Search

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35–52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that ca, These authors contributed equally: Evgeniya Mickols, Alina Meyer and Niklas Handin.

Cancer patients often suffer from cancer symptoms, treatment complications and concomitant diseases and are, therefore, often treated with several drugs in addition to anticancer drugs. Whether such drugs, here denoted as 'concomitant drugs', have anticancer effects or interact at the tumor cell level with the anticancer drugs is not very well known. The cytotoxic effects of nine concomitant drugs and their interactions with five anti-cancer drugs commonly used for the treatment of colorectal cancer were screened over broad ranges of drug concentrations in vitro in the human colon cancer cell line HCT116wt. Seven additional tyrosine kinase inhibitors were included to further evaluate key findings as were primary cultures of tumor cells from patients with colorectal cancer. Cytotoxic effects were evaluated using the fluorometric microculture cytotoxicity assay (FMCA) and interaction analysis was based on Bliss independent interaction analysis. Simvastatin and loperamide, included here as an opioid agonists, were found to have cytotoxic effects on their own at reasonably low concentrations whereas betamethasone, enalapril, ibuprofen, metformin, metoclopramide, metoprolol and paracetamol were inactive also at very high concentrations. Drug interactions ranged from antagonistic to synergistic over the concentrations tested with a more homogenous pattern of synergy between simvastatin and protein kinase inhibitors in HCT116wt cells. Commonly used concomitant drugs are mostly neither expected to have anticancer effects nor to interact significantly with anticancer drugs frequently used for the treatment of colorectal cancer.

Quiescent cancer cells in malignant tumors can withstand cell-cycle active treatment and cause cancer spread and recurrence. Three-dimensional (3D) cancer cell models have led to the identification of oxidative phosphorylation (OXPHOS) as a context-dependent vulnerability. The limited treatment options for advanced hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) metastatic to the liver include the multikinase inhibitors sorafenib and regorafenib. Off-target effects of sorafenib and regorafenib are related to OXPHOS inhibition; however the importance of this feature to the effect on tumor cells has not been investigated in 3D models. We began by assessing global transcriptional responses in monolayer cell cultures, then moved on to multicellular tumor spheroids (MCTS) and tumoroids generated from a CRC patient. Cells were treated with chemotherapeutics, kinase inhibitors, and the OXPHOS inhibitors. Cells grown in 3D cultures were sensitive to the OXPHOS inhibitor nitazoxanide, sorafenib, and regorafenib and resistant to other multikinase inhibitors and chemotherapeutic drugs. Furthermore, nitazoxanide and sorafenib reduced viability, regrowth potential and inhibited mitochondrial membrane potential in an additive manner at clinically relevant concentrations. This study demonstrates that the OXPHOS inhibition caused by sorafenib and regorafenib parallels 3D activity and can be further investigated for new combination strategies.

Nitazoxanide is a Food and Drug Administration-approved antiprotozoal drug recently demonstrated to be selectively active against quiescent and glucose-deprived tumour cells. This drug also has several characteristics that suggest its potential as a radiosensitizer. The present study aimed to investigate the interaction between nitazoxanide and radiation on human colon cancer cells cultured as monolayers, and to mimic key features of solid tumours in patients, as spheroids, as well as in xenografts in mice. In the present study, colon cancer HCT116 green fluorescent protein (GFP) cells were exposed to nitazoxanide, radiation or their combination. Cell survival was analysed by using total cell kill and clonogenic assays. DNA double-strand breaks were evaluated in the spheroid experiments, and HCT116 GFP cell xenograft tumours in mice were used to investigate the effect of nitazoxanide and radiation in vivo. In the clonogenic assay, nitazoxanide synergistically and selectively sensitized cells grown as spheroids to radiation. However, this was not observed in cells cultured as monolayers, as demonstrated in the total cell kill assays, and much less with the clinically established sensitizer 5-fluorouracil. The sensitizing effect from nitazoxanide was confirmed via spheroid gamma-H2A histone family member X staining. Nitazoxanide and radiation alone similarly inhibited the growth of HCT116 GFP cell xenograft tumours in mice with no evidence of synergistic interaction. In conclusion, nitazoxanide selectively targeted quiescent glucose-deprived tumour cells and sensitized these cells to radiation in vitro. Nitazoxanide also inhibited tumour growth in vivo. Thus, nitazoxanide is a candidate for repurposing into an anticancer drug, including its use as a radiosensitizer.