Drug Screening Research Articles

A Novel Bioluminescent Biosensor Quantifying Intramolecular Interaction and Levels of Pyroptosis Effector GSDMD.

Gasdermin D (GSDMD) is a key executor of pyroptosis, a form of inflammation-induced programmed cell death. Recently, GSDMD has been shown to play important roles in the development of various inflammatory-related human diseases including heart failure and cancer, suggesting that it is a promising therapeutic target for these diseases. While extensive studies on GSDMD's role in pyroptosis have been reported, it is challenging to study its function due to the lack of enzymatic activity of GSDMD. In this study, we used the NanoBiT technology to develop a novel GSDMD bioluminescent biosensor (GSDMD-BS) that detects the amount of non-cleaved GSDMD. This sensor allows us to quantify GSDMD's intramolecular interactions, the amounts of uncleaved GSDMD after caspase-1 cleavage, and expression levels in living cells. In vitro experiments using purified GSDMD-BS also confirmed the sensor's accuracy in reporting GSDMD levels and cleavage. Moreover, the potential for in vivo application was demonstrated in a xenograft mouse model. In conclusion, we have developed a GSDMD biosensor that is a valuable tool for real-time monitoring of GSDMD dynamics and pyroptosis. This biosensor will significantly expedite pyroptosis research and can be used for high-throughput screening for drugs targeting GSDMD for the therapy of many inflammation-related diseases.

A light-addressable potentiometric sensor-based extracellular calcium dynamic monitoring and imaging platform for cellular calcium channel drug evaluation

Disruption and dysregulation of cellular calcium channel function can lead to diseases such as ischemic stroke, heart failure, and arrhythmias. Corresponding calcium channel drugs typically require preliminary efficacy evaluations using in vitro models such as cells and simulated tissues before clinical testing. However, traditional detection and evaluation methods often encounter challenges in long-term continuous monitoring and lack calcium specificity. In this study, a dynamic monitoring system based on ion-sensitive membranes for light-addressable potentiometric sensor (LAPS) was developed to meet the demand for monitoring changes in extracellular calcium ion (Ca2+) concentration in live cells. The effects of Ca2+ channel agonists and blockers on 2D and 3D HL-1 cells were investigated, with changes in extracellular Ca2+ concentration reflecting cellular calcium metabolism, facilitating drug evaluation. Additionally, calcium imaging technology with optical addressing capability complemented the LAPS system's ability to perceive 3D cell morphology, enhancing its drug evaluation capabilities. This work provides a novel, label-free, specific, and stable technique for monitoring cellular calcium metabolism. It achieves both continuous monitoring at single points and custom sensing area calcium imaging, holding significant implications for drug screening and disease treatment related to human calcium homeostasis.

Transgenic Drosophila Expressing Active Human LH Receptor in the Gonads Exhibit a Decreased Fecundity: Towards a Platform to Identify New Orally Active Modulators of Gonadotropin Receptor Activity.

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their receptors are major regulators of reproduction in mammals and are absent in insects. We previously established transgenic Drosophila lines expressing a constitutively active human LH receptor variant (LHRD578Y) and the wild-type receptor (LHRwt; inactive in the absence of an agonist). That study showed that ubiquitously expression of LHRD578Y-but not of LHRwt-resulted in pupal lethality, and targeted expression in midline cells resulted in thorax/bristles defects. To further study the Drosophila model for an in vivo drug screening platform, we investigated here whether expressing LHRD578Y in the fly gonads alters reproduction, as shown in a transgenic mice model. The receptor was expressed in somatic cells of the gonads using the tissue-specific traffic jam-Gal4 driver. Western blot analysis confirmed receptor expression in the ovaries. A fecundity assay indicated that the ectopic expression of LHRD578Y resulted in a decrease in egg laying compared to control flies carrying, but not expressing the transgene (~40% decrease in two independent fly lines, p < 0.001). No significant reduction in the number of laid eggs was seen in flies expressing the LHRWT (<10% decrease compared to non-driven flies, p > 0.05). The decreased egg laying demonstrates a phenotype of the active receptor in the fly gonads, the prime target organs of the gonadotropins in mammals. We suggest that this versatile Drosophila model can be used for the pharmacological search for gonadotropin modulators. This is expected to provide: (a) new mimetic drug candidates (receptor-agonists/signaling-activators) for assisted reproduction treatment, (b) blockers for potential fertility regulation, and (c) leads relevant for the purpose of managing extra gonadotropic reported activities.

Micromixer driven by bubble-induced acoustic microstreaming for multi-ink 3D bioprinting.

Recently, the 3D printing of cell-laden hydrogel structures, known as bioprinting, has received increasing attention owing to advances in tissue engineering and drug screening. However, a micromixing technology that efficiently mixes viscous bioinks under mild conditions is needed. Therefore, this study presents a novel method for achieving homogeneous mixing of multiple inks in 3D bioprinting through acoustic stimulation. This technique involves generating an acoustic microstream through bubble oscillations inside a 3D bioprinting nozzle. We determined the optimal hole design for trapping a bubble, hole arrangement, and voltage for efficient mixing, resulting in a four-fold increase in mixing efficiency compared to a single bubble arrangement. Subsequently, we propose a nozzle design for efficient mixing during bioprinting. The proposed nozzle design enabled the successful printing of line structures with a uniform mixture of different viscous bioinks, achieving a mixing efficiency of over 80% for mixing 0.5-1.0 wt% sodium alginate aqueous solutions. Additionally, acoustic stimulation had no adverse effects on cell viability, maintaining a high cell viability of 88% after extrusion. This study presents the first use of a bubble micromixer in 3D bioprinting, demonstrating gentle yet effective multi-ink mixing. We believe this approach will broaden 3D printing applications, particularly for constructing functional structures in 3D bioprinting.

Drug Discovery and Screening Tool Development for Tauopathies by Focusing on Pathogenic Tau Repeat 3 Oligomers.

Comprehending early amyloidogenesis is essential for the development of effective therapeutic strategies. In tauopathies like Alzheimer's disease (AD), the abnormal accumulation of tau protein is initiated by pathological tau seeds. Mounting evidence implies that the microtubule binding domain, consisting of three to four repeats, plays a pivotal role in this process, yet the exact region driving the formation of pathogenic species needs to be further scrutinized. Here, we chemically synthesized individual tau repeats to identify those exhibiting pathogenic prion-like characteristics. Notably, repeat 3 (R3) displayed a remarkable propensity to polymerize, form toxic filaments, and induce cognitive impairment, even in the absence of an aggregation-promoting inducer, highlighting its physiological relevance. Additionally, oligomeric R3 was identified as a particularly pathological form, prompting the establishment of a screening platform. Through screening, tolcapone was found to possess therapeutic efficacy against pathological tau aggregates in PS19 transgenic mice. This screening platform provides a valuable avenue for identifying compounds that selectively interact with peptides implicated in the progression of tauopathies.

The cutting-edge progress in bioprinting for biomedicine: principles, applications, and future perspectives.

Bioprinting is a highly promising application area of additive manufacturing technology that has been widely used in various fields, including tissue engineering, drug screening, organ regeneration, and biosensing. Its primary goal is to produce biomedical products such as artificial implant scaffolds, tissues and organs, and medical assistive devices through software-layered discrete and numerical control molding. Despite its immense potential, bioprinting technology still faces several challenges. It requires concerted efforts from researchers, engineers, regulatory bodies, and industry stakeholders are principal to overcome these challenges and unlock the full potential of bioprinting. This review systematically discusses bioprinting principles, applications, and future perspectives while also providing a topical overview of research progress in bioprinting over the past two decades. The most recent advancements in bioprinting are comprehensively reviewed here. First, printing techniques and methods are summarized along with advancements related to bioinks and supporting structures. Second, interesting and representative cases regarding the applications of bioprinting in tissue engineering, drug screening, organ regeneration, and biosensing are introduced in detail. Finally, the remaining challenges and suggestions for future directions of bioprinting technology are proposed and discussed. Bioprinting is one of the most promising application areas of additive manufacturing technology that has been widely used in various fields. It aims to produce biomedical products such as artificial implant scaffolds, tissues and organs, and medical assistive devices. This review systematically discusses bioprinting principles, applications, and future perspectives, which provides a topical description of the research progress of bioprinting.

Small Molecular Approaches for Cellular Reprogramming and Tissue Engineering: Functions as Mediators of the Cell Signaling Pathway.

Utilizing induced pluripotent stem cells (iPSCs) in drug screening and cell replacement therapy has emerged as a method with revolutionary applications. With the advent of patient-specific iPSCs and the subsequent development of cells that exhibit disease phenotypes, the focus of medication research will now shift toward the pathology of human diseases. Regular iPSCs can also be utilized to generate cells that assess the negative impacts of medications. These cells provide a much more precise and cost-efficient approach compared to many animal models. In this review, we explore the utilization of small-molecule drugs to enhance the growth of iPSCs and gain insights into the process of reprogramming. We mainly focus on the functions of small molecules in modulating different signaling pathways, thereby modulating cell fate. Understanding the way small molecule drugs interact with iPSC technology has the potential to significantly enhance the understanding of physiological pathways in stem cells and practical applications of iPSC-based therapy and screening systems, revolutionizing the treatment of diseases.

Establishment and Characterization of Three Human Ocular Adnexal Sebaceous Carcinoma Cell Lines.

Ocular adnexal sebaceous carcinoma (SebCA) represents one of the most clinically problematic periocular tumors, often requiring aggressive surgical resection. The pathobiology of this tumor remains poorly understood, and few models exist that are suitable for preclinical testing. The aim of this study was to establish new cell lines to serve as models for pathobiological and drug testing. With patient consent, freshly resected tumor tissue was cultured using conditional reprogramming cell conditions. Standard techniques were used to characterize the cell lines in terms of overall growth, clonogenicity, apoptosis, and differentiation in vitro. Additional analyses including Western blotting, short tandem repeat (STR) profiling, and next-generation sequencing (NGS) were performed. Drug screening using mitomycin-C (MMC), 5-fluorouricil (5-FU), and 6-Diazo-5-oxo-L-norleucine (DON) were performed. JHH-SebCA01, JHH-SebCA02, and JHH-SebCA03 cell lines were established from two women and one man undergoing surgical resection of eyelid tumors. At passage 15, they each showed a doubling time of two to three days, and all could form colonies in anchorage-dependent conditions, but not in soft agar. The cells contained cytoplasmic vacuoles consistent with sebaceous differentiation, and adipophilin protein was present in all three lines. STR profiling confirmed that all lines were derived from their respective patients. NGS of the primary tumors and their matched cell lines identified numerous shared mutations, including alterations similar to those previously described in SebCA. Treatment with MMC or 5-FU resulted in dose-dependent growth inhibition and the induction of both apoptosis and differentiation. MYC protein was abundant in all three lines, and the glutamine metabolism inhibitor DON, previously shown to target high MYC tumors, slowed the growth of all our SebCA models. Ocular adnexal SebCA cell lines can be established using conditional reprogramming cell conditions, and our three new models are useful for testing therapies and interrogating the functional role of MYC and other possible molecular drivers. Current topical chemotherapies promote both apoptosis and differentiation in SebCA cells, and these tumors appear sensitive to inhibition or MYC-associated metabolic changes.

Terahertz meta-biosensor for subtype detection and chemotherapy monitoring of glioma cells

Rapid and precise identification of glioma subtypes can assist surgeons in quickly determining patients’ conditions and developing treatment plans. However, current methods face challenges such as high cost and limited sensitivity, thereby significantly hindering diagnostic efficiency. In this paper, we proposed a spectral sensing strategy by introducing terahertz biosensors for glioma subtype recognition and therapeutic efficacy monitoring. Our findings demonstrated that both the resonance frequency and amplitude of the transmission spectra varied as the cell increased. The two-dimensional fingerprint diagram could help us rapidly recognize cell subtypes. Additionally, we conducted a theoretical analysis of the sensing performance. We confirmed that the high Q-factor (∼27.8) results from toroidal dipole resonance and examined the impact of dielectric loss and the refractive index of the analyte on the resonance dip. Furthermore, we applied the biosensor in therapeutic drug screening and showed that as the concentration of tubastatin A increased, the resonance frequency shifted from 183.1 GHz to 76.2 GHz. This result indicates that the toroidal dipole constructed by the metasurface enables ultrasensitive light-matter interactions that amplify subtle cellular changes to the resonance spectrum. The proposed THz meta-biosensor demonstrates significant potential for rapid intraoperative recognition of glioma cell subtypes and therapeutic efficacy monitoring.

Reliability of AlphaFold2 Models in Virtual Drug Screening: A Focus on Selected Class A GPCRs.

Protein three-dimensional (3D) structure prediction is one of the most challenging issues in the field of computational biochemistry, which has overwhelmed scientists for almost half a century. A significant breakthrough in structural biology has been established by developing the artificial intelligence (AI) system AlphaFold2 (AF2). The AF2 system provides a state-of-the-art prediction of protein structures from nearly all known protein sequences with high accuracy. This study examined the reliability of AF2 models compared to the experimental structures in drug discovery, focusing on one of the most common protein drug-targeted classes known as G protein-coupled receptors (GPCRs) class A. A total of 32 representative protein targets were selected, including experimental structures of X-ray crystallographic and Cryo-EM structures and their corresponding AF2 models. The quality of AF2 models was assessed using different structure validation tools, including the pLDDT score, RMSD value, MolProbity score, percentage of Ramachandran favored, QMEAN Z-score, and QMEANDisCo Global. The molecular docking was performed using the Genetic Optimization for Ligand Docking (GOLD) software. The AF2 models' reliability in virtual drug screening was determined by their ability to predict the ligand binding poses closest to the native binding pose by assessing the Root Mean Square Deviation (RMSD) metric and docking scoring function. The quality of the docking and scoring function was evaluated using the enrichment factor (EF). Furthermore, the capability of using AF2 models in molecular docking to identify hits with key protein-ligand interactions was analyzed. The posing power results showed that the AF2 models successfully predicted ligand binding poses (RMSD < 2 Å). However, they exhibited lower screening power, with average EF values of 2.24, 2.42, and 1.82 for X-ray, Cryo-EM, and AF2 structures, respectively. Moreover, our study revealed that molecular docking using AF2 models can identify competitive inhibitors. In conclusion, this study found that AF2 models provided docking results comparable to experimental structures, particularly for certain GPCR targets, and could potentially significantly impact drug discovery.

Multi-output prediction of dose–response curves enables drug repositioning and biomarker discovery

Drug response prediction is hampered by uncertainty in the measures of response and selection of doses. In this study, we propose a probabilistic multi-output model to simultaneously predict all dose–responses and uncover their biomarkers. By describing the relationship between genomic features and chemical properties to every response at every dose, our multi-output Gaussian Process (MOGP) models enable assessment of drug efficacy using any dose–response metric. This approach was tested across two drug screening studies and ten cancer types. Kullback-leibler divergence measured the importance of each feature and identified EZH2 gene as a novel biomarker of BRAF inhibitor response. We demonstrate the effectiveness of our MOGP models in accurately predicting dose–responses in different cancer types and when there is a limited number of drug screening experiments for training. Our findings highlight the potential of MOGP models in enhancing drug development pipelines by reducing data requirements and improving precision in dose–response predictions.

Drosophila melanogaster as a model organism for screening acetylcholinesterase reactivators

ABSTRACT The widely used insecticide chlorpyrifos (CP) is known to inhibit acetylcholinesterase (AChE) activity attributed to result in various neurological disorders and acetylcholine-dependent organ functions including heart, skeletal muscle, lung, gastrointestinal tract, and central nervous systems. Enzyme reactivators, such as oximes, are known to restore AChE activity and mitigate adverse effects. The identification of compounds that reactivate AChE constitute agents with important therapeutic beneficial effects in cases of pesticide poisoning. However, the screening of novel drugs using traditional models may raise ethical concerns. This study aimed to investigate the potential of Drosophila melanogaster as a model organism for screening AChE reactivators, with a focus on organophosphate poisoning. The efficacy of several oximes, including pralidoxime, trimedoxime, obidoxime, methoxime, HI-6, K027, and K048, against CP-induced AChE activity inhibition in D. melanogaster was determined in silico, in vitro, and in vivo experiments. Molecular docking studies indicated a strong interaction between studied oximes and the active-site gorge of AChE. Data showed that selected oximes (100 μM) are effective in the reactivation of AChE inhibited by CP (10 μM) in vitro. Finally, in vivo investigations demonstrated that selected oximes, pralidoxime and K048 (1.5 ppm), reversed the locomotor deficits, inhibition of AChE activity as well as lowered the mortality rates induced by CP (0.75 ppm). Our findings contribute to utilization of D. melanogaster as a robust model for determination of actions of identified new AChE inhibitory agents with more effective therapeutic properties that those currently in use in the clinical practice in treatment of AChE associated disorders.

Luminescent Terbium Probe for Time‐Resolved FRET and NSET Binding Assays with Quantum Dots and Gold Nanoparticles

Time‐gated or time‐resolved FRET (TR‐FRET) assays are important tools in biosensing, bioimaging, drug screening, and molecular diagnostics. Efficient TR‐FRET assays require stable lanthanide complexes with high absorption cross sections, high quantum yields, and long photoluminescence lifetimes. Owing to their challenging synthesis, such complexes are relatively rare and new components are of potential interest when developing TR‐FRET probes. Here, we evaluate the recently developed Tb complex CoraFluor‐1 concerning its analytical performance in terbium‐to‐quantum dot FRET and terbium‐to‐gold nanoparticle NSET assays using the prototypical biological recognition system of biotin and streptavidin. Biological binding was quantifiable at sub‐picomolar concentrations in small sample volumes, with broad applicability demonstrated across three commercial fluorescence plate readers used for time‐resolved, spectrally‐resolved, and clinical bioanalysis. Overall, CoraFluor‐1 provided excellent analytical performance as both FRET and NSET donor, validating its potential for developing new TR‐FRET probes for biosensing and bioimaging.

Deciphering Chemical Rules for Drug Penetration into Strongyloides.

Background: Strongyloidiasis, a parasitic infection, presents a significant public health challenge in tropical regions due to the limited repertoire of effective treatments. The screening of chemical libraries against the therapeutically relevant third-stage larvae (L3) of the model parasite Strongyloides venezuelensis yielded meager success rates. This situation is reminiscent of Gram-negative bacteria, where drug entry is a limiting factor. Methods: Here, we set out to determine whether similar barriers are in place and establish whether structural and property requirements exist for anti-strongyloides drug discovery. We focused on dyes as their uptake and effects on viability can be independently assessed in the multicellular parasite, thus providing a means to study the possibility of similar entry rules. We tested different dyes in in vitro assays on L3s. Results: We found that staining was necessary to reduce parasite viability, with some dyes achieving anti-strongyloides effects at concentrations similar to those of the reference drug, ivermectin (IV). Some dyes also showed activity against female adults at concentrations well below that of ivermectin. Unfortunately, the most potent dye, Methylene Blue, was unable to prevent the infection in a preliminary in vivo mouse model assay, presumably due to fast dye clearance. Structural analysis showed that positive charges facilitated the access of the compounds to the L3 tissue, thus providing a structural tool for the introduction of activity. For female adults, low globularity is additionally required. As a proof of concept, we added a positive charge to an inactive compound of one of our chemical libraries and re-determined the activity. Conclusions: These findings allow us to establish structural rules for parasite entry that could be of interest for future drug screening or drug development campaigns. These rules might also be applicable to other related parasites.

High-throughput drug screening identifies novel therapeutics for Low Grade Serous Ovarian Carcinoma

Low grade serous carcinoma (LGSOC) is a rare epithelial ovarian cancer with unique molecular characteristics compared to the more common tubo-ovarian high-grade serous ovarian carcinoma. Pivotal clinical trials guiding the management of epithelial ovarian cancer lack sufficient cases of LGSOC for meaningful subgroup analysis, hence overall findings cannot be extrapolated to rarer chemo-resistant subtypes such as LGSOC. Furthermore, there is a need for more effective therapies for the treatment of relapsed disease, as treatment options are limited. To address this, we conducted the largest quantitative high-throughput drug screening effort (n = 3436 compounds) in 12 patient-derived LGSOC cell lines and one normal ovary cell line to identify unexplored therapeutic avenues. Using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines, our data set identified 60 high and 19 moderate confidence hits which induced cancer cell specific cytotoxicity at the lowest compound dose assessed (0.1 µM). We also revealed a series of known (mTOR/PI3K/AKT) and novel (EGFR and MDM2-p53) drug classes in which LGSOC cell lines showed demonstrable susceptibility to.

Fluorescent Iron-Doped Polymer Dot Nanozyme-Based Cascade System for Dual-Mode Detection of Acetylcholinesterase Activity and Its Inhibitors.

The advancement of acetylcholinesterase (AChE) activity and its inhibitor assays is crucial for clinical diagnosis, drug screening, and environmental monitoring. A nanozyme-mediated cascade reaction system could offer promising prospects for a wide range of applications in such biosensing; however, the creation of nanozyme catalysts with diverse functionalities remains a significant challenge. Herein, we have proposed a multifunctional iron-doped polymer dots (Fe-PDs) nanozyme possessing excellent fluorescence and peroxidase (POD)-mimicking activity. Notably, the Fe-PDs nanozyme is capable of catalyzing H2O2 to produce a series of reactive oxygen species, which can simultaneously quench the fluorescence of Fe-PDs and induce a chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB), enabling the dual-mode detection of H2O2 through both fluorescence turn-off and absorbance turn-on signals. Furthermore, by integrating acetylcholine (ACh) and choline oxidase (ChOx), we have developed a three-enzyme (AChE-ChOx-POD) cascade-based fluorometric and colorimetric dual-mode sensing platform for monitoring AChE activity and its inhibitors. The sensitive and convenient dual-mode sensor has achieved low limits of detection with 0.5 mU/mL (fluorometry) and 0.014 mU/mL (colorimetry) for AChE, respectively, which are superior to the traditional Ellman's assay. More significantly, this sensor can also be extended to detect the reversible and irreversible inhibitors of AChE, such as tacrine (IC50 = 23.3 nM) and carbaryl (LOD = 0.8 nM). We firmly believe that this innovative dual-mode nanozyme-involved multienzyme cascade system-based sensing strategy will stimulate further exploration and serve as a versatile and practical tool for biochemical sensing applications.

Substrate-Mediated Regulation of Src Expression Drives Osteoclastogenesis Divergence.

Glass, bone, and dentin are commonly applied substrates for osteoclast cultures; however, the impact of these substrates on osteoclastogenesis remains underexplored. This study aimed to address a significant gap in understanding how different substrates influence the process of osteoclastogenesis. RAW 264.7 cells were cultured and induced with RANKL on glass, bone, and dentin slides. Histological and molecular techniques were used to identify patterns and differences in osteoclast behavior on each substrate. Osteoclasts cultured on glass slides possessed the greatest number of nuclei and the highest expression levels of ACP5 (TRAP) and CTSK, with osteoclasts on bone and dentin slides displaying progressively lower levels. Src expression was also most pronounced in osteoclasts on glass slides, with decreased levels observed on bone and dentin. This variation in Src expression likely contributed to differences in cytoskeletal remodeling and oxidative phosphorylation (OXPHOS), resulting in substrate-dependent divergences in osteoclastogenesis. Glass slides were the most favorable substrate for inducing osteoclastogenesis, while bone and dentin slides were less effective. The substrate-induced expression of Src played a fundamental role in shaping the phenotypic divergence of osteoclasts. These insights fill important knowledge gaps and have significant implications for the development and selection of in vitro models for bone-related diseases and drug screening platforms.

Development of two layer fibrous scaffolds for 3D in vitro modelling: Effects of morphology and surface properties on cell proliferation, adhesion and drug sensitivity

Three-dimensional (3D) in vitro cell models are gaining popularity in cancer research compared to two-dimensional (2D) cell culture models because of their ability to more accurately represent the tissue microenvironment. This study introduces a novel approach for the fabrication of 3D cell culture models by combining modified melt-electrospinning technology and solution electrospinning to create 3D two-layer fibrous scaffolds. By manipulating electrospinning parameters (voltage 11–12 kV and feed rate 0.85–1.55 mg/min), we engineered poly (ε-caprolactone) (PCL) scaffolds with varying fiber diameters (8.9 ± 2.2 to 23.1 ± 3.4 μm) and pore sizes (32.7 ± 12.8 to 122.5 ± 21.1 μm). Non-thermal plasma (NTP) treatment at energy density of 0.23 J/cm2 enhanced surface hydrophilicity, evident in a significant reduction in water contact angles from 113 ± 0° to 64 ± 0°. A comprehensive set of characterization techniques, including X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) confirmed that plasma treatment altered the surface wettability without compromising the scaffold's chemical stability or crystalline structure. Biological assays involving human breast cancer MDA-MB-231 cells and human glioblastoma U-87 MG cells demonstrated high cell viability, indicating biocompatibility of the scaffolds. A pronounced difference in proliferation patterns was noted between MDA-MB-231 and U-87 MG, with the latter fully utilizing voids for the formation of cell aggregates, especially in the “fine” scaffold, indicating the dependence of cell behavior on scaffold architecture, which must be adjusted according to the modelled system and duration. Furthermore, investigation of the therapeutic efficacy of doxorubicin revealed a distinct drug response in 3D cultures, EC50 values of doxorubicin in 3D culture was approximately twice higher compared to 2D. These findings underscore the potential of tailored PCL scaffolds to create more physiologically relevant models for cancer research, tissue engineering, and drug screening applications, highlighting the necessity for careful consideration of scaffold design to achieve the desired in vitro model.

Developing patient-derived organoids to demonstrate JX24120 inhibits SAMe synthesis in endometrial cancer by targeting MAT2B

Endometrial cancer (EC) is one of the most common gynecologic malignancies, which lacking effective drugs for intractable conditions or patients unsuitable for surgeries. Recently, the patient-derived organoids (PDOs) are found feasible for cancer research and drug discoveries. Here, we have successfully established a panel of PDOs from EC and conducted drug repurposing screening and mechanism analysis for cancer treatment. We confirmed that the regulatory β subunit of methionine adenosyltransferase (MAT2B) is highly correlated with malignant progression in endometrial cancer. Through drug screening on PDOs, we identify JX24120, chlorpromazine derivative, as a specific inhibitor for MAT2B, which directly binds to MAT2B (Kd = 4.724 μM) and inhibits the viability of EC PDOs and canonical cell lines. Correspondingly, gene editing assessment demonstrates that JX24120 suppresses tumor growth depending on the presence of MAT2B in vivo and in vitro. Mechanistically, JX24120 induces inhibition of S-adenosylmethionine (SAMe) synthesis, leading to suppressed mTORC1 signaling, abnormal energy metabolism and protein synthesis, and eventually apoptosis. Taken together, our study offers a novel approach for drug discovery and efficacy assessment by using the PDOs models. These findings suggest that JX24120 may be a potent MAT2B inhibitor and will hopefully serve as a prospective compound for endometrial cancer therapy.

Exploring the impact of microfluidic chip structure on the efficacy of three-dimensional tumor microspheres cultivation.

Three-dimensional (3D) tumor microspheres can simulate the interaction and growth dynamics of tumor cells, and have been used as a new in vitro model for drug screening and tumor biology related research. The scaffold-free culture of 3D tumor microspheres on microfluidic chips has many advantages, including low cost, high throughput, convenience and flexibility. However, it is still unclear how various factors, such as chip structure, influence the culture effect of tumor microspheres. The lack of standardized evaluation and characterization of the culture effect hinders the further optimization and development of chip function. This study presents numerical simulations of multiple parts or processes of the proposed 3D culture chips with two different structural parameters based on computational fluid dynamics (CFD) methods. An evaluation system for tumor microspheres was established. The prediction of the CFD simulation was consistent with the culture results of the chips, reflecting the important role of the structural parameters of the microtrap in the formation of uniform tumor microspheres. Furthermore, the velocity of cell suspension also had a significant impact on the retention of tumor cells. Additionally, the drug screening results of tumor microspheres indicated that tumor microspheres exhibit greater drug resistance, which may be attributed to their size. These results offer valuable insights into the factors that influence the characteristics of tumor microspheres. This research provides a reference and direction for the optimal design and functional evaluation of scaffold-free 3D culture chips, and holds promise for promoting the development of novel drug research platforms.