Model For Evaluation Of Drugs Research Articles

Dynamic Liver Chip Based on Well-Coupled Microfluidics: An Accurate NASH Model for Drug Evaluation.

The convenient liver model in vitro recapitulating the hepatic functions, metabolism, and even steatohepatitis to perform the accurate drug evaluation is still challenging because of the unattainable hominine physiological microenvironment in vitro. Here, the progressed stages of nonalcoholic steatohepatitis (NASH) disease were precisely modeled to accurately evaluate the performance of antilipemic based on the dynamic liver chip adopting the well-coupled microfluidics, which well recapitulated the normal and steatohepatitis of liver in vitro. In brief, the mild nutrient flow and sufficient oxygen supply for parenchymal liver cells could be well supplied through the endothelial cells layer that mimicked the real physiological barrier of endothelium, while the loading of drugs might be obtained by directly adding drug into the running nutrient flow to mimic the intravenously administrable. The progressed degree of steatohepatitis could be directly reflected by the amount of intramyocellular lipid content (IMLC) of the HepG2 cell hepatocyte layer in wells that were induced by different concentrations of free fatty acids (FFA). To prove the concept of the liver chip in drug evaluation, an accurate assessment of the performance of firsocostat, the acetyl-CoA carboxylase (ACC) inhibitor of hepatic mitochondria of hepatocytes, was carried out. The subtle time dependence of firsocostat treatment to different progressed stages of NASH was clearly figured out. Therefore, we prospect the liver chip that adopted well-coupled microfluidics could be an accurate and standard liver model in vitro to carry out the antilipemic evaluation and screening, which significantly enlightens the drug evaluation by liver on chip in vitro.

Construction of a lung cancer 3D culture model based on alginate/gelatin micro-beads for drug evaluation.

Lung cancer is one of the most common malignant tumors worldwide. Despite advances in lung cancer treatment, patients still face challenges related to drug resistance and recurrence. Current methods for evaluating anti-cancer drug activity are insufficient, as they rely on two-dimensional (2D) cell culture and animal models. Therefore, the development of an in vitro drug evaluation model capable of predicting individual sensitivity to anti-cancer drugs would greatly enhance the success rate of drug treatments for lung cancer patients. The purpose of this research is to utilise conditional reprogramming technology to cultivate patient-derived lung cancer cells and to construct an in vitro 3D culture model using sodium alginate (SA) and gelatin. The aim is to study the biological characteristics of cells in the 3D culture model and to further investigate the sensitivity of anti-cancer drugs based on the alginate-gelatin 3D culture model. This approach provides new means and insights for personalized precision anti-cancer therapy and the development of new anti-cancer drugs. Conditional reprogramming technology was used to generate conditionally reprogrammed lung adenocarcinoma cells (CRLCs). Alginate-gelatin hydrogel micro-beads were created to explore their potential use in the assessment of anti-cancer drugs. Cell proliferation was also examined using the MTS assay method. Live/dead staining was performed to estimate cell distribution and viability using calcein acetoxymethyl ester/propidium iodide (calcein-AM/PI) double staining. Protein expression was assessed by Western blot. The cells grown in the three-dimensional (3D) culture were in a state of continuous proliferation, and there was an obvious phenomenon of cell mass growth. The drug sensitivity assay results demonstrated that compared with the 2D-grown cells, the CRLCs grown in the alginate-gelatin hydrogel micro-beads exhibited more resistance to anti-cancer drugs. The results also showed that the 3D-cultured CRLCs showed greater protein expression levels of stem cell hallmarks, such as Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX-2), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1), than the 2D-grown cells. These findings suggest that the 3D hydrogel cell culture models more closely mimicked the in vivo biological and clinical behavior of cells, and demonstrated higher innate resistance to anti-cancer drugs than the 2D cell culture models, and thus could serve as valuable tools for diagnosis, drug screening, and personalized medicine.

3D bioprinting platform development for high-throughput cancer organoid models construction and drug evaluation

The evaluation of anti-tumor drugs is critical for their development and clinical guidance. Tumor organoid models are gaining increased attention due to their ability to better mimic real tumor tissues, as well as lower time and economic costs, which makes up for the shortcomings of cell lines and xenograft models. However, current tumor organoid cultures based on the Matrigel have limitations in matching with high-throughput engineering methods due to slow gelation and low mechanical strength. Here, we present a novel composite bioink for culturing colorectal cancer organoids that provides an environment close to real tissue growth conditions and exhibits excellent photocrosslinking properties for rapid gel formation. Most importantly, the tumor organoids viability in the composite bioink after printing was as high as 97%, which also kept multicellular polar structures consistent with traditional culture methods in the Matrigel. Using 3D bioprinting with this composite bioink loaded with organoids, we demonstrated the feasibility of this drug evaluation model by validating it with clinically used colorectal cancer treatment drugs. Our results suggested that the composite bioink could effectively cultivate tumor organoids using 3D bioprinting, which had the potential to replace less reliable manual operations in promoting the application of tumor organoids in drug development and clinical guidance.

Development of an alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell-derived liver organoids.

Alcoholic liver disease (ALD) poses a significant health challenge, so comprehensive research efforts to improve our understanding and treatment strategies are needed. However, the development of effective treatments is hindered by the limitation of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we use a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subject to ethanol treatment to induce ALD, thus establishing a disease model. A rigorous assessment through a series of experiments reveals that this model partially recapitulates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential use in screening drugs for ALD treatment. Overall, the liver organoid model of ALD, which is derived from hiPSC differentiation, has emerged as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

Human Motor System-Based Biohybrid Robot-On-a-Chip for Drug Evaluation of Neurodegenerative Disease.

Biohybrid robots have been developed for biomedical applications and industrial robotics. However, the biohybrid robots have limitations to be applied in neurodegenerative disease research due to the absence of a central nervous system. In addition, the organoids-on-a-chip has not yet been able to replicate the physiological function of muscle movement in the human motor system, which is essential for evaluating the accuracy of the drugs used for treating neurodegenerative diseases. Here, a human motor system-based biohybrid robot-on-a-chip composed of a brain organoid, multi-motor neuron spheroids, and muscle bundle on solid substrateis proposed to evaluate the drug effect on neurodegenerative diseases for the first time. The electrophysiological signals from the cerebral organoid induced the muscle bundle movement through motor neuron spheroids. To evaluate the drug effect on Parkinson's disease (PD), a patient-derived midbrain organoid is generated and incorporated into a biohybrid robot-on-a-chip. The drug effect on PD is successfully evaluated by measuring muscle bundle movement. The muscle bundle movement of PD patient-derived midbrain organoid-based biohybrid robot-on-a-chip is increased from 4.5 ± 0.99µm to 18.67 ± 2.25µm in response to levodopa. The proposed human motor system-based biohybrid robot-on-a-chip can serve as a standard biohybrid robot model for drug evaluation.

Outlook of PINK1/Parkin signaling in molecular etiology of Parkinson's disease, with insights into Pink1 knockout models.

Parkinson's disease (PD) relates to defective mitochondrial quality control in the dopaminergic motor network. Genetic studies have revealed that PINK1 and Parkin mutations are indicative of a heightened propensity to PD onset, pinpointing mitophagy and inflammation as the culprit pathways involved in neuronal loss in the substantia nigra (SNpc). In a reciprocal manner, LRRK2 functions in the regulation of basal flux and inflammatory responses responsible for PINK1/Parkin-dependent mitophagy activation. Pharmacological intervention in these disease-modifying pathways may facilitate the development of novel PD therapeutics, despite the current lack of an established drug evaluation model. As such, we reviewed the feasibility of employing the versatile global Pink1 knockout (KO) rat model as a self-sufficient, spontaneous PD model for investigating both disease etiology and drug pharmacology. These rats retain clinical features encompassing basal mitophagic flux changes with PD progression. We demonstrate the versatility of this PD rat model based on the incorporation of additional experimental insults to recapitulate the proinflammatory responses observed in PD patients.

Advances in Tumor Organoids for the Evaluation of Drugs: A Bibliographic Review.

Tumor organoids are defined as self-organized three-dimensional assemblies of heterogeneous cell types derived from patient samples that mimic the key histopathological, genetic, and phenotypic characteristics of the original tumor. This technology is proposed as an ideal candidate for the evaluation of possible therapies against cancer, presenting advantages over other models which are currently used. However, there are no reports in the literature that relate the techniques and material development of tumor organoids or that emphasize in the physicochemical and biological properties of materials that intent to biomimicry the tumor extracellular matrix. There is also little information regarding the tools to identify the correspondence of native tumors and tumoral organoids (tumoroids). Moreover, this paper relates the advantages of organoids compared to other models for drug evaluation. A growing interest in tumoral organoids has arisen from 2009 to the present, aimed at standardizing the process of obtaining organoids, which more accurately resemble patient-derived tumor tissue. Likewise, it was found that the characteristics to consider for the development of organoids, and therapeutic responses of them, are cell morphology, physiology, the interaction between cells, the composition of the cellular matrix, and the genetic, phenotypic, and epigenetic characteristics. Currently, organoids have been used for the evaluation of drugs for brain, lung, and colon tumors, among others. In the future, tumor organoids will become closer to being considered a better model for studying cancer in clinical practice, as they can accurately mimic the characteristics of tumors, in turn ensuring that the therapeutic response aligns with the clinical response of patients.

Abstract 5988: Intra-carotid artery brain metastasis models for evaluation of lung and breast cancer drugs

Abstract Non-CNS metastatic brain cancer is about 10 times more common than CNS cancer. Lung cancer and breast cancer account for most brain metastasis. KRAS-mutant NSCLC compose a third lung adenocarcinoma, among which 17% to 55% will develop brain metastases. Likewise, more than a third HER2-positive breast cancer will develop brain metastasis. Among existing animal models, the ectopic injection in brain cannot reflect the mechanism of tumor invasion and metastasis, while tail vein and intra-cardiac injection usually produces extra-cerebral metastatic disease and the animals have to be sacrificed before brain metastases appeared. Here we developed several metastasis models injected by intra-carotid artery (ICA) to address these problems. Two KRAS-mutant lung cancer cells and two HER2-positive breast cancer cells selected by their metastasize potential were luciferase labeled for in vivo imaging and drug evaluation. The tumor pathology in brain was evaluated by H&E staining. Compared to ectopic models, these ICA models have natural dissemination and progression in the brain, which recapitulate the different pattern of metastatic lesions in the clinic. With synchronized growth rate and little variation compared to tail vein and intra-cardiac models, these tumor growth kinetics allow for more appropriate animal efficacy study. Both KRAS-sensitive and insensitive models have been developed and validated for the next generation drug development; HER-2 positive breast models with ICA method also showed a good discriminative power on anti-HER2 modalities including antibodies, small molecules and ADC. Meanwhile, ICA models with human immune cell reconstitution were also developed to support immune therapies in pre-clinical stage. In summary, we have developed a panel of intra-carotid artery brain metastatic models for drug evaluation, and demonstrated their superiority in growth kinetics, brain microenvironment, drug selectivity and immune component. These models can be a promising predictive tool for novel drug/modality discovery to target brain metastasis. Citation Format: Fuyang Wang, Yuying Yang, Haoxia Zhao, Chunlei Dai, Bingrui Han, Mengyao Zhang, Xia Xiong, Chao Zhang, Xuzhen Tang, Qunsheng Ji. Intra-carotid artery brain metastasis models for evaluation of lung and breast cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5988.

Role of Glycogen Synthase Kinase-3 in the Etiology of Type 2 Diabetes Mellitus: A Review.

The risk of type 2 diabetes mellitus (T2DM) is increasing abundantly due to lifestyle-related obesity and associated cardiovascular problems. Presently, Glycogen synthase kinase-3 (GSK-3) has gained considerable attention from biomedical scientists to treat diabetes. Phosphorylation of GSK-3 permits a number of cellular activities like regulation of cell signaling, cellular metabolism, cell proliferation and cellular transport. Inhibiting GSK-3 activity by pharmacological intervention has become an important strategy for the management of T2DM. This review focuses on the schematic representation of fundamental GSK-3 enzymology and encompasses the GSK-3 inhibitors as a future therapeutic lead target for the management of T2DM that may significantly regulate insulin sensitivity to insulin receptor, glycogen synthesis and glucose metabolism. The various signaling mechanisms of inhibiting the GSK-3 by describing insulin signaling through Insulin Receptor Substrate (IRS-1), Phosphatidylinositol-3 Kinase (PI3K) and Protein Kinase B (PKB/ AKT) pathways that may hopefully facilitate the pharmacologist to design for antidiabetic drug evaluation model in near future have also been highlighted.

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

Abstract LB250: Development of organoid raft cultures of cervical, breast and glioblastoma tumors as quick economical ex vivo human cancer models for pre-clinical drug evaluation

Abstract Background: In the past few years, 3D organoid cultures of patient-derived tumors or patient-derived xenografts have gained significant attention as faster and more economical ex vivo alternatives to animal models for the pre-clinical evaluation of therapeutics. We reported previously that raft cultures of ex vivo epithelial warts, normal human epithelia from various anatomic sites, as well as cancer cell lines (cervical and melanoma) grown at the liquid:air interface recapitulate parental tissue phenotypes. Here we describe adaptation of the above technique to develop Organoid Raft Culture (ORC) of tumors of various origin and validation as preclinical models for drug evaluation. Methods: A stromal equivalent (SE) consisting of buffered rat-tail collagen and J2 mouse fibroblasts was prepared in 24-well tissue culture plates. Freshly harvested tumors from patients or patient-derived xenografts of cervical cancers were minced to <2 mm pieces, mixed with buffered rat tail collagen mixture, assembled on the SE and cultured submerged for 2 days in simple raft culture media (DMEM, F12, 10% FBS, growth factors). This assembly was then raised on stainless steel grids in a 6-well plate and cultured at liquid:air interface for variable times (10 days to over 2-3 months). Tumors were harvested, formalin-fixed and paraffin-embedded for analyses by histology and immunohistochemistry. Results: The cervical ORCs maintained the heterogeneity of the parental tumor or xenografts. We evaluated cytotoxicity of vorinostat and CFI402257 on ORCs of cervical cancer-derived PDX models by TUNEL assays and immunohistochemistry. The ex-vivo data was validated in experiments with the in vivo PDXs of cervical cancers. We also successfully developed ORCs of mouse xenografts of syngeneic breast tumor cell lines, and organoids from glioblastomas. Conclusions: Organoid Raft Cultures of patient- or mouse-derived tumors and xenografts of cervical, breast and glioblastoma, recapitulate the morphology of parent tumors, can be passaged multiple times, are suitable for bio-banking and expedite drug evaluation. This method does not require costly growth components and multiple cultures could be easily performed. The PDX derived ORC platform would allow rapid evaluation of drug candidates against many types of solid tumors. Acknowledgement: R21CA216789 grant to NSB. Citation Format: Nilam Sanjib Banerjee, Dianne W. Moore, Abhisek Gangrade, Donald J. Buchsbaum, Luise Burt Nabors, Thomas R. Broker, Louise T. Chow. Development of organoid raft cultures of cervical, breast and glioblastoma tumors as quick economical ex vivo human cancer models for pre-clinical drug evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB250.

Abstract 3005: Establishment and characterization of a series of lymphoma PDX models representing varied clinical behaviors and treatment responses

Abstract Background: Lymphomas are comprised of a heterogeneous group of hematologic malignancies based on different origins with diverse patterns of clinical behavior and treatment response. Pathologically, lymphomas are divided into two main subtypes, Hodgkin (HL) and non-Hodgkin (NHL), which account for around 10% and 90% of cases, respectively. NHL is further divided into B cell, T cell and natural killer (NK) cell subtypes. Besides histopathology, cancer is increasingly recognized as a genetic disease with specific molecular alterations, such as ALK translocation in anaplastic large cell lymphoma, MYC/BCL2/BCL6 translocation in B cell lymphoma (often referred to as ‘double-hit' or ‘triple-hit') and other specific mutations or overexpression. Current clinical therapeutic options for lymphoma therefore need to consider not only the histological type, clinical aggressiveness and cancer stage, but also the molecular markers, cytokine signaling, epigenetic aberrations and lymphoma microenvironment. Preclinical modeling with diverse types of lymphomas and deep characterization are important for the drug discovery of new lymphoma treatments. Methods: We have established and characterized a number of different subtypes of lymphoma patient-derived xenograft (PDX) models including both mature T/NK cell neoplasms and B cell neoplasms. Immunodeficient mice were used for establishment of these lymphoma PDX models with subcutaneous or tail vein engraftment. Model classification and characterization were determined by histopathology and IHC, as well as genomic profiling by next-generation sequencing (NGS) and response to chemo- or targeted therapies. Results: We have developed and characterized a large panel of lymphoma PDX models with various subtypes, including mantle cell lymphoma, angioimmunoblastic T cell lymphoma, ALK negative anaplastic large cell lymphoma, adult T cell leukemia/lymphoma, peripheral T cell lymphoma, extranodal NK/T cell lymphoma and also other common B cell lymphomas such as DLBCL and follicular lymphoma. Histopathology analysis of these models is in line with clinical pathology diagnosis. Additionally, cancer type-specific biomarkers, such as CD3, CD20, BCL2, BCL6 and MYC, were used for characterization of these lymphoma models by IHC. The response to subtype-specific chemotherapy or targeted therapies, such as CHOP, BTK inhibitors, CD38 antibodies etc., are now tested in some of these lymphoma PDX models and will be presented at the meeting. Conclusions: Lymphomas have diverse subtypes, represented by different clinical features and treatment responses. We have established and characterized a series of lymphomas with varied types including a large cohort of B cell lymphoma and T/NK cell lymphoma PDX models for drug evaluation. Citation Format: Jessie J. Wang, Yanrui Song, Jitai Zheng, Guiqing Wu, Likun Zhang, Henry Q.X. Li, Davy Xuesong Ouyang. Establishment and characterization of a series of lymphoma PDX models representing varied clinical behaviors and treatment responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3005.

Method for rapid synchronization of different growth cycles of Plasmodium falciparum in vitro and application in differential gene expression profile of 3D7 after dihydroartemisinin treatment

Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log_2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer's clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape; the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites' rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.

Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening.

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient’s disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish (Danio rerio) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC50) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC50 fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.

Differentiation of Intrafusal Fibers from Human Induced Pluripotent Stem Cells

Human-based "body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers, sensory neurons) and efferent (extrafusal fibers, motoneurons) cells, in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling.

Abstract 5016: A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels

Abstract Background; Two dimensional single-layer culture still remains the preferred platform for most laboratory preclinical studies, although interaction between cancer and stromal cells has been well reported to be important in tumor progression and resistance against therapies. In order to mimic the patient tumor tissues, ex vivo model which recaptures the tumor microenvironment is required. Methods; Layered 3D stromal tissues were produced by culturing normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) coated with extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and overlaid tumors were morphologically characterized by HE stain, immunohistochemistry (IHC) and immunofluorescence (IF). Furthermore, drug sensitivity assays were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of Japanese Foundation for Cancer Research. Cancer cell viability was evaluated by fluorescent labeling, enzymatic dissociation and cell counting analysis. IF with cancer specific markers and imaging analyses were also performed. Results; The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and formation of microvascular network was observed within several days. In comparison with 2D mono-culture or 3D mono/co-cultured spheroid model, decreased drug sensitivities were represented in our 3D co-cultured model. In the simultaneous treatment with cytotoxic anticancer agents and molecular targeted drugs, dose-responses were significantly different between the2D and 3D models. Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug evaluation with the co-cultured tumors may reflect the drug sensitivity of cancer cells in vivo. Our unique 3D ex vivo model represents a valuable tool for drug development in a fully human cell and matrix microenvironment, and thus testing patient-derived cells and approved compounds also enable better prediction their efficacy. Citation Format: Yuki Takahashi, Kei Tsukamoto, Shiro Kitano, Shinji Irie, Michiya Matsusaki, Satoshi Nagayama, Ryohei Katayama, Eiji Shinozaki, Naoya Fujita. A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5016.

Disruption of PTPS Gene Causing Pale Body Color and Lethal Phenotype in the Silkworm, Bombyx mori.

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene or by defects in the tetrahydrobiopterin (BH4) synthesis pathway. Here, by positional cloning, we report that the 6-pyruvoyl-tetrahydropterin synthase (PTPS) gene, encoding a key enzyme of BH4 biosynthesis, is responsible for the alc (albino C) mutation that displays pale body color, head shaking, and eventually lethality after the first molting in silkworm. Compared to wild type, the alc mutant produced more substrates (phenylalanine (Phe) and tyrosine (Tyr)) and generated less DOPA and dopamine. Application of 2,4-diamino-6-hydroxypyrimidine (DAHP) to block BH4 synthesis in the wild type effectively produced the alc-like phenotype, while BH4 supplementation rescued the defective body color and lethal phenotype in both alc and DAHP-treated individuals. The detection of gene expressions and metabolic substances after drugs treatments in alc and normal individuals imply that silkworms and humans have a high similarity in the drugs metabolic features and the gene pathway related to BH4 and the dopamine biosynthesis. We propose that the alc mutant could be used as an animal model for drug evaluation for BH4-deficient PKU.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

Method Development for Clinical Comprehensive Evaluation of Pediatric Drugs Based on Multi-Criteria Decision Analysis: Application to Inhaled Corticosteroids for Children with Asthma.

Establishing a comprehensive clinical evaluation system is critical in enacting national drug policy and promoting rational drug use. In China, the 'Clinical Comprehensive Evaluation System for Pediatric Drugs' (CCES-P) project, which aims to compare drugs based on clinical efficacy and cost effectiveness to help decision makers, was recently proposed; therefore, a systematic and objective method is required to guide the process. An evidence-based multi-criteria decision analysis model that involved an analytic hierarchy process (AHP) was developed, consisting of nine steps: (1) select the drugs to be reviewed; (2) establish the evaluation criterion system; (3) determine the criterion weight based on the AHP; (4) construct the evidence body for each drug under evaluation; (5) select comparative measures and calculate the original utility score; (6) place a common utility scale and calculate the standardized utility score; (7) calculate the comprehensive utility score; (8) rank the drugs; and (9) perform a sensitivity analysis. The model was applied to the evaluation of three different inhaled corticosteroids (ICSs) used for asthma management in children (a total of 16 drugs with different dosage forms and strengths or different manufacturers). By applying the drug analysis model, the 16 ICSs under review were successfully scored and evaluated. Budesonide suspension for inhalation (drug ID number: 7) ranked the highest, with comprehensive utility score of 80.23, followed by fluticasone propionate inhaled aerosol (drug ID number: 16), with a score of 79.59, and budesonide inhalation powder (drug ID number: 6), with a score of 78.98. In the sensitivity analysis, the ranking of the top five and lowest five drugs remains unchanged, suggesting this model is generally robust. An evidence-based drug evaluation model based on AHP was successfully developed. The model incorporates sufficient utility and flexibility for aiding the decision-making process, and can be a useful tool for the CCES-P.

Establishment of a drug evaluation model against light-induced retinal degeneration using adult pigmented zebrafish

Age-related macular degeneration (AMD) is a major cause of irreversible loss of central vision in the elderly. Zebrafish is an attractive animal model in some respects, lower cost, smaller housing facilities and easier genetic manipulation compared to rodents. The present study aimed to establish a drug evaluation method against light irradiation, as a dry AMD disease model, using adult pigmented zebrafish. Intravitreal administration of an antioxidant, N-acetylcysteine, protected against light-induced retinal degeneration in a concentration-dependent manner. We established a new drug evaluation model against light-induced retinal degeneration that can provide new knowledge about dry AMD pathology and therapy.