Fatty acids are not a significant contributor to the TCA cycle in cancer cell lines: evidence of incomplete fatty acid oxidation.

Fatty acid (FA) oxidation (FAO) is upregulated in many cancers, which has been contextualised to thereby be significantly fuel the TCA cycle and generate vast amounts of ATP to support cancer cell viability. However, direct evidence of this is lacking. Here, we set out to determine the capacity of FAO in a pan-cancer setting and if this relates to the amount of carbon FAO contributes to the TCA cycle relative to other substrates. We profiled the baseline FAO rate and capacity of 27 cancer cell lines from 10 tissue origins, and then selected 6 cancer cell lines that represented the diversity of this panel. Despite the diverse range of FAO rates, we consistently found that exogenous long-chain FAs (LCFAs) were a minor (<10%) TCA cycle substrate in all cells compared to glucose and glutamine. Glucose withdrawal significantly increased FAO rates, while glucose and/or glutamine deprivation marginally increased incorporation of 13C-palmitate into TCA cycle metabolites. Palmitate alone modestly assisted cataplerosis but did not sustain TCA cycle fluxes, leading to reduced cell viability. The low contribution of fatty acids to the TCA cycle in cancer cells may be explained by incomplete oxidation of LCFAs of varying chain lengths of saturations to produce shortened acyl-carnitines. Overall, our results provide significant insights into mitochondrial FA metabolism in cultured cancer cells and challenges the belief that the oxidation of FAs is a complete process, contributing significant fuel for the TCA cycle and ATP generation.