Using 3D spheroids to evaluate mTORC1-mediated drug toxicity

Abstract: Mammalian target of rapamycin complex 1 (mTORC1) is a key mediator of protein synthesis and cell growth. Its inappropriate activation through loss of the tumour suppressors TSC1 or TSC2 in Tuberous Sclerosis Complex (TSC) leads to cyst and tumour formation in multiple organs. mTORC1 is also seen to be overactive in several sporadic cancers. The clinically approved mTORC1 inhibitor, rapamycin, can shrink tumours in TSC, but they regrow following drug withdrawal [1], indicating a cytostatic rather than cytotoxic response.

Currently, research into improved therapeutics for TSC utilises 2D cell screens and mouse models. These methodologies have their limitations as 2D models poorly mimic the tumour, while mouse models cannot be used to screen large numbers of drugs. By utilising 3D spheroids, we can overcome some of these limitations and screen drugs in a way that more faithfully reflects their interaction with a 3D tumour.

Using TSC-deficient cell lines grown as 3D spheroids, we aimed to evaluate cytotoxic drug combinations that specifically target mTORC1 active cells. As mTORC1 activation leads to enhanced endoplasmic reticulum stress, we used nelfinavir, an HIV-1 protease inhibitor and inducer of ER stress, in combination with other drugs in order to further stress TSC-deficient cells and induce cell death. Mirroring what is seen in mouse models, rapamycin treatment shrank the spheroids, but the cells remained viable as when replated onto adherent plates, the cells from the spheroid successfully attached to the plastic and migrated from the spheroid. In contrast, nelfinavir drug combinations caused cell death within the spheroid, as visualised via DRAQ7 staining. When replated onto an adherent surface, these spheroids did not attach and no viable cells grew out from the spheroid. Our work introduces 3D models to the TSC field and shows that mTORC1-driven cells can be specifically eliminated through rapamycin-independent treatments, thus highlighting potential new avenues for therapy for patients.

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