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Description
PROJECT TITLE: Interaction of natural killer cells with infected cancer cell population: microfluidics-based
experiments and single-cell-level mathematical modeling
OBJECTIVES. The aim of the project is to analyze interactions of natural killer (NK) cells with an infected cell
population at the single-cell resolution. Specifically, we will investigate the interactions of primary NK cells, two
respiratory epithelial cell lines: cancerous A549 and non-cancerous BEAS-2B, and two respiratory viruses:
respiratory syncytial virus (RSV) and influenza A virus (IAV). The A549 cell line and RSV will be the primary
subjects of research. The main objectives are as follows:
a) Elucidation of the impact of NK cell-induced cell death on the dynamics of propagation and eradication of
RSV and IAV infections. Characterization of distinct specificities of NK cells towards the infected and the noninfected
subpopulation of the cell culture.
b) Determination of the capacity of NK cells to induce immunogenic cell death in infected cells.
c) Development of a droplet-based microfluidic system to perform experiments on co-cultures of adherent
and suspended cells, that will enable data acquisition at the single-cell resolution.
d) Development of an agent-based, spatial stochastic model capturing interactions of NK cells with an
infected cell population.
IMPACT. We envision that outcomes of the project will increase our understanding of (i) the role of NK cells in
elimination or suppression of virus propagation, (ii) their potential role in cancer immunotherapies, (iii)
applicability of droplet microfluidics technologies for the adherent cells studies.
Summary of project results
The aim of the project was to characterize the infection dynamics of airway epithelium with common respiratory viruses - RSV and IAV, and especially the role of interferon signaling and NK cells-induced cytotoxicity on the outcomes of such infections. As interferons of three distinct types (β,λ and γ) influence the fate of infected cell population, it is important to elucidate the minutiae of their modes of action, especially in relation to different cell death types occurring during the infection, like apoptosis and potentially detrimental, pro-inflammatory pyroptosis. Adding to that, we wanted to check how viral infection influences NK cells capability to recognize and eliminate cancer cells.
Our aim was also to create better tools to observe interactions of NK and epithelial cells in a single cell level. We planned to do this by developing a droplet microfluidic chip, allowing for segregation of cells into discrete microvolume compartments, precise administration of interferon and virus doses, as well as for convenient confocal microscopic observations coupled with automatic quantification. Finally, we wanted to use our experience in mathematical modeling to create better models describing mechanisms of interferon signaling and antagonistic cell-virus interactions.
During the project, we were able to establish effective protocols for co-culturing of airway epithelial cells together with NK cells and infect them with RSV or IAV. This allowed us to perform experiments to compare the dynamics of viral infection - viral proliferation, activation of NFкB/IRF3 and Jak/STAT signaling pathways, production of antiviral proteins, secretion of interferons and activation and execution of cellular death pathways. We used advanced methods for gathering data - development of knockout cell lines using CRISPR technology, confocal microscopy, Real Time and digital PCR, ELISA assays, cytometry and western blotting, among others. The wealth of gathered data allowed us to establish crucial parameters in developing mathematical models, which in turn guided the planning of the follow up experiments.
In parallel, our microfluidic team and SINTEF researchers worked on development and perfecting the microfluidic droplet device. The key aspects in this work were providing effective means of gas supply to the cells, choosing appropriate material and coating allowing for proper cell adherence, scaling the geometries of the chip to ensure proper volumes and pressures and adjusting flow rate to prevent cell detachment. We have also designed a second microfluidic chip, with the aim to alleviate th problem of NK cells forming floating clumps, which prevented us from observing interactions of NK and their targets at single-cell level. This chip which would be used to co-encapsulate unclamped NK cells with target cancer or virus-infected cells and allow for convenient analysis using a confocal microscope.
Motivated by providing adequate gas supply for cells growing inside microfluidic devices, we have researched the use of polymethylpentene (PMP), a gas-permeable thermoplastic polyolefin, as a promising candidate material for cell culture inside the microfluidic devices. PMP has been modified (polished) by the SINTEF team, making it more transparent and thus compatible with both transmitted light and confocal microscopy procedures. We have planned and performed a series of experiments, testing its applicability for long term cell culture. To this end, we have devised and manufactured cell culture devices with air-tight lids with integrated PresSens oxygen sensors and replaceable bottom parts for cell culture, made alternately from glass, polished PMP or PDMS.
Our experiments allowed us to describe the mechanism of a switch in cellular expression of immune response genes (from STAT1/STAT2 regulated genes to NF-κB/IRF3 regulated genes) when subjected to nonself RNA, as well differential mechanisms of Type I and Type III interferons for inducing cell death pathways in infected cells, including a novel, non-transcriptional mode of IFN-λ action. We especially present that interferon λ is a more potent initiator of cell death during infection with IAV. We have also shown that NK cells protect cells from RSV and IAV infection through both IFN-γ-dependent and independent mechanisms, including initiation of apoptosis (preferentially) and pyroptosis (when apoptosis is being blocked by viral proteins). The data obtained led to constructing a novel mathematical model, which allows for predicting the progression of RSV virus infection in a monolayer of respiratory epithelial cells. The experience of our experts in modeling has also resulted in an analysis of SARS-CoV-2 progression in the population, especially the Alpha and Omicron variants.
We have also demonstrated that distinct strategies of RSV and IAV viruses (avoiding interferon effects vs. inhibiting interferon production) lead to an interference effect between these viruses. Prior RSV infection is capable of preventing subsequent influenza virus infection by inducing the production of interferons β and λ, putting epithelial cells into an antiviral state where IAV cannot effectively replicate.
We have also successfully constructed two versions of droplet microfluidic chips, which were successfully tested for culturing epithelial and NK cells. Finally, our tests with transparent PMP have shown that it can provide, in contrast to glass, a very good supply of oxygen to the cultured cells, while having comparable optical qualities, allowing for convenient microscopic observations.
In summary, our research provides significant insights into understanding the mechanism of respiratory viral infections, pointing towards directions for the development of potential new antiviral therapies. The microfluidic droplet technologies we utilized can help create better solutions in cell culture and numerous other biological and chemical analytics areas.
Summary of bilateral results
The bilateral collaboration was satisfactory. SINTEF offered PhD students an international educational experience through research visits in SINTEF laboratories. As a result of the cooperation the aplication “Inflammatory modes of regulated cell death in respiratory viral infections” was submitted on 2023-12-15 and it is recommended for funding by National Science Centre.