Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

Evaluation of Human Liver Microtissues for Drug Screening on Schistosoma mansoni Schistosomula.

Schistosomiasis is a major neglected tropical disease with more than 200 million infections annually. Despite only one drug, praziquantel, being available, the drug pipeline against schistosomiasis is empty, and drug screening tools have limitations. We evaluated the potential of human liver microtissues (hLiMTs) in antischistosomal drug discovery. Because hLiMTs express all human P450 enzymes, they are an excellent tool to evaluate compounds' bioinactivation, bioactivation, and toxicity. To validate the metabolic conversion capacity of hLiMTs, we first quantified (R)- and (S)-praziquantel and the main metabolite trans-OH-praziquantel following incubation with 0.032-50 µM (0.01-15.62 µg/mL) praziquantel for up to 72 h by a validated LC-MS/MS method. We cocultured hLiMTs with newly transformed schistosomula (NTS) and evaluated the antischistosomal activity and cytotoxicity of three prodrugs terfenadine, tamoxifen citrate, and flutamide. HLiMTs converted 300-350 ng (R)-praziquantel within 24 h into trans-OH-praziquantel. We observed changes in the IC50 values for terfenadine, flutamide, and tamoxifen citrate in comparison to the standard NTS assay in vitro. Cytotoxicity was observed at high concentrations of flutamide and tamoxifen citrate. An in vitro platform containing hLiMTs could serve as an advanced drug screening tool for Schistosoma mansoni, providing information on reduced or increased activity and toxicity.

Parallelized Impedance-Based Platform for Continuous Dose-Response Characterization of Antischistosomal Drugs.

Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma, which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance-based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose-response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug-induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose-response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance-based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.

Activity and pharmacokinetics of a praziquantel crystalline polymorph in the Schistosoma mansoni mouse model.

Schistosomiasis is a global disease of significant public health relevance. Only one racemic drug, praziquantel, characterized by low bioavailability, low water solubility and extensive first pass metabolism, is currently available. We studied a new praziquantel formulation (polymorph B), which is based on a racemic praziquantel crystalline polymorph (TELCEU01). Its in vitro activity was tested on newly transformed schistosomula (NTS) and adult Schistosoma mansoni. In vivo studies were conducted in mice harboring chronic S. mansoni infections. Pharmacokinetic (PK) profiles of R- and S-praziquantel and R- and S- polymorph B following oral administration with both formulations were generated by sampling mice at 30, 60, 240 min and 24 h post-treatment, followed by LC-MS/MS analysis. PK parameters were calculated using a non-compartmental analysis with a linear trapezoidal model. In vitro, commercial praziquantel and the polymorph B performed similarly on both NTS (IC50 = 2.58 and 2.40 µg/mL at 72 h) and adults (IC50 = 0.05 and 0.07 µg/mL at 72 h). Praziquantel showed higher in vivo efficacy with an ED50 of 58.75 mg/kg compared to an ED50 of 122.61 mg/kg for the polymorph B. The PK profiles of the two drugs exhibited differences: R-praziquantel showed an overall 40% higher area under the plasma drug concentration-time curve (AUC0→24) (R-praziquantel = 3.42; R-polymorph B = 2.05 h*µg/mL) and an overall 30% lower apparent clearance (Cl/F) (R-praziquantel = 70.68 and R-polymorph B = 97.63 (mg)/(µg/mL)/h). Despite the lack of improved activity and PK properties of polymorph B against S. mansoni, here presented; research on pharmaceutical polymorphism remains a valid and cost-effective option for the development of new praziquantel formulations with enhanced properties such as increased solubility and/or dissolution.

Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni.

Drug discovery for schistosomiasis is still limited to a handful of academic laboratories worldwide, with only a few novel antischistosomal lead compounds being actively researched. Despite recent international mobilization against the disease to stimulate and promote antischistosomal drug discovery, setting up a drug-screening flow with schistosome parasites remains challenging. Whereas numerous different protocols to obtain and cultivate schistosomes have been published, those describing the drug-screening process are scarce, and none gather together parasite cultivation and early drug discovery procedures. To help overcome this hurdle, we provide here a set of integrated methods either adapted from already-published protocols or based on our long-term experience in schistosomiasis research. Specifically, we detail the establishment and maintenance of the complex and several-week-long Schistosoma mansoni life cycle in a laboratory setting, as well as the means of retrieving and culturing the parasites at their relevant life stages. The in vitro and in vivo assays that are performed along the drug-screening cascade are also described. In these assays, which can be performed within 5 d, the effect of a drug is determined by phenotypic assessment of the parasites' viability and morphology, for which stage-specific scoring scales are proposed. Finally, the modalities for testing and evaluating a compound in vivo, constituting a procedure lasting up to 10 weeks, are presented in order to go from in vitro hit identification to the selection of early lead candidates.

Impedance-Based Microfluidic Assay for Automated Antischistosomal Drug Screening.

Schistosomiasis is a neglected tropical disease, caused by parasitic worms, which affects almost 200 million people worldwide. For over 40 years, chemotherapeutic treatment has relied on the administration of praziquantel, an efficacious drug against schistosomiasis. However, concerns about developing drug resistance require the discovery of novel drug compounds. Currently, the drug-screening process is mostly based on the visual evaluation of drug effects on worm larvae in vitro by a trained operator. This manual process is extremely labor-intensive, has limited throughput, and may be affected by subjectivity of the operator evaluation. In this paper, we introduce a microfluidic platform with integrated electrodes for the automated detection of worm larvae viability using an impedance-based approach. The microfluidic analysis unit consists of two sets of electrodes and a channel of variable geometry to enable counting and size detection of single parasite larvae and the collective evaluation of the motility of the larvae as an unbiased estimator for their viability. The current platform also allows for multiplexing of the analysis units resulting in increased throughput. We used our platform to record size and motility variations of Schistosoma mansoni larvae exposed to different concentrations of mefloquine, a drug with established in vitro antischistosomal properties. The developed platform demonstrates the potential of integrated microfluidic platforms for high-throughput antischistosomal drug screening.