Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.
Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols.
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.
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