High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

Abstract

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in druginduced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals

“…There is evidence to suggest that HepG2 cells cultured in 3D spheroids results in decreased proliferation and reduced reliance on glucose (Hiemstra et al 2019 ). Thus, we investigated if HepG2 3D spheroids had an increased sensitivity to the ETC inhibitors.…”

“…Interestingly, culturing HepG2 as 3D spheroids sensitised the cells to OCR inhibition in the presence of glucose. Previous studies have demonstrated that 3D spheroid culture of HepG2 cells decreases proliferation, increases differentiation and increases sensitivity to various compounds with human drug-induced liver injury liability (Ramaiahgari et al 2014 ; Hiemstra et al 2019 ). Taken together, the data suggest that proliferating cells are less reliant on oxidative phosphorylation as an energy source where glucose is not limiting and that 3D HepG2 cells switch towards an oxidative phosphorylation-mediated energy source.…”

“…Transcriptomic data of a previous HepG2 study from the de Water lab were used to interrogate potential differences in glycolysis gene expression in 3D cultured cells (Hiemstra et al 2019 ). The study compared 2D at day 3 and 3D for 3, 7, 14, 21, and 28 days.…”

Setting the stage for next-generation risk assessment with non-animal approaches: the EU-ToxRisk project experience

“…Simpler test systems were combined to obtain advanced complex test systems to better resemble the in vivo situation. These approaches include, for instance, the use of mixed cell cultures (Gutbier et al 2018 ), 3D organoids and neurospheres (Brull et al 2020 ; Hiemstra et al 2019 ; Kobolak et al 2020 ) and microphysiological systems (MPS), e.g. four-organ-chips developed to interconnect miniaturized human intestine, liver, brain and kidney equivalents (Ramme et al 2019 ) and improved iPSC cell differentiation to hepatocytes (Boon et al 2020 ).…”

Self‐Organizing Human Induced Pluripotent Stem Cell Hepatocyte 3D Organoids Inform the Biology of the Pleiotropic TRIB1 Gene

“…Supporting our observation in hiPSC hepatic organoids, recent reports in HepG2 and rat primary hepatocytes have shown that hepatic genes such as PROX1, HNF4α, and C/EBPα are induced in 3D hepatocyte cultures. (48,49) TRIB1 liver-specific KO mice has increased C/EBPα protein (due to reduced protein degradation) and decreased C/EBPα transcript (due to negative feedback from the increased protein). (23) We also know that TRIB1 is a transcriptional target of C/EBPα, leading to a negative feedback loop in which excess C/EBPα induces TRIB1, which then promotes the degradation of C/EBPα.…”