Profiling and Targeting of Energy and Redox Metabolism in Grade 2 Bladder Cancer Cells with Different Invasiveness Properties

Abstract

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.

An Optimized Workflow for the Analysis of Metabolic Fluxes in Cancer Spheroids Using Seahorse Technology

“…Seahorse assays on 2D cultures were performed according to the manufacturer’s instructions, as reported in Pasquale et al [ 15 ]. MDA-MB-231 and MCF7 cell lines were seeded in Seahorse XF plates at a density of 2 × 10 4 cells per well and cultured for 24 h in standard culture medium.…”

“…For instance, the Mito stress test includes modulators of the electron transport chain to investigate their role in sustaining cell metabolism. This technology has been exploited, for instance, to perform metabolic phenotyping of cancer cells in standard conditions and under perturbed conditions [ 15 ], to discover the metabolic switches responsible for the acquisition of malignant features (e.g., metastasis) [ 16 ], to observe metabolic heterogeneity in cancer through the identification of cell subpopulations harboring different metabolic profiles (e.g., cancer stem cells) [ 17 , 18 ], and many other studies of cancer metabolism [ 19 , 20 , 21 , 22 ].…”

RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment

“…The exchange of material through TNT connections gives cancer cells, in the context of the tumoral microenvironment, specific traits (e.g., enhanced metabolic plasticity, migratory phenotypes, angiogenic ability and therapy resistance). In this paper, we demonstrate that TNTs formed by bladder cancer cells of mid and high-stages with invasive properties [ 108 ] are functionally active and transport organelles, such as mitochondria, and proteins involved in bladder cancer malignancy and in nanotubes formation, such as RalA GTPase and LST1. Furthermore, we characterized the molecular machinery underlying TNTs formation in bladder cancer and kidney cells and unveiled the crucial role played by RalGPS2, which promotes tunneling nanotubes formation through interaction with Akt and PDK1.…”

Overexpression of NREP Promotes Migration and Invasion in Gastric Cancer Through Facilitating Epithelial-Mesenchymal Transition

“…The protocol used for the wound healing assay was based on earlier studies ( Pasquale et al, 2020 ). Cell migration toward the scratch zone was photographed using an inverted fluorescence microscope (Olympus CKX-41, Japan; ×200 magnification) every 12 h.…”