000 population for men and 10.48/100,000 for women The 5-year survival rate for lung cancer is 14.8% 1.1.3 Causes and risk factors for lung cancer - Family history and gene variants - Gene polymorphism - Smoke - Diet and alcohol consumption - Chronic infections and other diseases - Ionizing radiation - Occupational exposure - Air pollution 1.1.4 Classification of Lung Cancer - Small cell lung cancer: Often associated with smoking, very malignant - Non-small cell lung cancer: divided into types + Adenocarcinoma + Squamous cell carcinoma + Large cell lung carcinoma - Some other types of lung cancer + Bronchial carcinoids + Supportive tissue cancer in the lung 1.1.5 Pathogenesis of lung cancer 1.1.5.1 Pathogenesis of lung adenocarcinoma There is a lot of evidence that there are at least two molecular pathways, KRAS and EGFR, that cause lung cancer EGFR mutations, especially short deletion mutations in exon 19, L858R and L861Q in exon 21 Other common point mutations and copy number alterations (CNA) have been reported described in LUAD and examined in other regions of the gene A great deal of evidence to date has clearly shown surface-active protein C expressed on LUAD-associated type II alveolar cells Recent work has shown that AAH is associated with the linear progression of cells in the alveoli (respiratory terminal components) to adenocarcinoma in situ and then to invasive LUAD, due to the expression of genes common to alveolar cells and AAH Loss of heterozygosity (LOH) in chromosomes 3p (18%), 9p (CDKN2A), 9q (1/TSC1 tuberous sclerosis complex), 17q and 17p (TP53) and decreased expression of the block inhibitor tumor, serine threonine kinase 11 (STK11 also known as LKB1) Epigenetic alterations including DNA methylation of the CDKN2A and PTPRN2 genes were also seen in AAH In LUAD disease, the NKX2-1 gene is frequently overexpressed or amplified (position 14q13.3), which has a type II alveolar cell line-specific oncogenic role for the transcription factor NKX2-1 in LUAD 1.1.5.2 Pathogenesis of lung squamous cell carcinoma (LUSC) Epithelial cells that lose alleles at multiple sites on chromosomes 3p (3p21, 3p14, 3p22–24 and 3p12) and 9p21 (CDKN2A) in the bronchial epithelium are the earliest detected changes in the bronchial epithelium pathogenesis of LUSC Allele imbalances at 8p21–23, 13q14 (RB1) and 17p13 (TP53) were also detected in pre-invasive squamous cell lesions CDKN2A is methylated DNA in precancerous squamous lesions Vascular endothelial growth factor (VEGF) and VEGF receptors are upregulated in bronchial squamous dysplasia lesions Fatty acids and retinoic acid, are also associated between pre-invasive squamous lesions and LUSC The oncogene SOX2 is amplified at position 3q26.3 1.1.5.3 Pathogenesis of small cell lung carcinoma The LOH at some chromosomal sites, the microscopic instability of SCLC cells was significantly higher than that of neighboring epithelium SCLC is initiated by the inactivation of the TP53 and RB1 genes, which activate the hedgehog signaling pathway SCLC is caused by specific changes in TP53 and RB1, repeat mutations in the histone regulators CREBBP, EP300 and MLL as well as amplification in the oncogene FGFR1 1.1.6 Current treatment methods for lung cancer in Vietnam 1.1.6.1 Surgery In recent years, there have been modern surgeries, including laparoscopic video-assisted thoracic surgery (VATS) 1.1.6.2 Irradiation - Stereotactic radiotherapy: Radiation therapy continues to evolve and there are many modern techniques used to treat lung cancer - Radiofrequency/microwave: Used for early-stage peripheral lung tumors or metastases in patients not indicated for surgery 1.1.6.3 Targeted therapies and immune checkpoint inhibitors Erlotinib inhibits EGFR, gefitinib inhibits PI3K/AKT/mTOR, everolimus and entrectinib are NTRK/ROS1 inhibitors Immune checkpoint inhibitors (nivolumab and pembrolizumab) 1.2 Measles vaccine virus therapy treats human lung cancer 1.2.1 Measles Virus Biology Measles virus is a single-stranded, negative, enveloped RNA virus of the morbillivirus genus and the family Paramyxovirus (Figure 1) Figure 1.1 Structure of measles virus Source: Engeland C.E et al (2021) (a) Measles virus; (b) Measles virus genome nucleoprotein (N), Large Protein (L), Phosphoprotein (P), Matrix (M), Haemagglutinin protein (H), Fusion protein (F), V and C nonstructural proteins 1.2.2 Measles vaccine virus specifically infects lung cancer cells 1.2.2.1 Lung cancer cells have specific receptors for the measles virus Since NSCLC cell lines strongly express MeV-specific CD46 and Nectin-4 receptors, there is ample immunohistochemical evidence that there is up to 40% of human NSCLC cell samples strongly express CD46 and Nectin-4 receptors 1.2.2.2 Mechanism of specific infection of cancer cells Recent studies have demonstrated that MV‐Edm strains are effective against lung cancer cells correlated with CD46 and Nectin-4 specific receptor expression levels * Cancer cells responds defectively to interferon Lung tumor cells have defects in the antiviral interferon response (IFN) pathway, making cancer cells more susceptible to viral infeection than normal cells 1.2.3 Mechanism lysing lung cancer cells of measles vaccine virus 1.2.3.1 Formation of syncytia (multinucleated giant cells) MeV has F and H receptor-attaching proteins that involved in the process of membrane fusion, the fusion between virus-infected cells and neighboring normal cells to form syncytial cells (multinucleated giant cells) A virus-infected cell can fuse with 50-100 neighboring cells to form a syncytium 1.2.3.2 MeV generates specific immunity against tumor cells * Tumor cell death due to immune causes MeV infection activates the innate immune system and lysis MeV-mediated cancer cell has that has been confirmed in many studies There is growing evidence that MeV infection activates the immune system to induce MeV-mediated cancer cell lysis Pathogenassociated molecular pattern (PAMP) and Danger-associated molecular pattern (DAMP), they trigger an specific immune response against tumor cells MeV infection promotes antigen-presenting cell function and effective initiation, activation of anti-tumor T-cell response MeV activates other components of the immune system against cancer cells (Figure 1.2) Figure 1.2 MeV activates the immune system against tumor cells * Source: G Pidelaserra-Martí and C.E England (2020) 1.2.3.3 Modificating MeV enhances immunity against cancer cells Modificating MeV was encoded by GM-CSF, FmIL-12, FmIL-15 (BiTEs… showed significant anti-tumor efficacy 1.2.4 Host immune response against MeV Anti-MeV antibodies have been shown to prevent MeV-mediated tumor cell lysis, including local injection of MeV However, more extensive testing is needed than further evaluation 10 TCID50 formula: I: Proportionate distance (PD): (% positive above 50%) – (50%) (% positive above 50%) – (% positive below 50%) II: (Log dilution% above 50%) (for example: log 10-3 is 3) TCID50/ml = 10I+II/ml 2.2.3 Evaluation of MeV cytotoxicity by using MTT assay Infecting MeV into HT-29 cells on 96-well plates with diluted concentrations of 1MOI and 0.1MOI On the 3rd, 4rd and 5rd day postinfection, putting MTT solution into the wells on 96-well plates, measure the optical absorption (OD) at wavelength of 570 nm to read the results and calculating the ratio of living cells in the wells 2.2.4 Preparation of samples of virus-infected H460 and A549 cells to evaluate apoptosis using flow cytometry analysis Proliferating and seeding H460 and A549 cell lines into of 6wells plates Infecting MeV into HT-29 cell wells on 6-well plates with 1.MOI Collecting virus-infected cell samples in the wells on the 6-wells plates at 3rd, 4rd and 5rd day post-infection 2.2.5 Evaluation of apoptotic H460 and A549 cells using flow cytometry analysis MeV-infected H460 and A549 cells are processed and stained according to the procedure instructions of the Fluorescein isothiocyanate Annexin V Apoptosis Detection Kit Evaluation of apoptosis and necrosis cell rates on FACS CANTO II system (BD) 2.2.6 Nude mice husbandry Nude mice- BALB/c strain were brought up in sterile rooms 2.2.7 Transplantation of H460 cell tumors on nude mouse thighs and calculating tumor sizes Proliferation of HT-29 cells: preparing healthy nude mice, 6-8 weeks old of age Injecting HT-29 cells with a concentration of 106 cells/mouse in subcutaneous mouse thighs Measuring tumor size: length and width by standard caliper Formula: V=D x R2/2 (mm3) V: volume; D: length; R: width 11 2.2.8 Treatment of nude mice bearing H460 cell tumor with MeV There are study groups (10 nude mice/1 group), including: MeVtreated group; PBS-treated control group Injecting MeV directly into tumors with doses of 107 PFU/time, twice a week for weeks 2.2.9 Assessment of efficacy of treatment with MeV Monitoring healthy status of post-treatment nude mice: weight, movement, stimulating response, skin color of nude mice, nude mice droppings Comparing the mean tumor sizes of study groups after treatment Mean survival time, survival and death rates between the study groups 2.2.10 Dissection for getting tumor tissues of post-treated nude mice Dissecting to get the spleen and external tumor tissues (2-3 mm) of MeV, MuV-post-treated nude mice, washing with PBS, (the tumor tissues is put into the cell immobilizing solution is glutaraldehyte 2% in the buffer cacodylate, pH = 7.3 to make ultrastructure specimens) 2.2.11 Process H460 cell tumor tissue into cell samples to evaluate the rate of immune cells and apototic cell death by flow cytometry Crush the tumor tissue, add 1X PBS solution Incubate the solution with Trypsin-EDTA 1X solution at room temperature for 2030 minutes, centrifuge at 5000 rpm for minutes Add 1X PBS solution to the cell mass, mix well and filter through a 70 µm filter, we get H460 cell solution of tumor tissue Use specific antibodies to assess the percentage of immune cells in the tumor tissue 2.2.12 Ultrastructural analysis of H460 tumor cell Using transmission electron microscopy (JEM 1400, JEOL, Japan) to Analyze HT-29 tumor cell ultrastructure 2.2.13 Histopathological analysis of H460 cells transplanted on nude mice Dissection of samples of H460 cell line tumor transplanted on nude mice in each MeV treatment group and control group Sending specimens to the Pathology Department, 103 Military Hospital for analysis according to the following steps: - Pass the patient through different chemicals: 12 - Casting specimens in faraffin: - Hematoxylin - Eosin staining: 2.13 Statistical analysis Data were analyzed using the GraphPad Prism 5.0 and SPSS software Statistical significance was defined as p value 0,05 Chart 3.5 Comparison of viable H460 cell rates according to infected times Chart 3.6 Comparison of viable A549 cell rate according to infected times Figure 3.7 Apoptotic cell results of MeVinfected H460 cells on day 3rd post-infection (Q4) early apoptosis region; (Q2) late apoptosis area; (Q1) necrotic cell region Chart 3.7 The apoptotic cell rate of MeV-infected H460 cells on the 3rd day post-infection 15 Figure 3.8 Apoptotic cell results of MeVinfected H460 cells on day 4th post-infection (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Figure 3.9 Apoptotic cell results of infectedMeV H460 cells on day 5th post-infection (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Chart 3.8 Apoptotic cell rates of MeV-infectedH460 cells on the 4th day post-infection Chart 3.9 Apoptotic cell rates of MeV-infected H460 cells on the 5th day post-infection Chart 3.10 Comparison of apoptotic cell death of virus-infected H460 cells according to infected time 16 Figure 3.10 Apoptotic cell results of A549 MeV- infected cells on day 3rd post-infection (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Chart 3.11 Apoptotic rates of A549 MeV- infected cells on the 3rd day post-infection Figure 3.11 Apoptotic results of A549 MeVinfected cells on day 4th post-infection (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Chart 3.12 Apoptotic rates of A549 MeV- infected cells on the 4th day post-infection Figure 3.12 Apoptotic cell results of A549 MeVinfected cells on day 5th post-infection (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Chart 3.13 Apoptotic cell rates of A549 MeV- infected cells on the 5th day post-infection 17 Chart 3.14 Comparison of apoptotic cell death rates of virus-infected A549 cells according to infected time 3.4 MeV against H460 cell tumor in nude mouse xenograft models Table 3.4 The H460 cell tumor transplanted under nude mouse thigh skin The time after injection Monitoring indicators Number of tumor-baering nude mice Pecentage of tumor-bearing nude mice 1st day (n = 20) 4th day (n=20) 7th day (n=20) 10th day (n=40) 12th day (n=20) 12 20 20 20 60 100 100 100 Figure 3.13 Image of H460 cell tumors transplanted under nude mouse thigh skin and measuring tumor size Table 3.5 Monitoring health of nude mice groups Assessment levels Nomal Poor level 20 mice 20 mice 20 mice 20 mice Monitoring indicators Movement Stumulating responses Droppings Skine colour Weight (gram) 100 MeV groups p>0.05 Control groups 80 60 40 20 The day post-injection 1th 4th 8th 11th 15th 18th 22th 25th 28th Chart 3.15 Weight changes of nude mice groups after treatment with MeV 18 Chart 3.16 Mean tumor sizes after treatment with MeV and MuV (A): Comparing tumor size of the groups on the 1st day post-treatment (B): Comparing tumor size of the groups on the 28th day post-treatment Survival day 47±1.7 50 40 p=0.0001 30.3±2.41 30 20 10 MeV Control Chart 3.17 Survival time, survival-death rate of the study groups treated by MeV Figure 3.14 Apoptotic cell results of H460 MeV- injected tumor cells (Q4) early apoptosis region; (Q2) late apoptosis region; (Q1) necrotic cell region Chart 3.18 Apoptotic cell rates of H460 MeV- injected tumor cells Chart 3.19 The results of immune cells in MeV-injected H460 cell tumors 19 3.5 Ultrastructure Analysis of post-treated H460 tumor cells n hn n n n hn hn hn n n n bt bt bt bt n bt bt hn n hn n n bt Figure 3.15 Ultrastructural images of Virus-noninjected H460 tumor cells infection n Figure 3.16 Ultrastructural images of necrotic H460 tumor cells after MeV injection injection bq n bq n bq hb n bq hb kb n n n n n n n Figure 3.17 MeV entered into H460 10 Figure 3.18 Ultrastructural images of MeV-injected tumor cells 11.apoptosis of H460 MeV- injected tumor cells 3.6 Histopathological results of H460 cell tumors transplanted on nude mice after treatment with MeV 12 Figure 3.19 Histopathological images 13 of MeV-non injected H460 cell tumors 14 cells Figure 3.20 Histopathological images of necrotic H460 MeV-injected cell tumors CHAPTER 4: DISCUSSIONS 4.1 Proliferation of Vero, H460 and A549 cell lines in vitro 4.1.1 Vero cell line proliferation Vero cells were cultured and maintained in DMEM medium adding 10% FBS and 1% antibiotics (Figure 3.3) 4.1.2 Culture and proliferation of H460 and A549 cell lines This study, we used H460 and A549 cell lines These cell lines were cultured and maintained in DMEM medium supplemented with 10% FBS and 1% antibiotics The steps of sowing, maintaining and harvesting cells we followed by the right process H460 and A549 20 cells have been shown to retain the same CD46 and Nectin-4 surface receptors as human lung cells 4.2 Proliferatimg measles vaccine virus and TCID50 Titration Firstly, proliferating Vero cells, then, infecting MeV into Vero cells to proliferat MeV Enders J.F et al (1949) demonstrated that this method is widely used for the proliferation of viruses Virus titration is an important step in the research The most common is the TCID50 method Reed L.J (1938) firstly described the TCID50 assay In 2001, LaBarre D.D et al came up with a complete formula for calculating TCID50 4.3 Cytotoxic effect of MeV on H460 and A540 cells in vitro 4.3.1 MeV Directly lysed H460 and A540 cells by forming syncytia in vitro The study results showed that MeV lysised H460 and A549 cells via forming syncytia on 3rd-6th day of infection in vitro, most clearly at 3rd-5th day post-infection MeV-infected cells produce widespread syncytial (Figure 3.6) Syncytia usually die after a few days E-H Lin et al (2009) also demonstrated that the viral membrane fusion protein F induces fusion and lysis of human lung cancer cells in vitro 4.3.2 Evaluation of the cytotoxic effect of MeV on H460 and A549 cells by MTT assay The results showed that the percentage of H460 and A549 viable cells in the two MeV-infected groups was lower (p