Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 18.104.22.168 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: „Role of natural killer cells in the therapeutic efficacy of oncolytic measles virus”
Project No.: 22.214.171.124/VIAA/2/18/292
Period: 36 months (1st October 2018 – 30 st September 2021)
Project costs: 133 806,00 EUR
Project implementer: Dr.rer.nat. Rūta Veinalde
Oncolytic viruses (OVs) are emerging as a multimodal, clinically relevant therapeutic option for cancer treatment. Stimulation of anti-tumor immunity of the host is one of the mechanisms of action of OVs. Natural killer (NK) cells are cytotoxic lymphocytes which are involved in recognition of malignant and virus-infected cells. The role of NK cell activation in OV therapy remains controversial. In this project molecular mechanisms of NK cell activation in the context of oncolytic measles virus (MV) infection will be investigated. Moreover, the role of the NK cell activity in MV therapy will be examined depending on the tumor microenvironment, specifically the MHC-I status. Based on the obtained results, options for rational NK cell modulation during MV therapy will be investigated with the aim to develop novel combination treatment strategies with increased therapeutic efficacy.
Information published 01.10.2018.
Progress of the project
1 October 2018 – 31 December 2018
During the initiation phase of the project a primary melanoma cell line collection (11 cultures in total) available at the BMC has been validated for expression of melanoma specific markers on mRNA level using RT-PCR. Validated melanoma cell lines have been compared for susceptibility to oncolytic measles virus (oMV) infection using fluorescence microscopy. Cytotoxic effects of oMV in the same cultures have been assessed by a colorimetric cell viability assay. Based on these results two of the melanoma cell lines have been selected for further experiments. Protocols for human natural killer (NK) cell isolation from full blood of healthy donors and cultivation in vitro have been established. The two previously selected primary melanoma lines have been shown to be susceptible to IL-2 primed NK cell killing using a lactate dehydrogenase release assay. Furthermore, production of a laboratory grade oMV at the BMC for further experiments has been started.
Information published 28.12.2018.
Progress of the project
1 January 2019 – 31 March 2019
During this peridod production of a laboratory grade oncolytic measles virus (oMV) at the BMC has been finished. A protocol for natural killer (NK) cell isolation from human peripheral blood mononuclear cells has been optimised. NK cell isolation based on negative selection using magnetic beads has been determined to be the most appropriate for the planned applications. Experiments to compare NK cell-mediated killing of oMV-infected and uninfected primary melanoma cells (in two different cell lines) have been performed. In one of the cell lines a tendency for increased NK cell killing of oMV-infected melanoma cells has been observed, while in the other cell line this effect has not been detected. To confirm these results, a protocol to measure NK cell mediated cytotoxicity in real time using a digital bio-imaging system has been prepared.
Also, experiments to assess the direct impact of oMV on the activation status of NK cells have been performed. Results demonstrate that incubation of NK cells in the presence of an increasing amount of oMV leads to slightly elevated expression levels of the early activation marker CD69 on the surface of NK cells.
Finally, protocols to assess the impact of oMV infection on the dendritic cell and NK cell crosstalk are being prepared in collaboration with the Laboratory of Immunology at the National Cancer Institute in Vilnius, Lithuania.
Information published 29.03.2019.
Progress of the project
1 April 2019 – 30 June 2019
Protocol for measurement of NK cell-mediated cytotoxicity in real time using the Cytation 5 Cell Imaging system has been established and validated. Experiments to compare NK cell cytotoxicity against oMV-infected and uninfected melanoma cell lines have been finished using the real-time cytotoxicity assay protocol. Results demonstrate that activated NK cells rapidly kill melanoma cells in vitro, reaching up to 80% specific target cell lysis in four hours after start of the experiment. However, no significant differences have been observed in terms of NK cell killing efficiency of oMV-infected and uninfected melanoma cells using the tested cell lines. The real-time cytotoxicity assay protocol will further be used to assess if NK cell incubation with oMV has an impact on cytotoxic properties of NK cells.
Furthermore, pilot experiments to establish a protocol for production of monocyte-derived dendritic cells (DC) have been performed. The impact of oMV infection on DC and NK cell cross-talk will further be assessed using the established protocol. These experiments are performed in collaboration with the Laboratory of Immunology at the National Cancer Center in Vilnius, Lithuania.
Information published 28.06.2019.
1 July 2019 – 30 September 2019
During this phase of the implementation of the project, experiments to assess the impact of oMV infection on the NK and dendritic cell (DC) crosstalk are carried out in collaboration with the Laboratory of Immunology at the National Cancer Center in Vilnius, Lithuania. Flow cytometry results have demonstrated that maturation markers are upregulated on the surface of DCs following infection with oMV. Currently the work continues to establish oMV-infected DC and NK cell co-cultures to assess the impact on NK cell activation status.
Also, the planning of animal experiments to assess the role of NK cell activation in the therapeutic efficacy of oMV depending on the MHC-I status of the tumor has been started. The planned animal experiments will be performed using transplantable murine tumor models. Accordingly, a procedure plan for the use of animals in experimental procedures has been prepared to be assessed at the Food and Veterinary Service of Latvia.
Information published 30.09.2019.
1 October 2019 – 31 December 2019
Protocol for studies of cocultures of oncolytic measles virus (oMV)-infected dendritic cells (DCs) and natural killer (NK) cells has been established. The results demonstrate that NK cells upregulate early activation marker CD69 and degranulation marker CD107a when cocultivated with oMV-infected DCs. The results also show that the increased expression of these NK cell activation markers in the DC-NK coculture experiments is dependent on oMV replication, since in cocultures with UV-inactivated oMV-treated DCs no upregulation of the mentioned NK cell activation markers was observed. Also, the results demonstrate that direct contact between NK cells and DCs is not required for upregulation of the NK cell activation markers, as demonstrated when using transwell inserts in the coculture setting. This suggests that mainly soluble factors might be involved in NK cell activation in cocultures with DCs. Further, experiments to investigate the underlying mechanisms of NK cell activation in cocultures with oMV-infected DCs will be performed.
Also, protocols for the animal experiments that will be performed in the next phase of the project have been finished and all of the planned experimental procedures have been approved by the Food and Veterinary Service of Latvia.
Information published 30.12.2019.
1 January 2020 – 31 March 2020
Experiments with NK and DC cocultures have been continued during this phase of the implementation of the project. Coculture experiments have been repeated using cells from five different donors in total. The previously observed NK cell activation in cocultures with oMV-infected DCs has been confirmed for all analysed donors. Results from all experiments have also confirmed that mainly soluble factors are involved in the activation of NK cells in the coculture experiments. Concentrations of 13 different cytokines involved in antiviral response have been measured using multiplex cytokine bead array to identify factors that are involved in NK cell activation in the studied system. Results demonstrate that concentration of IFN-a2 and IFN-l1, as well as IL-6, TNF-a and IL-10 increase in the medium collected from oMV-infected DCs. High increase has also been observed for the chemokine CXCL10.
Furthermore, preparations are continued for experiments in which the role of NK cells in the therapeutic efficacy of oMV depending on the expression of the major histocompatibility complex I (MHC-I) on tumor cells will be investigated. An MHC-I negative murine colorectal adenocarcinoma MC38cea cell line (MC38cea-MHC-IKO) has been generated using the CRISPR-Cas9 genome editing technique. The MC38cea-MHC-IKO cell line will be further used for implantation into the immunocompetent C57BL/6 mice to generate an MHC-I negative tumor model. Also, since oMV is not able to naturally infect murine cells, a novel oMV genome has been generated in which a single chain variable fragment of an antibody against CEA is attached to the measles hemagglutinin (NSe Hbl-anti-CEA) to allow infection of the MC38cea cells. Laboratory grade NSe Hbl-anti-CEA viruses have been produced in the amount that is required for the further planned animal experiments.
Finally, a manuscript for a publication is being prepared in collaboration with colleagues from the National Center for Tumor Diseases in Heidelberg, Germany.
Information published 31.03.2020.
1 April 2020 – 8 June 2020
An experiment has been carried out during this project implementation phase to identify changes in the functionality of NATIVE NK cells infected with the oMV. In particular, the real-time cytotoxicity test shows that NK cells from coculturals with infected DMS kill primary melanoma cells in vitro more quickly.
During this project implementation phase, work has been continued on the characterization of the previously established MC38cea-MHCTI negative (MC38cea-MHC-I-KO) cell line. Namely, measles virus replication and cytotoxicity were compared in the new MC38cea-MHC-I-KO cell line and in the parental MC38cea cell line.
Information published 08.06.2020.