Arbitrarily Accessible 3D Microfluidic Device for Combinatorial High-Throughput Drug Screening

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 μM), Apicidin (10 μM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.

Dynamic microphysiological system chip platform for high-throughput, customizable, and multi-dimensional drug screening

The evolution of preclinical in vitro cancer models has led to the emergence of human cancer-on-chip or microphysiological analysis platforms (MAPs). Although it has numerous advantages compared to other models, cancer-on-chip technology still faces several challenges such as the complexity of the tumor microenvironment and integrating multiple organs to be widely accepted in cancer research and therapeutics. In this review, we highlight the advancements in cancer-on-chip technology in recapitulating the vital biological features of various cancer types and their applications in life sciences and high-throughput drug screening. We present advances in reconstituting the tumor microenvironment and modeling cancer stages in breast, brain, and other types of cancer. We also discuss the relevance of MAPs in cancer modeling and precision medicine such as effect of flow on cancer growth and the short culture period compared to clinics. The advanced MAPs provide high-throughput platforms with integrated biosensors to monitor real-time cellular responses applied in drug development. We envision that the integrated cancer MAPs has a promising future with regard to cancer research, including cancer biology, drug discovery, and personalized medicine.

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

Combination chemotherapies play an important role in clinical cancer treatment. We have previously developed a microfluidic high-throughput drug screening platform and used it for the screening of prostate cancer sensitizing agents. We have further developed the system into a fully automated live cell array platform with uniform cell seeding for better control, and used the platform to investigate the gene expression regulation of colorectal cancer cells in response to combination cancer drug treatment. The results showed significant drug effects on the proliferation of the HCT116 colorectal cancer cells, demonstrating the potential of this microfluidic device as a high-throughput combination chemotherapeutic drug-screening platform.

7060 Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and has an overall cure rate of approximately 60%. Previously, we observed high PIM1 mutation rates in DLBCL patients with poor outcome. However, the mechanism whether they lead to enhanced PIM1 kinase activity and contribute to lymphomagenesis is currently unknown. Methods: In this study, a multifaceted approach was employed to elucidate the functional consequences of PIM1 gene mutations and their implications in DLBCL. Recurrent PIM1 gene mutations that exhibited high frequencies across various lymphoma cohorts from public databases were screened and patient outcomes were stratified by PIM1 genen mutational sites. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis combined with Co-IP was used to identify proteins interacting with PIM1. Transcriptomics analysis was utilized to identify proteins involved in critical cellular pathways relevant to PIM1 mutation. A high-throughput drug screening platform was leveraged to find potential therapeutic vulnerabilities unique to PIM1 mutant cells. The antiproliferative effects of PIM1 and PI3K inhibitor were evaluated in DLBCL cell lines and further validated in NSG mouse xenograft models. Results: We have identified PIM1 mutations, specifically P81S, E135K, L184F, and S97N, as frequently occurring variants, with the former three significantly associated with poor outcome. In particular, the PIM1 L184F mutation promoted cell proliferation and inhibited cell apoptosis in vitro and showed faster tumor growth in vivo. Mechanistically, the PIM1 L184F mutation was found to interact with the annexin A2 (ANXA2) gene, activating it through phosphorylation of serine 26. The activated ANXA2 gene then translocated from the cytoplasm to the cell membrane, binding with the Toll-like receptor 4 (TLR4) gene and recruiting BCAP to the cell membrane to interact with p85α further activating the PI3K/AKT/mTOR signaling pathway. Additionally, the high-throughput drug screening demonstrated that the PIM1 L184F mutated cells were more sensitive to the PI3K inhibitor YY20394. PIM1 inhibitor SMI-4a combined with YY20394 showed synergistic antitumor effects both in vitro and in vivo. Conclusions: Taken together, these findings not only shed light on an innovative regulatory mechanism for how PIM1 L184F mutation contributes to the pathogenesis of DLBCL but also provide a potential therapeutic strategy for effectively managing DLBCL patients harboring PIM1 L184F mutation.

3D bioprinting for engineering complex tissues

BackgroundAmyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2–3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well suited for the study of diseasepathophysiology and also for high throughput (HT) drug screening. The mutant sod1 zebrafish model of ALS mimics the hallmark features of ALS. Using a fluorescence based readout of neuronal stress, we developed a high throughput (HT) screen to identify neuroprotective compounds.ResultsHere we show that the zebrafish screen is a robust system that can be used to rapidly screen thousands ofcompounds and also demonstrate that riluzole is capable of reducing neuronal stress in this model system. The screen shows optimal quality control, maintaining a high sensitivity and specificity withoutcompromising throughput. Most importantly, we demonstrate that many compounds previously failed in human clinical trials, showed no stress reducing activity in the zebrafish assay.ConclusionWe conclude that HT drug screening using a mutant sod1 zebrafish is a reliable model system which supplemented with secondary assays would be useful in identifying drugs with potential for neuroprotective efficacy in ALS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0122-3) contains supplementary material, which is available to authorized users.

Over the years, the field of drug discovery has seen significant progress, with technological advancements proving crucial in the identification of new therapeutic candidates. High-Throughput Screening (HTS) which combines large-scale data analysis with automated micro-quantitative experiments, and can rapidly analyse a large number of chemical compounds against the drug targets. This chapter explores the recent advancements in high-throughput screening platforms that significantly enhanced drug discovery process, and also includes the incorporation of miniaturization, automation, and image-based analysis to increase throughput and data quality. The significance of these advanced HTS platforms on accelerating the discovery and development of new therapeutic drugs is discussed, along with the challenges. This chapter, taken as a whole, will offer a comprehensive overview of the most advanced high-throughput screening (HTS) platforms available for use in the drug development industry and will address how they might completely transform the process.

Patient-derived tumor organoids (TOs) generated from tumor tissues within a few days are promising new models since under optimal conditions they preserve genotype, phenotype, the tumor microenvironment and the cellular heterogeneity of the parental tumor. This exciting new technology could provide representative avatars of the patient tumor in large numbers and thus can be exploited for drug discovery and drug screening purposes more efficiently than conventional cell lines or xenografted mouse models. We established standardized TOs from fresh glioblastoma (GBM) tissue after enzymatic dissociation into single cell suspensions and seeding cells into anti-adhesive 384-well plates. Over 1000 standardized TOs were generated per patient with a success rate of over 90%. Morphologically, TOs formed after two days. Size was stable over 10 days, with a slow outgrowth in some cases. A steady increase of the ATP signal over time indicated proliferative activity in TOs. Immunofluorescent stainings for tumor cells (GFAP), immune cells (macrophages and T-cells), and extracellular matrix (Tensascin C) confirmed a strong resemblance of TOs to their corresponding parental tumor, which was further substantiated by RNA-seq and DNA-methylation. Next, automated robot-based high-throughput drug screening (aHTS) on TOs was performed using a drug library of 166 FDA-approved antineoplastic drugs. Cell viability was assessed by CellTiter-Glo3D. In total, aHTS on TOs from 11 GBM patients including two recurrent GBMs showed a strong heterogeneity of drug responses. The top ten best-performing drugs consisted of 35 compounds targeting 14 different modes of action. Proteasome and HDAC-inhibitors demonstrated the highest efficacy. Respective targets were validated by knockdown experiments in GBM cells. Altogether, our data corroborate the future use of standardized GBM TOs as patient avatars for personalized drug testing by utilizing an automated high-throughput drug screening platform to better account for inter-patient-individual drug responses.

Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

An effective method to quantify the parasite loads is the key to the evaluation of anti-cryptosporidial drug efficacy in vitro. However, high-throughput screening (HTS) of drugs against Cryptosporidium parvum in vitro was impractical by the labor-intensive traditional assays. Here we describe a simplified quantitative RT-PCR assay suitable for HTS of compounds and for evaluating drug efficacy against the growth of C. parvum in vitro.

BackgroundMiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies.MethodsTFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs.ResultsThe results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination.ConclusionsThe results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

Background: A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. Currently, most assays for examining the steps of metastasis fail to incorporate the biophysical forces experienced by tumor cells due to blood flow, or are low throughput and therefore not amenable to drug screening and high throughput experimentation. Methods: We have developed a novel high throughput mesofluidic platform for assaying cell adhesion under flow in a 96-well format. This device functions like a cone and plate viscometer in each well by inducing shear stress on cells cultured in a standard 96-well plate. We validated the fluid flow and alignment of the device and studied the adhesion of cultured leukocytic monocytes (THP-1 cells) and multiple cancer cell lines (HCT116, MDA-MB-231 and MCF-7 cells) to purified extracellular matrix molecules (ECM), endothelial cells and immobilized platelets. All assays were carried out under flow (0.5 dynes/cm2 of shear stress) and static conditions. Results: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Treatment of breast cancer cells with a small library of integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells under static conditions, whereas under shear conditions many of these compounds reduced adhesion of cancer cells. In addition, a static adhesion assay of breast cancer cells to various types of ECM showed higher adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the MDA-MB-231 in comparison to the MCF-7 cell line. Finally, we performed a high throughput screening experiment using a kinase inhibitor library with 80 compounds and found that the shear based assay yielded notably different results from a similar screen under static conditions for cancer cell adhesion to endothelial cells, immune cell adhesion to endothelial cells and cancer cell adhesion to platelets. Conclusions: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Thus, this high-throughput screening platform may enable the development of novel compounds to inhibit cancer metastasis and facilitate the study of the systems level behavior of cancer-endothelium adhesion. Citation Format: Adrianne Shearer, Chris Spruell, Victoria Le, Mar Creixell, Seema Nandi, Aaron B. Baker. Mesofluidic platform for high throughput screening for inhibitors of metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-008. doi:10.1158/1538-7445.AM2015-LB-008

The Society of Biomolecular Imaging and Informatics First Annual Conference