Tumour sampling conditions perturb the metabolic landscape of clear cell renal cell carcinoma

Human isotopic tracer studies are fast becoming the gold standard model to study cancer metabolism in vivo. Analysed tissues are typically retrieved after surgical resection, which exposes them to a variable extent of warm ischaemia. Although standardised protocols are emerging, the effects of sampling conditions on the tissue metabolome remain understudied. Here, we perform a 13C-glucose study coupled with metabolomic, transcriptomic, and proteomic profiling in patients with clear cell renal cell carcinoma (ccRCC) to assess the metabolic profile of tissues sampled intraoperatively, when blood supply is intact, versus post-surgical resection, which exposes the tissue to ischaemia. We show that ischaemia significantly affects the metabolic landscape of ccRCC, masking some critical features, such as suppressed gluconeogenesis. Furthermore, in orthotopic ccRCC-derived xenografts, we identified that prolonged exposure to ischaemia disrupts the tissue metabolome stability. Therefore, minimising tissue ischaemia is pivotal in accurately profiling cancer metabolism in these important and resource-intense patient studies. To investigate the impact of ischaemia (induced by tissue sampling conditions) on the molecular characterisation of ccRCC, patient tissues (normal adjacent kidney and ccRCC) were sampled at two timepoints: in vivo (tissues perfused) and post-surgical resection (tissues ischaemic). Each patient, where feasible, underwent multiregional sampling (n= 1-3) of their tumour and kidney. We then performed differential gene expression analysis and gene set enrichment analysis of RNA-seq data between ccRCC and kidney tissues and between timepoints.