Comparison of high-throughput single-cell RNA-seq methods for ex vivo drug screening.

Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27 327 cells are freely available to extend to future scRNA-seq methodological comparisons.

Sorafenib and nitazoxanide disrupt mitochondrial function and inhibit regrowth capacity in three-dimensional models of hepatocellular and colorectal carcinoma.

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.

Conditions for maintenance of hepatocyte differentiation and function in 3D cultures.

Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research.