Spatiotemporally\nResolved Approach for Profiling Ferroptosis-Associated\nMetabolic Vulnerabilities in Tumors Using Mass Spectrometry Imaging\nand Stable Isotope Tracing

Ferroptosis, as an iron-dependent cell death mediated\nby lipid\nperoxidation, has sparked great interest in the tumor research community.\nTargeting ferroptosis has been proven to be a new therapeutic opportunity\nfor inhibiting tumor growth. However, it is challenging to precisely\ncharacterize the metabolic pattern of ferroptosis in heterogeneous\ntumors and further identify ferroptosis-associated metabolic vulnerabilities\nfor tumor treatment. In this work, we developed a spatiotemporally\nresolved method to image ferroptosis-associated metabolic alterations\nin 3D tumor spheroids by combining mass spectrometry imaging and stable\nisotope tracing techniques. The construction of a 3D tumor spheroid\nmodel allows for a more accurate simulation of ferroptosis, and the\nintroduction of MALDI-MSI enables in situ screening of abnormal molecules\nin tumor tissues. Using this method, we showed that the expression\nproportion of <i>n</i><sub>CC</sub> = 4 polyunsaturated\nfatty acids, including arachidonic acid (FA-20:4) and adrenic acid\n(FA-22:4), were upregulated in RSL3-induced 3D tumor ferroptosis models.\nAnd the isotope tracing experiment revealed that the absorption of\nfatty acids and the biosynthesis of polyunsaturated fatty acids were\nsignificantly increased during ferroptosis. In addition, we discovered\nthat arachidonic acid and adrenic acid supplementation render tumor\ncell sensitive to ferroptosis, thereby limiting the growth of tumor\ncells and the formation of 3D tumor spheroids. Such findings provide\nsignificant clues for understanding the metabolic signatures of tumor\nferroptosis and raise the possibility to screen potential metabolic\nvulnerabilities for better tumor treatment in combination with ferroptosis.