Profiling Metabolic and Signaling Phenotype of Bladder Cancer Cell Lines.

Bladder cancer (BC) is one of the most common malignancies worldwide[1]. Most patients are diagnosed with non-muscle invasive BC, which is associated with frequent recurrence. The prognosis for early-stage bladder tumors is generally good; however, patients often progress to invasive disease resulting in a much less favorable outcome[2,3]. 3D cultures constitute more clinically relevant models than monolayers for studying cancer as spheroids recapitulate in vivo structures, cell-cell interactions, nutrients and oxygen gradients[4]. In this study we conducted a comparative analysis of metabolism and cellular features of a panel of BC cell lines covering different stages/grades, grown in monolayer cultures and spheroids. Samples were analysed at different timepoints for cellular features, like spheroid morphology and viability, by quantitative imaging using Operetta CLS high-throughput microscope. Here we show that high-grade cell lines had an increased capacity to form viable spheroids. In addition, we used Seahorse technology to assess bioenergetic parameters, e.g. mitochondrial respiration and glycolysis, and demonstrated that high-grade cell lines grown in monolayer cultures present higher basal and maximal glycolysis compared to low-grade cells, while mitochondrial respiration showed no obvious correlation with pathological grade. No obvious correlation was observed in the use of either mitochondrial respiration or glycolysis and pathological grade during spheroid formation, although some high-grade cell lines, like HT1376, showed a preferential use of respiration for ATP production. Our laboratories have previously established the role of the progranulin/EphA2 axis in regulating motility, invasion and in vivo tumour formation in bladder cancer[6] but whether this pathway regulates the capacity of bladder cancer cells to form spheroids has not been established. Significantly, we provide now preliminary evidence that genetic targeting of either progranulin or EphA2 by CRISPR/Cas9 approaches modulated spheroid formation of invasive bladder cancer cells. Understanding the pathways and mechanisms regulating spheroid formation and metabolic changes of bladder cancer cells might contribute to the identification of novel targets and specific therapies for bladder cancer. 1. C. Yeung, et al (2014), Pharmacoeconomics, 32, 1093-1104. 2. M. Babjuk, et al. (2017), Eur. Urol., 71, 447-461. 3. J. Alfred Witjes, T. Lebret, E. M. Compérat, N. C. Cowan, et al. (2017), Eur. Urol., 71, 462-475. 4. I. Vasyutin, L. Zerihun, C. Ivan, A. Atala. (2019), Anticancer Res., 39, 1105-1118. 5. V. Pasquale, G. Ducci, G. Campioni, A. Ventrici, et al. (2020), Cells, 9, 1-26. 6. S. Buraschi, T. Neill, S.-Q. Xu, C. Palladino, et al. (2020), Matrix Biol., 93, 10-24.