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Wells had been casted into the agarose using a custom 3D-printed comb ( Figure 2B). The spheroids are then transferred to low-melting-point-agarose holders sitting in 35-mm glass-bottom dishes, each dish containing wells capable of holding up to 5 spheroids. After growing the cells for 72 hours at 37˚C and 5% CO 2, the HEK293 cells self-assemble into spheroids. To investigate AZD2014 within the spheroid environment, HEK293 cells were cultured in 96-well round -bottom plates (U-well) at a cell density of 10,000 cells per well. To investigate the working mechanism of AZD2014, Digital LightSheet ( DLS) Microscopy was used to observe the cellular uptake of AZD2014 within a living 3D-cell-culture-model environment, taking advantage of its naturally occurring fluorescence properties and its localization within living spheroids. Although AZD2014 is currently undergoing active clinical trials, its mode of action is unknown. Novel second-generation ATP-competitive inhibitors, such as AZD2014, provide improved antitumor activity by targeting both mTORC1 and mTORC2. As mTORC2 directly phosphorylates Akt, an important protein involved in cell survival, there is a likely need to inhibit both complexes for effective cancer treatments. Recent studies have also shown that tumors develop rapalog resistance to first generation mTOR inhibitors and exhibit effectiveness in few types of cancer. The broad functionality of the mTOR protein makes it an attractive drug target in diseases closely associated with dysfunctional or hyper-activated mTOR signaling like cancer.įirst generation mTOR inhibitors, such as rapamycin and clinically approved analogues (rapalogs), even though they initially showed effectiveness in previous preclinical models, turned out to only partially inhibit mTOR by targeting Complex 1. The mTOR protein exists in two complexes ( Figure 1): Complex 1 (mTORC1) and Complex 2 (mTORC2). In this cascade signaling pathway, these downstream substrates are responsible for regulating functions, such as cell proliferation, protein synthesis, autophagy, senescence, and apoptosis. The mammalian Target of Rapamycin (mTOR) pathway co-ordinates the availability of nutrients, growth factors, and the energy status of the cell with the activation of its downstream target proteins via phosphorylation events.