Objectives: The present study uses immune-deficient mice model bearing human colon cancer to investigate the change of the rate of immune cells which play a key role in cancer treatment by treating complex oncolytic virus (OLV) Measle/Mump.
Journal of military pharmaco-medicine No7-2016 INVESTIGATION OF THE ABILITY TO STIMULATE IMMUNE RESPONSE BY USING COMPLEX ONCOLYTIC VIRUS MEASLE/MUMP IN NUDE MOUSE MODEL BEARING HUMAN COLON CANCER Le Duy Cuong*; Nguyen Phuong Thanh**; Nguyen Dac Tu** Nguyen Linh Toan*; Ho Anh Son* SUMMARY Objectives: The present study uses immune-deficient mice model bearing human colon cancer to investigate the change of the rate of immune cells which play a key role in cancer treatment by treating complex oncolytic virus (OLV) Measle/Mump Subject and methods: Nude mice was divided into groups: control group, measle virus-treated group, mump virus-treated group and complex Measle/Mump virus-treated group with dose of 106 CFU/individual for single injection Population of macrophage, dendritic cell, NK cell in nude mice’s spleen were analyzed by flow cytometry method Results: The results showed differences between groups: the rate of immune cells in treated groups were clearly increased compared to control group while complex OLV-treated group had a higher rate of immune cells than single OLV-treated groups Conclusion: Using complex OLV bring a significant effects on stimulating immune response more than using single OLV * Key words: Colon cancer; Oncolytic virus Measle/Mump; Immune response; Nude mice INTRODUCTION Human colon cancer has been known for a long time as a common type of cancer In Vietnam, the prevalence of cancer patients tends to be increased annually Recently, scientists have created some new kinds of medicines in colon cancer treatment which are not only more effective but also decrease side effects However, they’re still looking for a new method with optimizing ability of selectively killing cancer cells and minimizing the damage to normal cells They give a theory that oncolytic virotherapy can solve this problem Oncolytic virotherapy have been used since the late 20 th century Dramatic achievements of molecular biology had shed light on the mechanism of virus’s infection and replication via specific receptors expressed on the cancer cell’s surface and opened a new direction of the effects and safety in cancer treatment Researches showed that, each OLV has a specific target and one type of cancer can be infected by many kinds of OLV in the same time Meanwhile, Measle virus (MeV) can infect to some different cancer cell types through CD46 receptor, which has a very high expression in cancer cell’s surface * Military Medical University ** VNU University of Science Corresponding author: Ho Anh Son (hoanhsonhp@gmail.com) 17 Journal of military pharmaco-medicine No7-2016 Though entry receptor of Mump virus (MuV) has been unkown, many studies showed that some cancer cell lines are more sensitive to the viral infection than nomal cell lines Furthermore, MeV and MuV vaccines have been used widely in the world and have been verified of high safety To shed light on the mechanism of complex OLV in cancer treatment, we use MeV/ MuV complex for research “Investigation of ability to stimulate immune response by using complex OLV Measle/Mump in nude mouse model bearing human colon cancer” to compare the tendency to changing percentages of some important immune cells (macrophage, natural killer cell, dendritic cell) in BALB/c nude mice bearing human collon cancer tumor when treated with single OLV and treated with complex OLV (this word was funded by NAFOSTED, grand number 106-YS.06-2013.22) MATERIALS AND METHODS Materials, chemicals - BALB/c nude mice, produced by Charlie River (USA) They were kept in aseptic conditions and were strictly controlled Human colon cancer tumor was created under the skin in right thigh with the dose of 106 HT29 cells for each organism Observation the appearance and development in size of tumor to the moment at which tumor gains the average size of 50 µL Then, nude mice were randomly divided into groups to treat with OLV: + Group (n = 3): treated with physiological salt 0.9% + Group (n = 2): treated with single MeV + Group (n = 2): treated with single MuV + Group (n = 2): treated with MeV/MuV complex Research method - MeV/MuV were infected to the body through tail vein with one dose of 106 PFU/organism - Anti-mouse CD11b antiboby, anti-mouse CD49b antibody, anti-mouse CD11c antibody, anti-mouse CD197 (CCR7) antibody - After week, gathering nude mice from groups, collecting their spleen and - Universal 320 Centrifuge, BD FACT Canto II - Crushing spleen, eliminating red blood cells (by using lysis buffer) and evaluating spleenic cells’ morphology by using trypanblue staining Research subjects - MeV/MuV vaccines cloning from Priorix vaccine (GlaxoSmithKline, U.K) contain sequences Schwarz MeV, RIT 4385 and Wistar RA 27/3 RV, cultured in single layer Vero cells line 18 evaluating spleen’s morphology - Staining spleenic immune cells with specific flourescent antibody to identify the existence and percentage of immune cell populations: Journal of military pharmaco-medicine No7-2016 + Macrophage: anti-mouse CD11b antibody (FITC) + Natural killer cell: anti-mouse CD49b antibody (FITC) + Dendritic cell: anti-mouse CD11c antibody (APC) + Mature dendritic cell: anti-mouse CD197 antibody (PerCP-Cye5-5) The results of observating spleenic patterns show that there’s no negative difference among spleenic patterns between the groups The spleenic patterns are in good enough conditions to perform the next step of research Result of evaluating mouse spleenic cell’s morphology RESULTS Result of evaluating mouse spleen’s morphology Figure 2: Evaluating spleenic cells’ morphology Control group Images of spleenic cells show that cells are globular, - µm There is a large amount of cells with high viability (> 95%) Spleenic cells are in good enough conditions to perform cell staining and counting immune cell populations Result of evaluating the changing rate of immune cell populations Teated groups Figure 1: Comparision spleen’s morphology between control and treated groups The results of counting spleenic immune cell populations show that the amount of macrophage, natural killer cells (NK), dendritic cell (DCs) and mature dendritic cell (mature DCs) are significantly higher in complex OLV MeV/MuV-treated group compared to those in single OLV-treated group and control group 19 Journal of military pharmaco-medicine No7-2016 A B C D Figure 3: Histograms describing the changing of the splenic immune cell Populations’ rate show a common trend: the proportion of immune cell populations in complex OLV MeV/MuV-treated group (IV) is significantly higher than single MuVtreated group (III), single MeV-treated group (II) and control group (I) (3A) Proportion of CD11b positive macrophage in groups are: (I) 3.27%; (II) 8.40%; (III) 16.30%; (IV) 25.65% (3B) Similarly, proportion of CD49b positive NK cell in groups are (I) 1.23%; (II) 9.35%; (III) 5.60%; (IV) 12.95% (3C) Proportion of CD11c positive DCs in groups are (I) 1.6%; (II) 9.45%; (III) 9.85%; (IV) 14.50% (3D) Finally, proportion of CD197 positive mature DCs in group I is approximately 0%, in others groups are 0.05% (II), 1.05% (III), 1.8% (IV) DISCUSSION This study focuses on evaluating the ability to stimulate the immune responses by using complex MeV/MuV, with the property of stimulating a range of immune cells such as macrophage, NK cell and DCs, 20 which play a key role in killing cancer cells [1, 4] The result of counting four immune cell populations described a tendency of increasing in number of those in complex OLV-treated group more than single Journal of military pharmaco-medicine No7-2016 OLV-treated group and control group Previous research utilized complex OLV MeV/MuV to investigate the effectiveness of killing cancer cells in vitro in some cancer cell lines U937, HuT-102, Jurkat… [3] suggested that the enhancement to killing cancer cells was result from the increase of apoptosis activation due to local inflammation inside tumor, but not of viral infection and replication Along with the close interaction between inflammatory responses and rising of immune cells’ rate, we suggested a theory that getting higher splenic immune cells have intimate relationship with enhancement to effects on killing cancer cells in complex OLVtreated group The more complex OLV’s immune response stimulating effectiveness gains, the more splenic immune cells increase Significantly, DCs and mature DCs populations were increased in number, but still have a very low rate It can be easily explained: the percentage of DCs in normal conditions of the body is very low, approximate to - 3% lymphocytes in total, while the amount of mature DCs is even lower since they only was immune response occurs Based on previous in vitro study of Jean-Baptiste Guillerme et al [2], infection of MeV in MOI = caused to the death of 100% plasmacytoid DCs (pDCs) after 72 hours while infection of virus in MOI = 50 added IL-3 caused to the maturity of pDCs This result gave support to the prediction that in a very high dose of infected virus can produce signals to the DCs’ mature progress before DCs’ apoptotic death begin Therefore, we suggest that in this research, with a dose of infected virus 106/BALB/c organism, it is just enough to stimulate the maturity of a small number of DCs CONCLUSION The very high proportion of immune cell populations in complex OLV MeV/MuVtreated group compared to those in single MeV-treated group, single MuV-treated group and control group shows that using complex OLV MeV/MuV brings significant effects on stimulating immune responses compared to those in using single OLV REFERENCE G Ferlazzo, D Thomas, SL Lin, Kiera Goodman, B Morandi, WA Muller, Alessandro, Moretta, C Münz The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic J Immunol 2004, 172, pp.1455-1462 JB Guillerme, N Boisgerault, D Roulois, J Ménager, C Combredet, F Tangy, JF Fonteneau, M Gregoire Measles virus vaccine-infected tumor cells induce tumor antigen cross-presentation by human plasmacytoid dendritic cells Clin Cancer Res 2012, 19 (5), pp.1147-1158 LF Zhang, D.Q.C Tan, AD Jeyasekharan, WS Hsieh, H A Son, K Ichiyama, M Ye, B Pang, K Ohba, X Liu, S.de Mel, B.K Cuong, WJ Chung, A Ryo, Y Suzuki, KG Yeoh, N.L Toan, N Yamamoto Combination of vaccine-strain measles and mumps virus synergistically kills a wide range of human hematological cancer cells: Special focus on acute myeloid leukemia Cancer Letters 2014, 354, pp.272-280 Michel A Poli, O Domingues, M Mauffray, M Thérésine, N.H.C Brons Mouse lung and spleen natural killer cells have phenotypic and functional differences, in part influenced by macrophages PLoS ONE 2012, (12) 21 ... MuV complex for research Investigation of ability to stimulate immune response by using complex OLV Measle/Mump in nude mouse model bearing human colon cancer to compare the tendency to changing... to stimulate the immune responses by using complex MeV/MuV, with the property of stimulating a range of immune cells such as macrophage, NK cell and DCs, 20 which play a key role in killing cancer. .. Canto II - Crushing spleen, eliminating red blood cells (by using lysis buffer) and evaluating spleenic cells’ morphology by using trypanblue staining Research subjects - MeV/MuV vaccines cloning