Culture of ZMEL1 cells, their adaptation to acidosis, exposition to fatty acids and formation of spheroids

Cancer represents a major challenge for researchers all over the world. According to WHO, this is the second leading cause of death worldwide, about 1 death out of 6 being due to cancer. Research is carried out to find new efficient treatments. It was proven that some fatty acids display many health benefits, including anti-cancer properties. This phenomenon has already been proven in vitro, on human cancer cells but it has to be assessed in vivo, on animals. A model of transparent zebrafish bearing fluorescent tumours, due to the injection of zebrafish melanoma (ZMEL1) fluorescently labelled cells, has been put in place in Richard White’s Lab in the Memorial Sloan Kettering Cancer Center in New-York. It would be interesting to verify if it is possible to treat the tumour developed by these fishes using anti-cancer fatty acids. To test the anti-cancer potential of targeted fatty acids, the first step is to grow ZMEL1 cells and then assess the impact of these fatty acids on their viability. The viability tests performed in this master’s thesis show that some fatty acids have strong cytotoxic effects while some others are only slightly cytotoxic for ZMEL1 cells. The most active fatty acids are however partly different from those known to be active on human cancer cells. Surprisingly, the saturated fatty acids, that are not cytotoxic for human cancer cells, are highly cytotoxic for zebrafish cancer cells. In contrast, docosahexaenoic acid, an omega-3 member with 22 carbons, is not cytotoxic for ZMEL1 cells, although it was known to be highly efficient on human cancer cells. As for punicic acid (PuA), a conjugated fatty acid present in pomegranate seed oil, with a strong cytotoxic effect on human cancer cell lines, displays in our tests also the highest cytotoxicity on ZMEL1 cells among all the fatty acids tested. This fatty acid can kill 100 % of the ZMEL1 cells at a concentration of 65 µM. Interactions between PuA (26 µM and 65 µM) and other fatty acids have been tested. It was shown that some fatty acids (linoleic, α-linolenic and oleic acids) are able to partly inhibit the cytotoxicity of PuA. This inhibition of toxicity was more intense at 26 µM than at 65 µM of PuA. Attempts were also made to adapt the ZMEL1 cells to acidic pH (6.5 and 6.9) in order to mimic tumour microenvironment pH conditions. Only the cells at pH 6.9 were able to survive. Another way to get the in vitro model closer to a real tumour is by the formation of spheroids, which are 3D-cell aggregates that display similar chemical gradients to those observed in tumours. The formation of spheroids from ZMEL1 cells was therefore tested under different experimental conditions. In some of these conditions, the cells were able to form aggregates but no real spheroids. In the future, it would be important to confirm if the results obtained on ZMEL1 cells are reproductible on other zebrafish cancer cell lines and are not cell line dependent. Spheroid formations should also be tested with these cell lines to obtain a strong in vitro model. Finally, it will be worth treating zebrafish fibroblasts with the different fatty acids in order to evaluate their impact on non-cancer cells.