VIETNAM MILITARY MEDICAL UNIVERSITYNGÔ THU HẰNG ANTICANCER EFFECTS OF VACCINE STRAIN MEASLES VIRUS IN COMBINATION WITH NIMOTUZUMAB IN TREATMENT OF LARYNGEAL CANCER IN VITRO AND IN VIVO M
Trang 1VIETNAM MILITARY MEDICAL UNIVERSITY
NGÔ THU HẰNG
ANTICANCER EFFECTS OF VACCINE STRAIN MEASLES VIRUS IN COMBINATION WITH NIMOTUZUMAB IN TREATMENT OF LARYNGEAL
CANCER IN VITRO AND IN VIVO
Major: Biomedical ScienceCode: 9720101
SUMMARY OF DOCTORAL THESIS IN MEDICINE
HA NOI - 2020
Trang 21 Prof Dr NGUYỄN LĨNH TOÀN
2 Assoc Prof Dr HỒ ANH SƠN
Reviewer 1: Prof Dr VĂN ĐÌNH HOA
Reviewer 2: Assoc Prof Dr TRỊNH TUẤN DŨNG
Reviewer 3: Assoc Prof Dr PHẠM TUẤN CẢNH
The thesis is defended in front of the scientific committee at theVietnam Military Medical University at on 2020
The thesis can be found at:
- National Library
- Library of the Vietnam Military Medical University
Trang 3Cancer is a major health problem and is increasingly concerned inall countries of the world Head and neck cancer (HNS) is a group ofmalignant tumors that develop in this part of the body, and 90% ofHNS has squamous cell carcinoma (HNSCC) Globally, HNS ranksseventh with 4.8% of all newly diagnosed cancers Oncolytic virus(OLV) therapy is based on the main mechanism that OLVs have theability to specifically enter and replicate in cancer cells of the tumorand cause cell lysis, promote cell apoptosis and stimulate immuneresponse against cancers
Nimotuzumab is a monoclonal antibody targeting the epidermalgrowth receptor (EGFR), which is effective against angiogenesis,inhibits cell proliferation, induces apoptosis, and promotes cellsensitivity to radiation and chemotherapy
We conducted the project “Anticancer effects of vaccine strainmeasles virus in combination with nimotuzumab in the treatment of
laryngeal cancer in vitro and in vivo” with two objectives.
1 To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab in vitro.
2 To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab on nude mouse model with head and neck cancer (in vivo).
Necessity of the project:
Investigation of the anticancer effect of measles vaccine virus
(MeV) in combination with Nimotuzumab both in vitro and on a
nude mouse model will serve as a basis for further studies on themechanism of combined antitumor effect of MeV and Nimotuzumab,
Trang 4as well as for clinical trials using a MeV and Nimotuzumabcombination to treat cancer patients in general and HNS in particular.
New contribution of the thesis:
This thesis is the first study to evaluate the anticancer effect of thecombination of MeV and Nimotuzumab against HNS on Hep2 cells
as well as on a nude mouse model with head and neck cancer This isthe basis for further experimental studies and clinical trials for cancertreatment
Layout of the thesis:
The thesis has 150 pages, including: Introduction (2 pages),Chapter 1: Literature review (36 pages), Chapter 2: Materials andMethods (27 pages), Chapter 3: Results (47 pages), Chapter 4:Discussion (35 pages), Conclusion (2 pages), Recommendations (1page) The thesis has 175 references (171 references in English)
CHAPTER 1: LITERATURE REVIEW
1.1 Head and neck cancer
Head and neck cancer (HNS) is a group of malignant tumors thatdevelop in this part of the body including mouth, nose,throat, larynx, sinuses, or salivary glands, cancer of the oral cavity,sinuses next to nose and tongue The incidence of HNS is increasingboth in Vietnam and in some parts of the world HNS has a badprognosis, is dangerous and has many major complications
The main risk factors for HNS include tobacco, alcoholconsumption, HPV infection (for oral cancer), EBV infection (forthroat cancer) The oncogenes in HNSCC are associated with fourmain functional pathways: cell proliferation, squamousepithelialization, cell survival and invasion/metastasis
Trang 51.2 Therapy targeting Epidermal Growth Factor Receptor
1.2.1 Role of EGFR in head and neck squamous cell carcinoma
Epidermal growth factor receptor (EGFR) has a molecular weight
of 170 kiloDaltons (kDa) When the epithelial growth factor (EGF)binds to its receptor (EGFR), two EGFR molecules bind to eachother (dimerization), the tyrosine kinase region is thenphosphorylated This phosphorylation leads to the activation ofspecific tyrosins and EGFR receptor-dependent intracellularsignaling proteins subsequently leads to the transcription of targetgenes that promote cell proliferation, survival (apoptosis), invasionand metastases
EGFR is highly important in the pathogenesis of HNSCC and itsexpression was found in 92% of the HNSCC tumors Moreover, theexpression of EGFR is high in tumors in the advanced stage or inless differentiated tumors
1.2.2 Nimotuzumab in treatment of Head and neck cancer
Nimotuzumab is a monoclonal antibody that specifically binds toEGFR and blocks the activation of this receptor Nimotuzumabrecognizes the EGFR extracellular domain and competes for the bindingsite of EGF, prevents EGF to bind to its receptor, therefore, prevents theactivation of EGFR, inhibits tyrosine kinase activity, consequentlyinhibiting the growth of tumor cells In order to respond to EGFRblockaded by Nimotuzumab, tumor cells reduce the secretion ofvascular proliferation factors, such as vascular endothelial growth factor(VEGF), which leads to reduced formation of vascular and increase thenumber of apoptotic cells Nimotuzumab (Cimaher) has been shown to
Trang 6be effective in the treatment of advanced HNSCC and Nimotuzumabhas 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 thatmediate viral binding and integration with the host cells In currentOLV therapy, the use of Edmonston vaccine strains includes a labstrains, which is closely related to a clinical strain isolated from thethroat of a baby named David Edmonston (1954) and wastransplanted 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 enterthe target cells, the most important receptor is the CD46, which is atype 1 transmembrane glycoprotein that is common in all cells TheCD46 receptor is found to be highly expressed in cancer cells Innormal cells with low CD46 expression, MeV is likely to beinfectious but the syncytial formation is negligible In cancer cellswith high CD46 expression, MeV infection leads to a strong synapticformation: MeV binds to the receptor to enter the cells, replicate andcause the cells to form the symplasm and consequently kill the cellsthrough a CD46 mediated mechanism
1.3.3 Safety of attenuated Measles vaccine
Trang 7MeV meets the standards of an ideal OLV, which must have ahigh selection of tumors, non-pathogenicity, genetic stability and nodisease 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 normalcells forms syncytia A virus-infected cell can merge 50-100neighbouring cells to form a syncytium This is a mechanism forspreading viruses without releasing viral particles from the host cells.The process of cell consolidation reduces the exposure of viruses toneutralizing antibodies of the host, that avoids the control andneutralization of the immune system
1.3.4.2 Lysis of tumor cells mediated by stimulating specific tumor immunity
anti-MeV produces two types of danger signals including associated molecular pattern molecules (DAMP) and pathogen-associated molecular patterns (PAMP), which trigger specificimmune responses that contribute to tumor cell lysis such as IFN,cytokines, activation of NK cells, macrophages, DCs, and Tlymphocytes
damage-1.4 Combination of measles vaccine virus and Nimotuzumab monoclonal antibody in the treatment of cancer
Trang 8There were 2 clinical trials using MeV targeting EGFR to invade andlyze neuroblastoma cells and one trial with HNSCC cells All threetrials 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 beeffective in the treatment of HNSCC From the above evidence, weconducted 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,
Trang 9kits 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
trypsin-b, Methods of proliferation MeV vaccine origin
Infected Vero cells with MeV, after 9-10 days of infection, theviruses were collected, determined viral concentration by CCID50titration method Added Vero cells into a 96-well plate with aconcentration of 104/ 200µl/well MeV infection with a dilutedconcentration of stock virus from 10-2-10-7 After 5 days of MeV
Trang 10infection, methylene blue was used as a colorant to calculateCCID50.
c, Evaluation of cell inhibition by MTT
Using MeV and Nimotuzumab to treat Hep2 cells on 96-wellplate 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 thewells, measure the absorbance (OD) at 570 nm and calculate theresult
d, Flow cytometry method for evaluation of cell apoptosis and necrosis
Enriched Hep2 cells and added to 6 6-well plates Used MeV andNimotuzumab to treat Hep2 cells on 6-well plates Divided into 4groups (control, MeV, Nimotuzumab, combination of MeV andNimotuzumab) Collect cells at 24, 48, 72 and 96 hours to assessapoptosis 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 andNimotuzumab to treat Hep2 cells on 6-well plates Divided into 4groups (control, MeV, Nimotuzumab, combination of MeV andNimotuzumab) 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
Trang 11- 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 standardprocedures
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 wererandomly 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, 2times/week, at 107pfu/time/mouse
- Group of Nimotuzumab: intravenous tail with 0.1 ml ofNimotuzumab solution single dose of 100μg/mouse.g/mouse
- The MeV+Nimotuzumab group: MeV was injected into thetumor 6 times, 2 times/week, the dose was 107pfu/time/animal and
Trang 12was administered intravenously from mouse’s tail of 0.1 ml ofNimotuzumab solution with a single dose of 100μg/mouse.g/mouse.
- Comparison of tumor volume in research groups over time
- Monitored the survival rate
d, Analysed cell apoptosis by flow cytometry: Separated cells fromtissues of 4 groups and used Annexin V / PI kit to analyze apoptosis
e, Methods of tumor histopathological analysis: Tumors wereseparated from mouse thighs, paraffin cast and HE stained and readunder a microscope
f, Methods of analysis of cell superstructure
Scanned and read the results on the electron microscope transmittedthrough JEM 1400, JEOL, Japan (Institute 69, Command of the HoChi Minh Mausoleum)
2.3 Data analyses
- Data were analyzed by using SPSS 20.0 and GraphPad Prism 6software
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
Trang 13Figure 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 welland reached about 80-90% of the culture surface after 6-7 days After
2 weeks of culture, sufficient numbers Hep2 cells were collected forexperiments
Figure 3.2 On day 6 after infection, the Vero cells were death and
peeled off the plate surface The cells were harvested, centrifuged tocollect the supernatant, filtered the supernatant with a 0.45 µm filter
to get the MeV particles
3.1.2 Virus titration by CCID 50
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
Ngày 0
Ngày 4 Ngày 3
Ngày 2 Ngày 1
Ngày 5
Trang 143.1.4.1 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
F
igure 3.9 The percentage of viable cells at different timepoints ofviral therapy in the MeV group and MeV and Nimotuzumabcombination group
Figure 3.10 The percentage
of viable cells at differenttimepoints of group treated withNimotuzumab
0 50 100
150
Nhóm chứng Nimotuzumab MeV Kết hợp (MeV+Nimo)
%Tế bào sống
p<0,0001
10-2 10-3 10-4 10-5 10-6 10-7 10-8
0 20 40 60 80
100
72h 96h
p<0,0001
%Tế bào sống