Study finds cancercells use a new fuel in absence of sugar umich nature
]uridine was injected directly into the tumours 3 weeks after implantation. Tumours were collected 1 h after uridine injection and processed for isotope tracing, as detailed below.For in vitro extracellular and intracellular metabolomic profiling, PDA cells were seeded in triplicates in a 6-well plate at 4–6 × 10cells per well in growth medium. A parallel plate for protein estimation and sample normalization was also set up.
For tumours, the samples were flash frozen in liquid nitrogen upon collection. Tumours of approximately equal weight were collected per sample per experimental group. The tumours were then put into 2 ml Eppendorf tubes to which 1 ml of ice-cold 80% methanol . Metallic beads were added to each tube and samples were shaken and homogenized on an Retsch TissueLyser II in intervals of 30 s until fully homogenized.
. Data were analysed with Agilent Masshunter Workstation Quantitative Analysis for QQQ version 10.1, build 10.1.733.0.]uridine was supplemented at 0.1 mM or 1 mM for in vitro assays. In brief, wild-type or UPP1-KO cells were cultured overnight in regular medium. Next day, cells were washed once followed by the introduction of medium containing the indicated amounts of glucose, dialysed FBS and supplemented with labelled uridine.
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