Profiling Urothelial Tumor Metabolism

Freshly resected patient tissue samples were obtained from transurethral resection samples with the approval of the Leeds East Research Ethics Committee and informed patient consent. Specimens were weighed on parafilm, washed in fresh DMEM to remove residual lactate, and then cultured overnight at 37°C in 5% CO₂ in a small volume of fresh DMEM containing 10% FBS. A small volume of culture medium was removed after 4 hours and after overnight culture for determination of lactate levels as described.²² (link) The specimens were histologically graded and staged according to the 1973 World Health Organization recommendations and TNM classification by a trained pathologist. For immunohistochemistry, serial sections of fixed tissue specimens were blocked following antigen retrieval and then stained for target antigens using standard IHC methodology as previously described.³⁷ (link) Antibodies used were: GLUT1 (ab15309, Abcam), LDH‐A (19987‐1‐AP, Proteintech), MCT4 (H90, Santa Cruz Biotechnology), and MCT1 (AB3538P, Merck Millipore). Total RNA from a panel of 263 urothelial tumors was analyzed for mRNA expression levels of LDH‐A, LDH‐B, GLUT1, MCT4, and MCT1 (Supplementary Methods) (Hurst et al, manuscript in revision).