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<p> Measurement of cellular carbon has been accomplished by gravimetric, titrimetric or directly measuring CO₂ produced in the combustion of organic carbon in the Carbon Analyzer Gravimetric and titrimetric methods are cumbersome, inaccurate at concentrations higher than 600mg/l. </p> <p> The purpose of this series of experiments is to find an alternative method for measuring cellular carbon. The proposed method is based in a carbon balance in which the soluble and gaseous forms of carbon are monitored. Cellular carbon is calculated by subtracting the sum of the soluble and gaseous forms of carbon from the initial total carbon found in the reactor. </p> / Thesis / Master of Engineering (MEngr)

There is intense interest in developing novel approaches to enhance immune-mediated cancer therapies with cells such as Natural Killer (NK) cells. Previously, a particle-based method was developed for in vitro expansion of highly cytotoxic human NK cells (PM21-NK cells). Here, we tested two approaches to further enhance the antitumor activity of PM21-NK cells. First, we tested the hypothesis that oncolytic Parainfluenza virus 5 (P/V virus) would combine with PM21-NK cells for enhanced killing of lung cancer cells in vitro. Flow cytometry, luminescence and real-time imaging-based methods were used to assay PM21-NK cell-mediated killing of P/V virus-infected lung cancer cells in 2-dimensional (2D) and 3-dimensional (3D) spheroid cultures. In 2D cultures, lung cancer cells were efficiently infected by the P/V virus and PM21-NK cell killing activity was enhanced against P/V virus-infected cancer cells compared to non-infected cells. By contrast, P/V virus infection of 3D lung cancer cell spheroids was restricted to only the outer-most layer of cells. Nevertheless, PM21-NK cells showed enhanced killing in both infected and non-infected spheroid cells. Antibody neutralization assays showed enhanced NK cell killing was due to both type I and III interferon signaling in lung cancer target cells, which increased their killing by PM21-NK cells. In a second approach to enhance NK cell anti-tumor activity, we designed a novel chimeric protein (NA-Fc) which positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. Real time viability assays revealed that stable expression or lentiviral delivery of NA-Fc to A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells increased their killing in vitro by PM21-NK cells, and this depended on CD16-Fc interactions. Our results lay the foundation for further development of oncolytic viruses and/or novel Fc-based molecules to deliver signals for enhanced NK cell mediated anti-cancer therapies.

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