Bosung Ku

High-Throughput Screening (HTS) of Anticancer Drug Efficacy on a Micropillar/Microwell Chip Platform

Contemporary cancer therapy refers to treatment based on genetic abnormalities found in patients tumor. However, this approach is faced with numerous challenges, including tumor heterogeneity and molecular evolution, insufficient tumor samples available along with genetic information linking to clinical outcomes, lack of therapeutic drugs containing pharmacogenomic information, and technical limitations of rapid drug efficacy tests with insufficient quantities of primary cancer cells from patients. To address these problems and improve clinical outcomes of current personalized gene-targeted cancer therapy, we have developed a micropillar/microwell chip platform, which is ideally suited for encapsulating primary cancer cells in nanoscale spots of hydrogels on the chip, generating efficacy data with various drugs, eventually allowing for a comparison of the in vitro data obtained from the chip with clinical data as well as gene expression data. As a proof of concept in this study, we have encapsulated a U251 brain cancer cell line and three primary brain cancer cells from patients (448T, 464T, and 775T) in 30 nL droplets of alginate and then tested the therapeutic efficacy of 24 anticancer drugs by measuring their dose responses. As a result, the IC50 values of 24 anticancer drugs obtained from the brain cancer cells clearly showed patient cell-specific efficacy, some of which were well-correlated with their oncogene overexpression (c-Met and FGFR1) as well as the in vivo previous results of the mouse xenograft model with the three primary brain cancer cells.

High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening

Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Application of the DataChip/MetaChip technology for the evaluation of ajoene toxicity in vitro.

The DataChip is a universal platform for three-dimensional (3D) cell cultures on a micropillar chip, which can be applicable to a variety of human cells to simulate organ-specific toxicity. In addition, the MetaChip is developed for various combinations of drug metabolizing enzymes that can be spotted into the microwell chip and incubated with 3D human cells to simulate systematic compound metabolism in the human liver on a microscale format. Ajoenes have been known for various therapeutics activities, including anticancer effects, but there was limited information available in regard to their metabolism and cytotoxicity. In the present work, the metabolism-mediated toxicity of ajoenes was evaluated on a DataChip/MetaChip platform. In detail, we tested cytotoxicity of E- and Z-ajoene on 3D cultured Hep3B human hepatoma cells coupled with mixtures of drug metabolizing enzymes. Metabolic profiles of ajoenes were assessed with 23 representative drug metabolizing enzymes on the MetaChip. As a result, cytotoxicity of E-ajoene was significantly augmented in the presence of cytochrome P450 (CYP) isoforms, such as CYP2E1 and CYP3A5. Both E- and Z-ajoene were drastically detoxified in the presence of Phase II enzymes, including major UGTs, SULTs, NATs, and GSTs. Interestingly, All Mix, an artificial human liver microsome containing representative P450 mixture and phase II enzyme mixture, attenuated P450-induced cytotoxicity of ajoenes. Conclusively, we were able to confirm the metabolism-medicated toxicity of ajoenes on the chip.

A study of electrochemical biosensor for analysis of three-dimensional (3D) cell culture.

Cell culture has a fundamental role not only in regenerative medicine but also in biotechnology, pharmacology, impacting both drug discovery and manufacturing. Although cell culture has been generally developed for only two-dimensional (2D) culture systems, three-dimensional (3D) culture is being spotlighted as the means to mimic in vivo cellular conditions. In this study, a method for cytotoxicity assay using an electrochemical biosensor applying 3D cell culture is presented. In order to strengthen the advantage of a 3D cell culture, the experimental condition of gelation between several types of sol-gels (alginate, collagen, matrigel) and cancer cells can be optimized to make a 3D cell structure on the electrode, which will show the reproducibility of electrical measurement for long-term monitoring. Moreover, cytotoxicity test results applying this method showed IC(50) value of A549 lung cancer cells to erlotinib. Thus, this study evaluates the feasibility of application of the electrochemical biosensor for 3D cell culture to cytotoxicity assay for investigation of 3D cell response to drug compounds.

Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway

ABSTRACT The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.

High-throughput, miniaturized clonogenic analysis of a limiting dilution assay on a micropillar/microwell chip with brain tumor cells.

The limiting dilution assay (LDA) is a clonogenic drug efficacy test designed to determine a value for drug efficacy based on an all-or-none (positive or negative) response within replicates. It also attempts to calculate minimum cell numbers for cells to form colony in each drugged conditions, wherein a large value implies high drug efficacy (as a large number of extant cells are required to start a colony). However, traditional LDAs are time-consuming to set up, often requiring many replicates for statistical analysis, and manual colony identification under a microscope to determine a positive or negative response is tedious and is susceptible to human error. To address these issues, a high-throughput miniaturized LDA assay is developed using a micropillar/microwell chip platform using an automatic colony identification method. Three glioblastoma multiforme (GBM) brain tumor isolates (448T, 464T, and 775T) are used to test this new assay, using the c-Met kinase inhibitors SU11274 and PHA665752 as the target drugs. The results show that the minimum cell number of 775T is larger than that of the other two cell types (SU11274 and PHA665752) in both the sampled drugs, a result that is in good agreement with the results of previous conventional experiments using 96 well plates.

Automatic 3D Cell Analysis in High-Throughput Microarray Using Micropillar and Microwell Chips

Area-based and intensity-based 3D cell viability measurement methods are compared in high-throughput screening in order to analyze their effects on the assay results (doubling time and IC50) and their repeatability. Many other 3D cell-based high-throughput screening platforms had been previously introduced, but these had not clearly addressed the effects of the two methods on the assay results and assay repeatability. In this study, the optimal way to analyze 3D cultured cells is achieved by comparing day-to-day data of doubling times and IC50 values obtained from the two methods. In experiments, the U251 cell line is grown in chips. The doubling time, based on the area of the 3D cells, was 27.8 ± 1.8 h (standard deviation: 6.6%) and 27.8 ± 3.8 h (standard deviation: 13.7%) based on the intensity of the 3D cells. The doubling time calculated by area shows a smaller standard deviation than one calculated by intensity. IC50 values calculated by both methods are very similar. The standard deviations of IC50 values for the two methods were within ±3-fold. The IC50 variations of the 12 compounds were similar regardless of the viability measurement methods and were highly related to the shape of the dose–response curves.

Estimation of bisphenol A-Human toxicity by 3D cell culture arrays, high throughput alternatives to animal tests.

Bisphenol A (BPA) has been widely used for manufacturing polycarbonate plastics and epoxy resins and has been extensively tested in animals to predict human toxicity. In order to reduce the use of animals for toxicity assessment and provide further accurate information on BPA toxicity in humans, we encapsulated Hep3B human hepatoma cells in alginate and cultured them in three dimensions (3D) on a micropillar chip coupled to a panel of metabolic enzymes on a microwell chip. As a result, we were able to assess the toxicity of BPA under various metabolic enzyme conditions using a high-throughput and micro assay; sample volumes were nearly 2,000 times less than that required for a 96-well plate. We applied a total of 28 different enzymes to each chip, including 10 cytochrome P450s (CYP450s), 10 UDP-glycosyltransferases (UGTs), 3 sulfotransferases (SULTs), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase 2 (ALDH2). Phase I enzyme mixtures, phase II enzyme mixtures, and a combination of phase I and phase II enzymes were also applied to the chip. BPA toxicity was higher in samples containing CYP2E1 than controls, which contained no enzymes (IC50, 184±16μM and 270±25.8μM, respectively, p<0.01). However, BPA-induced toxicity was alleviated in the presence of ADH (IC50, 337±17.9μM), ALDH2 (335±13.9μM), and SULT1E1 (318±17.7μM) (p<0.05). CYP2E1-mediated cytotoxicity was confirmed by quantifying unmetabolized BPA using HPLC/FD. Therefore, we suggest the present micropillar/microwell chip platform as an effective alternative to animal testing for estimating BPA toxicity via human metabolic systems.

Unnatural polyketide analogues selectively target the HER signaling pathway in human breast cancer cells.

Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin‐3‐galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N‐acetylcysteamine (SNAc) starter substrates and malonyl‐coenzyme A (CoA) and methylmalonyl‐CoA as extender substrates. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic‐2‐pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT‐474 cells resulted in identification of several trifluoromethylcinnamoyl‐based polyketides that showed strong suppression of the HER2‐associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF‐10A breast cells (IC50>100 μM). Specifically, 4‐trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC50<200 nM) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2 e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogues might prove to be useful drug candidates for potential breast cancer therapy.

Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.0) and an advanced metabolizing enzyme microarray (MetaChip 2.0). The DataChip is a functionalized micropillar chip that supports the Hep3B human hepatoma cell line in a 3D microarray format. The MetaChip is a microwell chip containing immobilized DMEs found in the human liver. As a proof of concept for generating compound metabolites in situ on the chip and rapidly assessing their toxicity, 22 model compounds were dispensed into the MetaChip and sandwiched with the DataChip. The IC50 values obtained from the chip platform were correlated with rat LD50 values, human Cmax values, and drug-induced liver injury categories to predict adverse drug reactions in vivo. As a result, the platform had 100% sensitivity, 86% specificity, and 93% overall predictivity at optimum cutoffs of IC50 and Cmax values. Therefore, the DataChip/MetaChip platform could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.