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

Upregulated fatty acid oxidation (FAO) is a hallmark of many aggressive cancers and is widely presumed to fuel the tricarboxylic acid (TCA) cycle for ATP production. However, the quantitative relationship between FAO capacity and its contribution to mitochondrial metabolism relative to other fuels remains unclear. Here, we combined parallel stable-isotope tracing with metabolic phenotyping across a diverse panel of 27 cancer cell lines to reveal a fundamental capacity-contribution paradox. Despite exhibiting FAO rates that varied over eight-fold, exogenous long-chain fatty acids consistently contributed minimally (<10%) to TCA cycle intermediates in all cancer cell lines. We demonstrated that FAO functions as a compensatory source of acetyl-CoA in cells with limited glucose-derived acetyl-CoA synthesis, rather than acting as a primary fuel source. In this context, high FAO rates do not primarily result from FAO-mediated suppression of glucose oxidation, but instead reflect the recruitment of fatty acid- and glutamine-derived carbons via a malic enzyme-dependent shunt to sustain the mitochondrial acetyl-CoA pool while preserving glucose-derived anaplerotic flux. These findings challenge the prevailing view that FAO serves as a primary bioenergetic fuel in cancer, instead identifying it as a compensatory rheostat that supplements acetyl-CoA supply in glucose-limited settings by working together with glutamine-driven, malic enzyme-dependent anaplerosis, providing a mechanistic framework to reinterpret the efficacy of FAO inhibitors beyond simple caloric starvation.