Anti-cancer efficacy of combination of vaccine-strain measles and mumps viruses against colorectal cancer in experiment

Anti-cancer efficacy of combination of vaccine-strain measles and mumps viruses against colorectal cancer in experiment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Anti-cancer efficacy of combination of vaccine-strain measles and mumps viruses against colorectal cancer in experiment Hung Trinh, Toan Linh, Le Cuong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6265416/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives Evaluation of oncolytic efficacy of measles and mumps virus combination against colorectal cancer cells (HT-29) in vitro and the nude mouse xenograft model. Materials and methods MTT assay and flow cytometry assay were used to evaluate post-infection viable HT29 cells and apoptosis in vitro . 40 nude mice 6–8 week old age, were divided into 4 groups (10 mice/group) and formed the nude mouse xenograft model for assessing colorectal oncolytic efficacy in vivo . Results The viable cell and apoptotic cell death rates of the viral combination-treated combination group were lower (p < 0.05) and higher (p < 0.01) more than those of the single virus-infected groups, respectively. The tumor sizes, survival time and death rates of the viral combination-treated combination group significantly slowly increased (p < 0.001), was longer (p < 0.05) and lower (p < 0.05) more than those of the single virus-treated groups, respectively. Conclusions the measles and mumps combination virotherapy had the better synergistic anti-cancer efficacy against colorectal than the single virus-treated therapy cancer in vitro and in vivo . Biological sciences/Cancer/Cancer therapy/Drug development Biological sciences/Cancer/Cancer therapy/Targeted therapies measles and mumps virus vaccine colorectal cancer and oncolytic virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Colorectal cancer (CRC) is a malignant disease that can occur in all ages and genders. In 2020, there were more than 1.9 million new cases of CRC and 935,000 CRC-related deaths worldwide, accounting for about 1/10 of total cancer cases and cancer-related deaths. CRC ranks third in terms of new cases and the second cause leading to death among cancers [ 34 ]. In Vietnam, annual statistics showed that CRC is among the top ten cancers with the highest new cases and deaths, causing a large burden of disease. In 2022, CRC ranked fourth in terms of both new cases and deaths among common cancers, with 16,835 new cases (9.3%) and 8,454 deaths (7.0%) [ 25 ]. The development of molecular biological techniques gave us much knowledge about cancer pathogenesis and virology. Researchers have designed strains of viruses that not only infect and selectively lyse cancer cells, but also stimulate specific host immune responses against cancer cells. Oncolytic virus (OV) is an approach that turns replication of virus into arms to kill cancer cells meanwhile they almost do not affect normal cells. OV has many advantages compared with the traditional cancer therapeutics including: reduced adverse effects, the possibility of broad applicability to many cancer types, and a self-amplifying mode of anti-tumor activity by producing more therapeutic viruses in tumors. Nowadays, the genome of OVs are further designed to increase viral ability of infection and oncolysis as well as control the level of viral replication in the host body. The OV that uses live attenuated measles vaccine virus (MeV) and mumps vaccine virus (MuV) to effectively treat human cancer cells has been demonstrated. Utilization of oncolytic MeV and MuV as the potential virotherapy for cancer treatment is being studied in many developed countries. MeV is known to preferentially infect various malignant cell types due to overexpression of CD46 [ 24 ], and Nectin-4 [ 9 ]. MuV uses sialic acid-containing sialoglycoproteins as the specific cancer cell surface receptors [ 16 ]. Many clinical trials using these viruses via different deliveries such as: intratumoral injection, intravenous injection, intraperitoneal injection and so on to treat effectively various cancers: MeV has demonstrated activity against lymphomas [ 37 ], multiple myeloma [ 11 ], ovarian cancer [ 29 ], glioblastoma multiforme [ 31 ], breast cancer [ 14 ], hepatocellular cancer [ 4 ], head and neck squamous cancer [ 17 ], prostate cancer [ 22 ] and pancreatic cancer [ 30 ]. Clinical evidence also indicates that MuV can suppress the growth of various cancers without causing serious complications [ 3 , 27 , 32 ]. In particular, some recent studies have demonstrated the effectiveness of the MeV and MuV combination against many cancer cell lines in vitro and in vivo [ 33 , 42 ]. From the above-mentioned information, we propose a novel strategy to potentially expedite preclinical and clinical evaluations: the combined use of different vaccine-strain viruses, including MeV and MuV (MM). From there, the study focused on evaluating their oncolytic effect as well as the mechanisms of cancer cell killing in vitro using HT-29 colorectal cells, and in vivo using an HT-29 colorectal xenograft tumor model. 2. Materials and Methods 2.1. Cell lines Vero cell line (kidney, African green monkey) and human colorectal cell, HT-29 cell line derived from American Type Culture Collection, (ATCC, Manassas, VA, USA). Vero cells were cultured in M199 medium (Biowest, Maine-et-Loire, France) supplemented with 10% Foetal Bovine Serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. HT-29 cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM), ATCC DMEM (ATCC 30-2002, Manassas, VA 20108 USA) medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a 75 cm2 culture flask. The cell lines were maintained at 37˚C in a humidified incubator with 5% CO 2 . The cell lines were harvested by using Trypsin EDTA, and after centrifuged to remove the culture medium. The concentration of 10 6 cells/ml of HT-29 cells was determined by using Neubauer counters and optical microscope in the experiments. 2.2. Propagation of vaccine-strain measles and mumps viruses MeV and MuV was plaque purified from Priorix vaccine (GlaxoSmithKline, UK) containing the attenuated measles virus (Schwarz MeV strain), mumps virus (RIT 4385 strain) and rubella virus (Wistar RA 27/3 RV strain) and was maintained in Vero cells. The procedure of MeV and MuV preparation for further experiments have been described in detail in our previous study [ 33 ]. 2.3. Viable cell evaluation using MTT colorimetric assay The MTT [3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide] assay was used to evaluate viable cell rates. MTT (yellow) is metabolized to purple formazan crystals (violet) by succinate dehydrogenase in the mitochondria of living cells, which can be quantified by measuring optical density (OD). This OD measurement is proportional to the number of living cells [ 21 ] and has been used in many previous studies [ 26 , 36 , 43 ]. HT-29 cells were harvested from cultured plates, they were seeded in 96-well plates (1x10 4 cells /ml, 200 µl/cell). After 24 h of incubation, when they adhered to the bottom of the wells, They were treated by MeV, MuV and MM combination with viral concentration of 1MOI, 0.5MOI and 0.25 MOI. The MTT assay was performed following 48 h, 72 h and 96 h incubation. The medium in the culture wells was completely removed and replaced with 100 µl of fresh culture medium containing 10 µl of MTT solution and incubated at 37˚C for 4 h to allow mitochondria to convert the MTT into purple formazan crystals. Then, the culture medium containing MTT solution was removed. 100 µl of dimethyl sulfoxide (DMSO) was added to each well and OD was measured at 570 nm. Data were the average of 6 wells, and the experiment was repeated 3 times with similar results. The control group (medium only-treated cells) served as the indicator of 100% cell viability. 2.4. Apoptosis Assays in vitro Annexin-V/Phycoerythrin (PE) and 7-Amino Actinomycin D (7-AAD)/PerCP-Cyanine5.5 (PerCP-Cy5.5) staining was used for early and late apoptosis detections, respectively using flow cytometry with the Annexin V/7AAD kit (Biolegend, CA, USA) according to the manufacturer’s instructions. Approximately 10 6 HT-29 cells were seeded in each 60 mm cell culture dish with EMEM, 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin for 24 h before treatment. After that, the HT-29 cells were treated with culture medium, MeV, MuV and MM combination. Apoptosis was analyzed at 48 h, 72 h and 96 h post-treatment. 2.5. Animal experiments Six to eight-week old male BALB/c nude mice were purchased from BioLASCO (Taiwan) and fed completely on sterile conditions according to Animal Center Guidelines. The procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore (NUS), Singapore (062/12) and Vietnam Military Medical University, Vietnam (072/13). All of the mouse surgery and euthanasia were performed using carbon dioxide inhalation and efforts were made to minimize sufferings. The mice were injected with 10 6 HT-29 cells in 50 µl FBS on the right rear flanks. One week after injection, the formation of tumor in mice was checked and the mice were then divided into 4 groups (10 mice/group). The groups were treated with multiple doses of PBS, MeV, MuV and MM combination (10 6 CFU/mouse/time, twice a week for 4 weeks) using intratumor injection. Tumor volume and survival time were checked and recorded for a time per week. A caliper was used to measure length and width of masses and tumor volume calculated by the formula: volume (mm3) = length × ½ × width 2 . Relative tumor volume was calculated as the volume at a given time divided by the volume on the indicated time points after initiation of treatment. 2.6. Evaluation of immune cells in the spleen of mice bearing HT-29 cell tumors In order to determine the effect of OV stimulating anti-cancer immune cells on mice bearing HT-29 tumors via a mechanism of innate immune response, nude mice were inoculated with 10 6 HT-29 cells in 50 µl FBS on the right rear flanks. After one-week injection, the formation of tumor in mice was checked and the mice were then divided into 4 groups (six mice per group). These mice were treated with PBS, MeV, MuV, and MM combination single dose injection. One week later, these mice were anaesthetized by carbon dioxide inhalation, and killed by bilateral thoracotomy of the euthanized animal and mouse spleens were collected to isolate immune cells. For detecting innate immune subpopulation cells, the cells were stained with anti-mouse antibodies: CD11b-FITC (myelomonocytic cells), (natural killer cells [NK cell]), CD11c-APC (dendritic cells [DC]) or CD197-PerCPCy5-5 (mature DC) and analyzed by flow cytometry with BD FACS Canto II. 2.7. Ultrastructure analysis of HT-29 tumor cell with Transmission electron microscopy Biopsy samples of post-treatment HT-29 tumors were cut into small pieces with size of 1×1×2 mm, washed 2–3 times with cacodylate buffer, kept in 1% osmic acid and cacodylate buffer (pH 7.3) for 1 h, after that washed again 2–3 times with cacodylate buffer (pH 7.3). Continuously, the samples were then dehydrated using consecutively transferring them for 15 min to different alcohol solutions of 50%, 60%, 70%, 80%, 90%, and 100%. After dehydration, the samples were transferred for 10 min to a solution of propylene + ethylene (1:1 ratio in volume), then passed to propylene solution for 10 min. The samples were moved to a mixture of propylene + epon 812 with a volume ratio of 1:1 for 15 min followed by a propylene + epon 812 mixture of 1: 2 in volume for 30 min. Finally, the samples were moved to epon 812 for 30 min, blocked and maintained at 37˚C for 24 h and polymerized at 60˚C for 48 h. Subsequently, the sample blocks were thinly cut into slides by a microtome (Walldorf, Germany) at thickness of 50 nm, and stained with toluidine blue. The slides were put in a 200-hole copper net, stained with 2% uranyl acetate for 5 min, washed twice with distilled water and stained with 5% lead citrate for 5 min, and washed twice with distilled water. The sections were observed under the transmission electron microscopy (TEM) JEM 1400 (JEOL, Japan) [ 36 ]. 2.8. Statistical analysis The GraphPad Prism 5.0 software (GraphPad Software, CA, USA) and SPSS v.20 (SPSS Statistics, IBM, Armonk, NY, USA) were used to analyze data. Mann-Whitney U -test, and Kruskal - Wallis H or Fisher’s exact tests were applied to compare between groups. 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. 3. Results 3.1. Anti-cancer efficacy of MeV and MuV against colorectal cancer in vitro On the 2nd post-infection day, we observed under the light microscope that virus-infected HT-29 cells shrank and suspended to form syncytia. On the 3rd and 4th day post-infection, syncytia enlarged and suspended around. Syncytial formation was almost entirely in culture plates and dead syncytium resulted in cell fragments (Fig. 1A). The syncytia were better observed at the larger objective (Fig. 1B). At post-infection 48h, the viable cell rates of the MM combination- and single virus-infected groups were significantly lower than those of the control group (p < 0.05) at the viral dilutions of 1MOI, 0.5M and 0.25MOI. The viable cell rates of the MM combination-infected group were significantly lower than those of the control group (p < 0.05) at the viral dilutions of 1MOI (Fig. 2A). At post-infection 72h and 96h, the viable cell rates of the MM combination- and single virus-infected groups were significantly lower than those of the control group (p < 0.05) at the viral dilutions of 1MOI, 0.5MOI and 0.25MOI. The viable cell rates of the MM combination-infected group were significantly lower than those of the single virus-infected groups (p < 0.05) at the viral dilution of 1MOI, 0.5MOI and 0.25MOI (Fig. 2B and 2C). Flowcytometry assay was used to assess apoptotic cells after virus infection. The results showed that the apoptotic cell rates of the virus-infected groups were significantly higher than those of control groups (p<0.01). The apoptotic cell rates of the MM combination-infected group were significantly higher than those of the virus-infected groups (p<0.01) at 48h, 72h and 96h post-infection, (Figure 3). 3.2. Anti-tumor efficacy of MeV and MuV against colorectal tumor in nude mice xenograft models After treatment, we measured tumor volume at follow-up time points (1st, 8th, 15 th, 22nd, 29th, 36th, 43rd and 50th days) to evaluate anti-tumor effect of MeV, MuV or MM combination in a colorectal cancer xenograft tumor model. The results showed that the mean tumor volume started to slowly increase from the 8th day to the 50th day post-treatment. Mean tumor volumes of the virus-treated groups significantly slowly increased more than those of the control group (p < 0.001). In particular, the mean tumor volumes of the MM combination-injected group significantly slowly increased more than those of the single virus-injected groups (p < 0.001) (Fig. 4). On the 50th day post-treatment, the mean survival times of the virus-treated groups were significantly longer than that of the control group (p < 0.05). The mean survival time of MM combination-treated group was significantly higher than those of the single virus-treated groups (p < 0.05) (Fig. 5A). The survival mouse rates of the virus-treated groups were significantly higher than that of the control group (p < 0.05) (100%; 60%; 70%; compared with 20%, respectively). The survival mouse rate of the MM combination-treated group was significantly higher than those of the single virus-treated groups (100% compared 60% and 70%, respectively) (Fig. 5B). The cell population in the mouse spleens were stained with anti-mouse antibodies and analyzed by flow cytometry assay. The rates of myelomonocytic cell, NK cell, DC and mature DC of the virus-treated groups were significantly higher than those of the control group with p = 0.001. Especially, the rates of myelomonocytic cell, NK cell, DC and mature DC of the MM combination group were significantly higher than the single virus-treated groups with p = 0.001 (Fig. 6). In addition, we also observed the morphologic ultrastructure of HT-29 tumor cells formed syncytia and underwent stages of apoptosis pathway including: loss of microvilli, chromatin condensation and changes of nuclear contours, nuclear fragmentation, appearance of vacuoles in cytoplasm and finally necrotizing cells (Fig. 7). 4. Discussion 4.1. Anti-cancer efficacy of MeV and MuV against colorectal cancer in vitro MuV and MeV are able to specifically infect CRC cells, because specific receptors of MeV and MuV frequently overexpress on the surface of CRC cells. MuV uses sialoglycoproteins containing sialic acid as specific receptors on the cell surface [ 16 ]. It has been shown that overexpression of sialic acid-rich glycoproteins on the surface of CRC cells [ 28 ] increases infection of the virus in this cell line. MeV-Edmonton strain uses CD46 molecular as a specific receptor, which is a transmembrane glycoprotein that regulates activation of complement, commonly expressed in all human nucleus cells, but is often overexpressed in CRC cells. In addition, Nectin-4 has recently been identified as a specific epithelial receptor of MeV [ 2 ]. Besides the replication of enveloped viruses such as MuV or MeV requires proteases to cleave viral and membrane glycoproteins to allow the viruses to effectively enter the host cells, these proteases are overexpressed in many cancer cell lines including CRC [ 7 ]. The results of our study showed that MeV and MuV strains effectively infected and caused Cytopathic effects (CPE) on the 2nd − 4th day post-infection in vitro , (Fig. 1 ). The more expression of H or HN and F membrane proteins they were higher the more membrane fusion between viral membrane and cell host membrane or the virus-infected cell membrane and neighboring membrane increased. The membrane fusion between the virus-infected cell membrane and neighboring membranes forms syncytium. The syncytia often survives no longer than 4–5 days [ 18 , 29 ]. Syncytial formation of virus-infected HT-29 cells showed that MeV and MuV directly lysed the cells via syncytial formation in vitro , this is the characteristic of oncolysis of MeV and MuV. Syncytial formation allowed the viruses to infect and spread from one cell to another very quickly without releasing viral particles from the cell. In addition, when the syncytia die, they released free viral particles, which infected the neighboring cells to accelerate infection and oncolytic ability. In this study, we also used MTT assay to assess the anti-proliferative properties against CRC cell lines in vitro of MeV and MuV on the 2nd, 3rd, and 4th day post-infection. To increase reliability of the MTT assay, we increased the dilution range of the virus stock from 10 − 2 to 10 − 8 MOI. Results of viable cell ratio using MTT assay in our study confirmed the anti-cancer effects of MeV and MuV against HT-29 cell line in vitro . The viable cell rates of the virus-infected groups were significantly lower than that of the control group, and the rates of viable cells of the MM combination-infected groups were significantly lower than those of single virus-infected groups, depending on the reduced dilution range of the virus concentration. MTT assay has been used in many previous studies and the results have showed that the OV treatment has significant anti-cancer activity against cancer cell lines in vitro [ 40 , 43 ]. Furthermore, MM combination had significant anti-cancer activity against cancer cell lines in vitro [ 33 , 42 ]. Although the molecular mechanism has not been clearly demonstrated, but these results showed that in addition to the anti-cancer efficacy of MeV or MuV single treatment, the MM combination therapy had a significantly greater anti-cancer synergistic efficacy against cancer cell lines, such as CRC cancer cell line in vitro. Apoptosis is an important physiological process to protect the body against infection, cytokines such as tumor necrosis factor (TNF)-α and IFNs are released by the host cells, these substances also can activate the apoptosis pathway in the cells against viral infection [ 13 ]. The apoptosis pathway provides an opportunity to eliminate the viruses by killing infected cells. We used flow cytometry analysis to evaluate the anti-cancer activity against colorectal cancer cell line in vitro . The results demonstrated that MeV and MuV have the ability to lyse HT-29 cell line mediated by activation of apoptosis pathway on the 2nd, 3rd, and 4th day post-infection. The apoptotic cell rate of virus-infected groups were significantly higher than those of control groups (p < 0.01). Many previous studies have demonstrated that anti-cancer activity of MeV and MuV against cancer cell lines via promotion of apoptosis pathway in vitro such as: MeV induced apoptotic cell death in ovarian cancer cell line SKOV-3 [ 44 ], ad breast cancer MCF-7 and CAL-51 cell lines [ 1 ], and ADK3, ADK117, A549 and ADK153 lung adenocarcinoma cell lines and Caco-2, HT29, SW480, and SW620 colorectal adenocarcinoma cell lines [ 5 ]; MuV induces apoptosis in renal carcinoma cells [ 19 ], and in SW480 human CRC cell line [ 20 ], and so on. Furthermore, the results showed that MM-combined anti-cancer activities against HT-29 colorectal cancer line were significantly higher than those of single virus-infected groups. Indeed, MM-combined anti-cancer efficacy against cancer cell lines via activating apoptosis pathway in vitro has been pointed out in some recent studies [ 33 , 42 ]. This suggests that there was synergistic oncolytic effect of the two viruses in killing HT-29 cell lines. The mechanism of MM combination is until now unclear, this requires more expansive research to discover it. However, perhaps both of these OVs have different specific mechanisms of activating apoptosis pathway, so the oncolysis via promoting apoptotic cell death of them resonated with each other. 4.2. Anti-tumor efficacy of MeV and MuV against colorectal cancer in nude mouse xenograft model Our study showed that MeV and MuV had the effective inhibition of growth of HT-29 cell tumors, in particular, the anti-tumor effect of MM-combined treatment significantly better than those single virus treatment on xenograft nude mice. The ability of the viruses spreading and infecting into tumor cells induced an anti-tumor activity. This was due to viral replication in the target tumor cells to suitably catch up tumor cell proliferation. Intratumoral injection with multi-dose caused early viral infection into tumor cells and the spread of the virus is mostly confined to the tumors, which increased oncolytic ability of the viruses. Furthermore, MeV and MuV had a synergic effectiveness to treat colorectal tumors in nude mouse xenograft model. Our results are consistent with those of previous studies that used live attenuated measles and mumps virus vaccine to treat tumors in both xenograft nude mice modes and clinical trials [ 23 , 33 , 42 ]. The results are unbelievably valuable to guide the use of MM combination with multi-dose for cancer treatment in future studies and clinical trials. MeV and MuV are capable of stimulating specific anti-cancer immune responses, which enhances immune-mediated oncolysis. In recent years, it has been demonstrated that this is the very important characteristic of OV in inducing anti-cancer effect [ 6 , 8 ]. Immunogenic cell death (ICD) and triggered immune system responses likely influence cancer treatment, and ICD is characterized by secretion, release or surface exposure of DAMPs, which include CRT, ATP, and HMGB1 [ 15 , 38 ]. Our study focused on assessing the ability of MeV and MuV to stimulate anti-tumor immune response in nude mouse xenograft model. After one-week treatment, we assessed the rate of myelomonocytic cells, NK cells, DC s and mature DC s . These cells play a very important role in systemic immune responses against tumor cells [ 10 , 12 , 35 ]. Our results showed that MeV or MuV treatment was able to stimulate the proliferation of anti-tumor immune cells (myelomonocytic cell, NK cell, DC and mature DC) in nude mouse xenograft model. It was worth mentioning that MM combination was able to stimulate the proliferation of anti-tumor immune cells significantly higher than single virus treatment. Previous studies also evaluated the anti-cancer cell effect of MM combination in vitro that the combination has the ability to synergistically enhance oncolysis both the direct pathway via syncytium formation and activating specific anti-tumor immune response [ 23 , 33 , 42 ]. Following the result with the correlated close association between the HT-29 cell antitumor efficacy and increased ratios of immune cells in the mouse spleen, we hypothesize that the MM-combined treatment has anti-HT-29 cell tumor mediated activation of specific anti-tumor cell immune response in nude mouse xenograft models. In this study, we used TEM to evaluate changes in HT-29 tumor cell ultra-structures in nude mice xenograft model after treatment with MeV and MuV. The results showed that the morphology of HT-29 tumor cells was undergoing apoptosis includes: pyknosis and chromatin condensation at the periphery of the nucleus, and fragmentation of nucleus and formation of apoptotic bodies; in the early apoptosis, the cell shrank, and the surface becomes smooth without microvilli; there are many vacuoles, forming foam cells; The vacuoles are surrounded by a double membrane, maybe due to the residues of the mitochondrial membrane. Thus, the results indicated that the treatment with MeV or MuV produced the anti- tumor effect in nude mice xenograft model mediated virus-induced apoptosis pathway. Being similar to our results, Yang L., et al. (2016) also evaluated HT-29 cell ultrastructure after treatment with quercetin by using TEM, quercetin is a factor that causes apoptosis [ 41 ]. Some previous studies also used TEM to analysed the changes of HT-29 cell ultrastructure during apoptosis [ 36 , 39 ]. Once again, we confirmed the role of MeV- and MuV-induced apoptosis in the anti-tumor cell efficacy in nude mouse xenograft model, which is an especially important mechanism being greatly interested in current cancer treatment. 5. Conclusion These results showed a strong anti-cancer effect of MeV, MuV vaccine-strain, especially MM combination against colorectal cancer in vitro and in vivo . Anti-cancer synergistic effects of MM combination were the direct cytolytic activity via syncytial formation, and stimulation of apoptosis pathway as well as mediated by activation of the anti-cancer immune response via ICD marker expression. The anti-cancer efficiency of MM combination should be considered for improvement of colorectal cancer treatment in future. Declarations Conflicts of Interest All Authors have no conflicts of interest to declare. Authors’ Contributions Trinh Xuan Hung, Nguyen Linh Toan, Le Duy Cuong supervised the study and contributed to the materials and reagents. Nguyen Linh Toan, Le Duy Cuong participated in the study design. Le Duy Cuong performed the experimental procedures. Trinh Xuan Hung, and Le Duy Cuong analyzed data and interpreted results as well as wrote the article. 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Sodium iodide symporter (NIS)-mediated radiovirotherapy for pancreatic cancer. American Journal of Roentgenology. 2010; 195(2):341-9. doi: Phuong Loi K, Cory Allen, Kah-Whye Peng, et al. Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme. Cancer research. 2003; 63(10):2462-9. doi: Shimizu Y, K Hasumi, Y Okudaira, et al. Immunotherapy of advanced gynecologic cancer patients utilizing mumps virus. Cancer detection and prevention. 1988; 12(1-6):487-95. doi: Son Ho Anh, LiFeng Zhang, Bui Khac Cuong, et al. Combination of vaccine-strain measles and mumps viruses enhances oncolytic activity against human solid malignancies. Cancer Investigation. 2018; 36(2):106-17. doi: Sung Hyuna, Jacques Ferlay, Rebecca L. Siegel, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 2021; 71(3):209-49. doi: https://doi.org/10.3322/caac.21660 Teng Michele WL, Jeremy B Swann, Catherine M Koebel, et al. Immune-mediated dormancy: an equilibrium with cancer. Journal of Leucocyte Biology. 2008; 84(4):988-93. doi: Toan Nguyen Linh, Ngo Thu Hang, Nguyen Kim Luu, et al. Combination of vaccine strain measles virus and nimotuzumab in the treatment of laryngeal cancer. Anticancer research. 2019; 39(7):3727-37. doi: Ungerechts Guy, Christoph Springfeld, Marie E Frenzke, et al. Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine. Cancer research. 2007; 67(22):10939-47. doi: Vandenberk Lien, Jochen Belmans, Matthias Van Woensel, et al. Exploiting the immunogenic potential of cancer cells for improved dendritic cell vaccines. Frontiers in immunology. 2016; 6:663. doi: Waziri Peter M, Rasedee Abdullah, Swee Keong Yeap, et al. Clausenidin induces caspase-dependent apoptosis in colon cancer. BMC complementary and alternative medicine. 2016; 16:1-12. doi: Yan Yulan, Bing Liang, Jin Zhang, et al. Apoptotic induction of lung adenocarcinoma A549 cells infected by recombinant RVG Newcastle disease virus (rL‑RVG) in vitro. Molecular Medicine Reports. 2015; 11(1):317-26. doi: Yang Lin, Yanqing Liu, Mei Wang, et al. Quercetin-induced apoptosis of HT-29 colon cancer cells via inhibition of the Akt-CSN6-Myc signaling axis. Molecular medicine reports. 2016; 14(5):4559-66. doi: Zhang Li Feng, Darren Qian Cheng Tan, Anand D Jeyasekharan, et al. 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(2):272-80. doi: Zhao Danhua, Ping Chen, Huiqiang Yang, et al. Live attenuated measles virus vaccine induces apoptosis and promotes tumor regression in lung cancer. Oncology Reports. 2013; 29(1):199-204. doi: Zhou Shengtao, Yuhua Li, Fuqiang Huang, et al. Live-attenuated measles virus vaccine confers cell contact loss and apoptosis of ovarian cancer cells via ROS-induced silencing of E-cadherin by methylation. Cancer letters. 2012; 318(1):14-25. doi: Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6265416","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431427669,"identity":"dbdc6f17-0771-42ef-87fb-9cc0353c419e","order_by":0,"name":"Hung Trinh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hung","middleName":"","lastName":"Trinh","suffix":""},{"id":431427670,"identity":"298b7bfb-d1a7-4cb3-9297-a023928b451a","order_by":1,"name":"Toan Linh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Toan","middleName":"","lastName":"Linh","suffix":""},{"id":431427671,"identity":"6faefe79-0429-4b20-817c-82354197fcab","order_by":2,"name":"Le Cuong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACPgYGAyiT/+ODD0CKjZ2AFjaEFgZjwxkgEWYStJgJ84AoglrYm7dJ89QcljPnX5DGbPNrmzwfMwPjh485eLTwHCuT5jl22NhyxoNjj3P7bhu2MTMwS87chkeLRI6ZNG/D4cQNNw62G+f23GYEamFj5sWnRf4NTMthNmnLntv2hLVI8EC1nG9jk2b4cTuRsBaetGLLOcfSjQ1u8DAb9jbcTm5jZmzG6xd+9sMbb7ypsZYzOH+G8cGPP7dt57c3H/zwEY8WEGACR4dEAgMDYxuIxdiAXz1IyQ+wfQeAxB+CikfBKBgFo2AEAgDzhk2La9yXsgAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Le","middleName":"","lastName":"Cuong","suffix":""}],"badges":[],"createdAt":"2025-03-20 02:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6265416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6265416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78956967,"identity":"a4338673-01be-4b51-8754-f112ccc2e696","added_by":"auto","created_at":"2025-03-21 10:25:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250725,"visible":true,"origin":"","legend":"\u003cp\u003eSyncytial formation of MeV and MuV-infected HT-29 cells with The viral concentration of 1MOI.\u003c/p\u003e\n\u003cp\u003e(A) HT-29 infected-virus cells observed under 20X lens according to time study; (B) HT-29 infected-virus cells observed under 40X lens.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/609c9ace1efad54c5ecab192.jpg"},{"id":78957228,"identity":"2da78351-0555-4f3c-b912-9f78b17efe6f","added_by":"auto","created_at":"2025-03-21 10:33:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189166,"visible":true,"origin":"","legend":"\u003cp\u003eThe viable rates of the virus-infected groups.\u003c/p\u003e\n\u003cp\u003e(A): The viable rates of the virus-infected groups at 48h; (B): The viable rates of the virus-infected groups at 72h; (C): The viable rates of the virus-infected groups at 96h.\u003c/p\u003e\n\u003cp\u003eNote:\u003cem\u003e HT-29 cells were cultured and treated with MeV, MuV alone, and MM combination. Non-infected cells were used as the control. MTT assay was used to evaluate the viable rates of HT-29 virus-infected cells\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eat 48h, 72h and 96 h with the viral dilutions of 1MOI, 0.5MOI and 0.25MOI.\u003c/em\u003e \u003cem\u003eAll experiments were repeated 4 times.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/228dde8a333c1ae42109d965.jpg"},{"id":78956090,"identity":"60d941de-cf08-4bd0-be7c-2f3b4345e089","added_by":"auto","created_at":"2025-03-21 10:09:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":141970,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis induction by MeV- and MuV-treated HT-29 cells with the concentration of 1MOI.\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003eColorectal cancer cells (HT-29 Cells) were cultured and treated with MeV, MuV alone, or MM combination. Non-treated cells were used as the control group. Apoptotic cells were detected after 48h, 72h and 96h after treatment. Original dot plots of apoptotic cells (Annexin V/7AAD – HT-29 cells, in upper panels), including: early apoptosis region (Q1)and late apoptosis region (Q2) were detected after treatment by flow cytometry assay. Arithmetic means±SD of the percentage of Annexin V/7AAD – cells in untreated cells (the control), cells treated with MeV, MuV alone, or MM combination for 48 h, 72 h and 96 h in the lower panel.\u003c/em\u003e \u003cem\u003eAll experiments were repeated 5 times. (**), p \u0026lt; 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/ab6b6abc4f3e01decbc032b5.jpg"},{"id":78956087,"identity":"46e70822-fa63-4a4c-92a0-52ecaf714967","added_by":"auto","created_at":"2025-03-21 10:09:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86676,"visible":true,"origin":"","legend":"\u003cp\u003eMean tumor sizes after treatment with MeV and MuV.\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003eMice bearing colorectal tumors were divided into four groups and treated intratumorally with multiple doses of PBS, MeV, MuV or MM combination (twice per week for 3 weeks). Tumor volume and survival time were recorded after 50-day post-treatment.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/e8e0c075c28e7c7299aa6487.jpg"},{"id":78956091,"identity":"ccc99383-86b0-417c-8cb0-ded48b0e20ee","added_by":"auto","created_at":"2025-03-21 10:09:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95360,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival mouse rate and survival times after treatment with MeV and MuV\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003eAnti tumor effect of MeV, MuV or MM combination in the colorectal cancer xenograft tumor model. Mice bearing colorectal tumors were treated intratumorally with multiple doses of PBS, MeV, MuV or MeV+MuV combination (twice per week for 3 weeks). Tumor volume and survival time were recorded after treatment and during a 50 days of follow-up.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/701c887bddc10e117f9a99fd.jpg"},{"id":78956399,"identity":"900de029-8222-41a2-bf3a-c1ac61f19d43","added_by":"auto","created_at":"2025-03-21 10:17:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":283865,"visible":true,"origin":"","legend":"\u003cp\u003eThe result of immune cellrates in nude mice spleen bearing HT-29 cell tumor after treatment with MeV, MuV and MM combination analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eNote:\u003cem\u003e In left panels, (A): CD11b/FITC - expressing P3 was the distribution region of myelomonocytic cells; (B): CD49b /FITC - expressing P3 was the distribution region of NK cells; (C): CD11c/APC - expressing P5 was the distribution region of DC; (D): CD197/ PerCP-Cy5-5-A - expressing P7 was the distribution region of mature DC.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/2d12d960d7c45175830b0801.jpg"},{"id":78956094,"identity":"b371fa65-4d9c-4a51-97d9-484685d35a49","added_by":"auto","created_at":"2025-03-21 10:09:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":300456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEvaluation of the morphologic ultrastructure of colorectal tumor cells before and after treatment using transmission electron microscope.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNote: (\u003cem\u003eA), normal structure of HT-29 tumor cells before treatment; (B), Virion in cell membrane; (C), HT-29 tumor cells infected with Viruses; (D), syncytia of cells that result from cell fusions; (E), HT-29 tumor cells with chromosome fragmentation; (G), HT-29 tumor cells with many vacuoles; (H), necrotizing cells.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/289a892c9b9d4ed22c8de713.jpg"},{"id":85516638,"identity":"4b3bc4cf-b0b0-43be-923d-f0635874d828","added_by":"auto","created_at":"2025-06-26 18:16:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2091123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6265416/v1/2c795302-0c18-4882-8bfa-afcb3c981f23.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-cancer efficacy of combination of vaccine-strain measles and mumps viruses against colorectal cancer in experiment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is a malignant disease that can occur in all ages and genders. In 2020, there were more than 1.9\u0026nbsp;million new cases of CRC and 935,000 CRC-related deaths worldwide, accounting for about 1/10 of total cancer cases and cancer-related deaths. CRC ranks third in terms of new cases and the second cause leading to death among cancers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In Vietnam, annual statistics showed that CRC is among the top ten cancers with the highest new cases and deaths, causing a large burden of disease. In 2022, CRC ranked fourth in terms of both new cases and deaths among common cancers, with 16,835 new cases (9.3%) and 8,454 deaths (7.0%) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of molecular biological techniques gave us much knowledge about cancer pathogenesis and virology. Researchers have designed strains of viruses that not only infect and selectively lyse cancer cells, but also stimulate specific host immune responses against cancer cells. Oncolytic virus (OV) is an approach that turns replication of virus into arms to kill cancer cells meanwhile they almost do not affect normal cells. OV has many advantages compared with the traditional cancer therapeutics including: reduced adverse effects, the possibility of broad applicability to many cancer types, and a self-amplifying mode of anti-tumor activity by producing more therapeutic viruses in tumors. Nowadays, the genome of OVs are further designed to increase viral ability of infection and oncolysis as well as control the level of viral replication in the host body. The OV that uses live attenuated measles vaccine virus (MeV) and mumps vaccine virus (MuV) to effectively treat human cancer cells has been demonstrated. Utilization of oncolytic MeV and MuV as the potential virotherapy for cancer treatment is being studied in many developed countries. MeV is known to preferentially infect various malignant cell types due to overexpression of CD46 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and Nectin-4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. MuV uses sialic acid-containing sialoglycoproteins as the specific cancer cell surface receptors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Many clinical trials using these viruses via different deliveries such as: intratumoral injection, intravenous injection, intraperitoneal injection and so on to treat effectively various cancers: MeV has demonstrated activity against lymphomas [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], multiple myeloma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], glioblastoma multiforme [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], breast cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], hepatocellular cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], head and neck squamous cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and pancreatic cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Clinical evidence also indicates that MuV can suppress the growth of various cancers without causing serious complications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In particular, some recent studies have demonstrated the effectiveness of the MeV and MuV combination against many cancer cell lines \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom the above-mentioned information, we propose a novel strategy to potentially expedite preclinical and clinical evaluations: the combined use of different vaccine-strain viruses, including MeV and MuV (MM). From there, the study focused on evaluating their oncolytic effect as well as the mechanisms of cancer cell killing \u003cem\u003ein vitro\u003c/em\u003e using HT-29 colorectal cells, and \u003cem\u003ein vivo\u003c/em\u003e using an HT-29 colorectal xenograft tumor model.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell lines\u003c/h2\u003e \u003cp\u003eVero cell line (kidney, African green monkey) and human colorectal cell, HT-29 cell line derived from American Type Culture Collection, (ATCC, Manassas, VA, USA). Vero cells were cultured in M199 medium (Biowest, Maine-et-Loire, France) supplemented with 10% Foetal Bovine Serum (FBS), 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin. HT-29 cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM), ATCC DMEM (ATCC 30-2002, Manassas, VA 20108 USA) medium supplemented with 10% FBS, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin in a 75 cm2 culture flask. The cell lines were maintained at 37˚C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cell lines were harvested by using Trypsin EDTA, and after centrifuged to remove the culture medium. The concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/ml of HT-29 cells was determined by using Neubauer counters and optical microscope in the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Propagation of vaccine-strain measles and mumps viruses\u003c/h2\u003e \u003cp\u003eMeV and MuV was plaque purified from Priorix vaccine (GlaxoSmithKline, UK) containing the attenuated measles virus (Schwarz MeV strain), mumps virus (RIT 4385 strain) and rubella virus (Wistar RA 27/3 RV strain) and was maintained in Vero cells. The procedure of MeV and MuV preparation for further experiments have been described in detail in our previous study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3. Viable cell evaluation using MTT colorimetric assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe MTT [3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide] assay was used to evaluate viable cell rates. MTT (yellow) is metabolized to purple formazan crystals (violet) by succinate dehydrogenase in the mitochondria of living cells, which can be quantified by measuring optical density (OD). This OD measurement is proportional to the number of living cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and has been used in many previous studies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. HT-29 cells were harvested from cultured plates, they were seeded in 96-well plates (1x10\u003csup\u003e4\u003c/sup\u003e cells /ml, 200 \u0026micro;l/cell). After 24 h of incubation, when they adhered to the bottom of the wells, They were treated by MeV, MuV and MM combination with viral concentration of 1MOI, 0.5MOI and 0.25 MOI. The MTT assay was performed following 48 h, 72 h and 96 h incubation. The medium in the culture wells was completely removed and replaced with 100 \u0026micro;l of fresh culture medium containing 10 \u0026micro;l of MTT solution and incubated at 37˚C for 4 h to allow mitochondria to convert the MTT into purple formazan crystals. Then, the culture medium containing MTT solution was removed. 100 \u0026micro;l of dimethyl sulfoxide (DMSO) was added to each well and OD was measured at 570 nm. Data were the average of 6 wells, and the experiment was repeated 3 times with similar results. The control group (medium only-treated cells) served as the indicator of 100% cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Apoptosis Assays in vitro\u003c/h2\u003e \u003cp\u003eAnnexin-V/Phycoerythrin (PE) and 7-Amino Actinomycin D (7-AAD)/PerCP-Cyanine5.5 (PerCP-Cy5.5) staining was used for early and late apoptosis detections, respectively using flow cytometry with the Annexin V/7AAD kit (Biolegend, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Approximately 10\u003csup\u003e6\u003c/sup\u003e HT-29 cells were seeded in each 60 mm cell culture dish with EMEM, 10% FBS, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin for 24 h before treatment. After that, the HT-29 cells were treated with culture medium, MeV, MuV and MM combination. Apoptosis was analyzed at 48 h, 72 h and 96 h post-treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Animal experiments\u003c/h2\u003e \u003cp\u003eSix to eight-week old male BALB/c nude mice were purchased from BioLASCO (Taiwan) and fed completely on sterile conditions according to Animal Center Guidelines. The procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore (NUS), Singapore (062/12) and Vietnam Military Medical University, Vietnam (072/13). All of the mouse surgery and euthanasia were performed using carbon dioxide inhalation and efforts were made to minimize sufferings. The mice were injected with 10\u003csup\u003e6\u003c/sup\u003e HT-29 cells in 50 \u0026micro;l FBS on the right rear flanks. One week after injection, the formation of tumor in mice was checked and the mice were then divided into 4 groups (10 mice/group). The groups were treated with multiple doses of PBS, MeV, MuV and MM combination (10\u003csup\u003e6\u003c/sup\u003e CFU/mouse/time, twice a week for 4 weeks) using intratumor injection. Tumor volume and survival time were checked and recorded for a time per week. A caliper was used to measure length and width of masses and tumor volume calculated by the formula: volume (mm3)\u0026thinsp;=\u0026thinsp;length \u0026times; \u0026frac12; \u0026times; width\u003csup\u003e2\u003c/sup\u003e. Relative tumor volume was calculated as the volume at a given time divided by the volume on the indicated time points after initiation of treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Evaluation of immune cells in the spleen of mice bearing HT-29 cell tumors\u003c/h2\u003e \u003cp\u003eIn order to determine the effect of OV stimulating anti-cancer immune cells on mice bearing HT-29 tumors via a mechanism of innate immune response, nude mice were inoculated with 10\u003csup\u003e6\u003c/sup\u003e HT-29 cells in 50 \u0026micro;l FBS on the right rear flanks. After one-week injection, the formation of tumor in mice was checked and the mice were then divided into 4 groups (six mice per group). These mice were treated with PBS, MeV, MuV, and MM combination single dose injection. One week later, these mice were anaesthetized by carbon dioxide inhalation, and killed by bilateral thoracotomy of the euthanized animal and mouse spleens were collected to isolate immune cells. For detecting innate immune subpopulation cells, the cells were stained with anti-mouse antibodies: CD11b-FITC (myelomonocytic cells), (natural killer cells [NK cell]), CD11c-APC (dendritic cells [DC]) or CD197-PerCPCy5-5 (mature DC) and analyzed by flow cytometry with BD FACS Canto II.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Ultrastructure analysis of HT-29 tumor cell with Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eBiopsy samples of post-treatment HT-29 tumors were cut into small pieces with size of 1\u0026times;1\u0026times;2 mm, washed 2\u0026ndash;3 times with cacodylate buffer, kept in 1% osmic acid and cacodylate buffer (pH 7.3) for 1 h, after that washed again 2\u0026ndash;3 times with cacodylate buffer (pH 7.3). Continuously, the samples were then dehydrated using consecutively transferring them for 15 min to different alcohol solutions of 50%, 60%, 70%, 80%, 90%, and 100%. After dehydration, the samples were transferred for 10 min to a solution of propylene\u0026thinsp;+\u0026thinsp;ethylene (1:1 ratio in volume), then passed to propylene solution for 10 min. The samples were moved to a mixture of propylene\u0026thinsp;+\u0026thinsp;epon 812 with a volume ratio of 1:1 for 15 min followed by a propylene\u0026thinsp;+\u0026thinsp;epon 812 mixture of 1: 2 in volume for 30 min. Finally, the samples were moved to epon 812 for 30 min, blocked and maintained at 37˚C for 24 h and polymerized at 60˚C for 48 h. Subsequently, the sample blocks were thinly cut into slides by a microtome (Walldorf, Germany) at thickness of 50 nm, and stained with toluidine blue. The slides were put in a 200-hole copper net, stained with 2% uranyl acetate for 5 min, washed twice with distilled water and stained with 5% lead citrate for 5 min, and washed twice with distilled water. The sections were observed under the transmission electron microscopy (TEM) JEM 1400 (JEOL, Japan) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe GraphPad Prism 5.0 software (GraphPad Software, CA, USA) and SPSS v.20 (SPSS Statistics, IBM, Armonk, NY, USA) were used to analyze data. Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test, and Kruskal - Wallis H or Fisher\u0026rsquo;s exact tests were applied to compare between groups. The Kaplan\u0026ndash;Meier method and the log-rank test were used to compare the survival time of nude mice between groups. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.1. Anti-cancer efficacy of MeV and MuV against colorectal cancer \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eOn the 2nd post-infection day, we observed under the light microscope that virus-infected HT-29 cells shrank and suspended to form syncytia. On the 3rd and 4th day post-infection, syncytia enlarged and suspended around. Syncytial formation was almost entirely in culture plates and dead syncytium resulted in cell fragments (Fig.\u0026nbsp;1A). The syncytia were better observed at the larger objective (Fig.\u0026nbsp;1B).\u003c/p\u003e\n \u003cp\u003eAt post-infection 48h, the viable cell rates of the MM combination- and single virus-infected groups were significantly lower than those of the control group (p \u0026lt; 0.05) at the viral dilutions of 1MOI, 0.5M and 0.25MOI. The viable cell rates of the MM combination-infected group were significantly lower than those of the control group (p \u0026lt; 0.05) at the viral dilutions of 1MOI (Fig.\u0026nbsp;2A). At post-infection 72h and 96h, the viable cell rates of the MM combination- and single virus-infected groups were significantly lower than those of the control group (p \u0026lt; 0.05) at the viral dilutions of 1MOI, 0.5MOI and 0.25MOI. The viable cell rates of the MM combination-infected group were significantly lower than those of the single virus-infected groups (p \u0026lt; 0.05) at the viral dilution of 1MOI, 0.5MOI and 0.25MOI (Fig.\u0026nbsp;2B and 2C).\u003c/p\u003e \u0026nbsp;Flowcytometry assay was used to assess apoptotic cells after virus infection.\u0026nbsp;\u0026nbsp;The results showed that the apoptotic cell rates of the virus-infected groups were significantly higher than those of control groups (p\u0026lt;0.01). The apoptotic cell rates of the MM combination-infected group were significantly higher than those of the virus-infected groups (p\u0026lt;0.01) at 48h, 72h and 96h post-infection, (Figure 3).\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.2. Anti-tumor efficacy of MeV and MuV against colorectal tumor in nude mice xenograft models\u003c/h2\u003e\n \u003cp\u003eAfter treatment, we measured tumor volume at follow-up time points (1st, 8th, 15\u003csup\u003eth,\u003c/sup\u003e 22nd, 29th, 36th, 43rd and 50th days) to evaluate anti-tumor effect of MeV, MuV or MM combination in a colorectal cancer xenograft tumor model. The results showed that the mean tumor volume started to slowly increase from the 8th day to the 50th day post-treatment. Mean tumor volumes of the virus-treated groups significantly slowly increased more than those of the control group (p \u0026lt; 0.001). In particular, the mean tumor volumes of the MM combination-injected group significantly slowly increased more than those of the single virus-injected groups (p \u0026lt; 0.001) (Fig.\u0026nbsp;4).\u003c/p\u003e\n \u003cp\u003eOn the 50th day post-treatment, the mean survival times of the virus-treated groups were significantly longer than that of the control group (p \u0026lt; 0.05). The mean survival time of MM combination-treated group was significantly higher than those of the single virus-treated groups (p \u0026lt; 0.05) (Fig.\u0026nbsp;5A). The survival mouse rates of the virus-treated groups were significantly higher than that of the control group (p \u0026lt; 0.05) (100%; 60%; 70%; compared with 20%, respectively). The survival mouse rate of the MM combination-treated group was significantly higher than those of the single virus-treated groups (100% compared 60% and 70%, respectively) (Fig.\u0026nbsp;5B).\u003c/p\u003e\n \u003cp\u003eThe cell population in the mouse spleens were stained with anti-mouse antibodies and analyzed by flow cytometry assay. The rates of myelomonocytic cell, NK cell, DC and mature DC of the virus-treated groups were significantly higher than those of the control group with p = 0.001. Especially, the rates of myelomonocytic cell, NK cell, DC and mature DC of the MM combination group were significantly higher than the single virus-treated groups with p = 0.001 (Fig.\u0026nbsp;6).\u003c/p\u003e\n \u003cp\u003eIn addition, we also observed the morphologic ultrastructure of HT-29 tumor cells formed syncytia and underwent stages of apoptosis pathway including: loss of microvilli, chromatin condensation and changes of nuclear contours, nuclear fragmentation, appearance of vacuoles in cytoplasm and finally necrotizing cells (Fig.\u0026nbsp;7).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Anti-cancer efficacy of MeV and MuV against colorectal cancer \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMuV and MeV are able to specifically infect CRC cells, because specific receptors of MeV and MuV frequently overexpress on the surface of CRC cells. MuV uses sialoglycoproteins containing sialic acid as specific receptors on the cell surface [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been shown that overexpression of sialic acid-rich glycoproteins on the surface of CRC cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] increases infection of the virus in this cell line. MeV-Edmonton strain uses CD46 molecular as a specific receptor, which is a transmembrane glycoprotein that regulates activation of complement, commonly expressed in all human nucleus cells, but is often overexpressed in CRC cells. In addition, Nectin-4 has recently been identified as a specific epithelial receptor of MeV [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Besides the replication of enveloped viruses such as MuV or MeV requires proteases to cleave viral and membrane glycoproteins to allow the viruses to effectively enter the host cells, these proteases are overexpressed in many cancer cell lines including CRC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The results of our study showed that MeV and MuV strains effectively infected and caused Cytopathic effects (CPE) on the 2nd \u0026minus;\u0026thinsp;4th day post-infection \u003cem\u003ein vitro\u003c/em\u003e, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The more expression of H or HN and F membrane proteins they were higher the more membrane fusion between viral membrane and cell host membrane or the virus-infected cell membrane and neighboring membrane increased. The membrane fusion between the virus-infected cell membrane and neighboring membranes forms syncytium. The syncytia often survives no longer than 4\u0026ndash;5 days [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Syncytial formation of virus-infected HT-29 cells showed that MeV and MuV directly lysed the cells via syncytial formation \u003cem\u003ein vitro\u003c/em\u003e, this is the characteristic of oncolysis of MeV and MuV. Syncytial formation allowed the viruses to infect and spread from one cell to another very quickly without releasing viral particles from the cell. In addition, when the syncytia die, they released free viral particles, which infected the neighboring cells to accelerate infection and oncolytic ability.\u003c/p\u003e \u003cp\u003eIn this study, we also used MTT assay to assess the anti-proliferative properties against CRC cell lines \u003cem\u003ein vitro\u003c/em\u003e of MeV and MuV on the 2nd, 3rd, and 4th day post-infection. To increase reliability of the MTT assay, we increased the dilution range of the virus stock from 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e MOI. Results of viable cell ratio using MTT assay in our study confirmed the anti-cancer effects of MeV and MuV against HT-29 cell line \u003cem\u003ein vitro\u003c/em\u003e. The viable cell rates of the virus-infected groups were significantly lower than that of the control group, and the rates of viable cells of the MM combination-infected groups were significantly lower than those of single virus-infected groups, depending on the reduced dilution range of the virus concentration. MTT assay has been used in many previous studies and the results have showed that the OV treatment has significant anti-cancer activity against cancer cell lines \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, MM combination had significant anti-cancer activity against cancer cell lines \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Although the molecular mechanism has not been clearly demonstrated, but these results showed that in addition to the anti-cancer efficacy of MeV or MuV single treatment, the MM combination therapy had a significantly greater anti-cancer synergistic efficacy against cancer cell lines, such as CRC cancer cell line \u003cem\u003ein vitro.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eApoptosis is an important physiological process to protect the body against infection, cytokines such as tumor necrosis factor (TNF)-α and IFNs are released by the host cells, these substances also can activate the apoptosis pathway in the cells against viral infection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The apoptosis pathway provides an opportunity to eliminate the viruses by killing infected cells. We used flow cytometry analysis to evaluate the anti-cancer activity against colorectal cancer cell line \u003cem\u003ein vitro\u003c/em\u003e. The results demonstrated that MeV and MuV have the ability to lyse HT-29 cell line mediated by activation of apoptosis pathway on the 2nd, 3rd, and 4th day post-infection. The apoptotic cell rate of virus-infected groups were significantly higher than those of control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Many previous studies have demonstrated that anti-cancer activity of MeV and MuV against cancer cell lines via promotion of apoptosis pathway \u003cem\u003ein vitro\u003c/em\u003e such as: MeV induced apoptotic cell death in ovarian cancer cell line SKOV-3 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], ad breast cancer MCF-7 and CAL-51 cell lines [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and ADK3, ADK117, A549 and ADK153 lung adenocarcinoma cell lines and Caco-2, HT29, SW480, and SW620 colorectal adenocarcinoma cell lines [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; MuV induces apoptosis in renal carcinoma cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and in SW480 human CRC cell line [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and so on. Furthermore, the results showed that MM-combined anti-cancer activities against HT-29 colorectal cancer line were significantly higher than those of single virus-infected groups. Indeed, MM-combined anti-cancer efficacy against cancer cell lines via activating apoptosis pathway \u003cem\u003ein vitro\u003c/em\u003e has been pointed out in some recent studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This suggests that there was synergistic oncolytic effect of the two viruses in killing HT-29 cell lines. The mechanism of MM combination is until now unclear, this requires more expansive research to discover it. However, perhaps both of these OVs have different specific mechanisms of activating apoptosis pathway, so the oncolysis via promoting apoptotic cell death of them resonated with each other.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Anti-tumor efficacy of MeV and MuV against colorectal cancer in nude mouse xenograft model\u003c/h2\u003e \u003cp\u003eOur study showed that MeV and MuV had the effective inhibition of growth of HT-29 cell tumors, in particular, the anti-tumor effect of MM-combined treatment significantly better than those single virus treatment on xenograft nude mice. The ability of the viruses spreading and infecting into tumor cells induced an anti-tumor activity. This was due to viral replication in the target tumor cells to suitably catch up tumor cell proliferation. Intratumoral injection with multi-dose caused early viral infection into tumor cells and the spread of the virus is mostly confined to the tumors, which increased oncolytic ability of the viruses. Furthermore, MeV and MuV had a synergic effectiveness to treat colorectal tumors in nude mouse xenograft model. Our results are consistent with those of previous studies that used live attenuated measles and mumps virus vaccine to treat tumors in both xenograft nude mice modes and clinical trials [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The results are unbelievably valuable to guide the use of MM combination with multi-dose for cancer treatment in future studies and clinical trials.\u003c/p\u003e \u003cp\u003eMeV and MuV are capable of stimulating specific anti-cancer immune responses, which enhances immune-mediated oncolysis. In recent years, it has been demonstrated that this is the very important characteristic of OV in inducing anti-cancer effect [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Immunogenic cell death (ICD) and triggered immune system responses likely influence cancer treatment, and ICD is characterized by secretion, release or surface exposure of DAMPs, which include CRT, ATP, and HMGB1 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our study focused on assessing the ability of MeV and MuV to stimulate anti-tumor immune response in nude mouse xenograft model. After one-week treatment, we assessed the rate of myelomonocytic cells, NK cells, DC\u003csub\u003es\u003c/sub\u003e and mature DC\u003csub\u003es\u003c/sub\u003e. These cells play a very important role in systemic immune responses against tumor cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our results showed that MeV or MuV treatment was able to stimulate the proliferation of anti-tumor immune cells (myelomonocytic cell, NK cell, DC and mature DC) in nude mouse xenograft model. It was worth mentioning that MM combination was able to stimulate the proliferation of anti-tumor immune cells significantly higher than single virus treatment. Previous studies also evaluated the anti-cancer cell effect of MM combination \u003cem\u003ein vitro\u003c/em\u003e that the combination has the ability to synergistically enhance oncolysis both the direct pathway via syncytium formation and activating specific anti-tumor immune response [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Following the result with the correlated close association between the HT-29 cell antitumor efficacy and increased ratios of immune cells in the mouse spleen, we hypothesize that the MM-combined treatment has anti-HT-29 cell tumor mediated activation of specific anti-tumor cell immune response in nude mouse xenograft models.\u003c/p\u003e \u003cp\u003eIn this study, we used TEM to evaluate changes in HT-29 tumor cell ultra-structures in nude mice xenograft model after treatment with MeV and MuV. The results showed that the morphology of HT-29 tumor cells was undergoing apoptosis includes: pyknosis and chromatin condensation at the periphery of the nucleus, and fragmentation of nucleus and formation of apoptotic bodies; in the early apoptosis, the cell shrank, and the surface becomes smooth without microvilli; there are many vacuoles, forming foam cells; The vacuoles are surrounded by a double membrane, maybe due to the residues of the mitochondrial membrane. Thus, the results indicated that the treatment with MeV or MuV produced the anti- tumor effect in nude mice xenograft model mediated virus-induced apoptosis pathway. Being similar to our results, Yang L., \u003cem\u003eet al.\u003c/em\u003e (2016) also evaluated HT-29 cell ultrastructure after treatment with quercetin by using TEM, quercetin is a factor that causes apoptosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Some previous studies also used TEM to analysed the changes of HT-29 cell ultrastructure during apoptosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Once again, we confirmed the role of MeV- and MuV-induced apoptosis in the anti-tumor cell efficacy in nude mouse xenograft model, which is an especially important mechanism being greatly interested in current cancer treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThese results showed a strong anti-cancer effect of MeV, MuV vaccine-strain, especially MM combination against colorectal cancer \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Anti-cancer synergistic effects of MM combination were the direct cytolytic activity via syncytial formation, and stimulation of apoptosis pathway as well as mediated by activation of the anti-cancer immune response via ICD marker expression. The anti-cancer efficiency of MM combination should be considered for improvement of colorectal cancer treatment in future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrinh Xuan Hung, Nguyen Linh Toan, Le Duy Cuong supervised the study and contributed to the materials and reagents. Nguyen Linh Toan, Le Duy Cuong participated in the study design. Le Duy Cuong performed the experimental procedures. Trinh Xuan Hung, and \u0026nbsp;Le Duy Cuong analyzed data and interpreted results as well as wrote the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the help and advice of the following in the study process: Department of Pathophysiology\u0026nbsp;and Institute of Biomedicine and Pharmacy, Vietnam Military Medical University .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah Sulaiman A, Ahmed Majeed Al-Shammari, Safaa A Lateef. Attenuated measles vaccine strain have potent oncolytic activity against Iraqi patient derived breast cancer cell line. 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Cancer letters. 2012; 318(1):14-25. doi: \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"measles and mumps virus vaccine, colorectal cancer and oncolytic virus","lastPublishedDoi":"10.21203/rs.3.rs-6265416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6265416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eEvaluation of oncolytic efficacy of measles and mumps virus combination against colorectal cancer cells (HT-29) \u003cem\u003ein vitro\u003c/em\u003e and the nude mouse xenograft model.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eMTT assay and flow cytometry assay were used to evaluate post-infection viable HT29 cells and apoptosis \u003cem\u003ein vitro\u003c/em\u003e. 40 nude mice 6\u0026ndash;8 week old age, were divided into 4 groups (10 mice/group) and formed the nude mouse xenograft model for assessing colorectal oncolytic efficacy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe viable cell and apoptotic cell death rates of the viral combination-treated combination group were lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) more than those of the single virus-infected groups, respectively. The tumor sizes, survival time and death rates of the viral combination-treated combination group significantly slowly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), was longer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) more than those of the single virus-treated groups, respectively.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ethe measles and mumps combination virotherapy had the better synergistic anti-cancer efficacy against colorectal than the single virus-treated therapy cancer \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Anti-cancer efficacy of combination of vaccine-strain measles and mumps viruses against colorectal cancer in experiment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 10:09:06","doi":"10.21203/rs.3.rs-6265416/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"890da544-00d0-4c4c-a78b-ca593ac9caa8","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45951304,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"},{"id":45951305,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"}],"tags":[],"updatedAt":"2025-06-26T18:08:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-21 10:09:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6265416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6265416","identity":"rs-6265416","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}