Drug Discovery Assays Research Articles

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a “testing funnel” of assays to identify genuine hits using high‐throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Abstract 1511: Differential expression analysis and biomarker identification in five salivary gland carcinoma types

Abstract Significant morphologic overlap can exist between distinct salivary gland neoplasms, making differential diagnosis challenging, even with the use of new immunohistochemistry stains and FISH probes (Griffith et al., Arch Pathol Lab Med 2017). RNA-sequencing (RNAseq) technology is increasingly being used as a tool for identify diagnostic molecular signatures in a wide variety of diseases (Byron et al., Nat Rev Genet 2016). Furthermore, transcriptomics data are being used to develop biomarkers that can be used in drug discovery assays. Using an unbiased RNAseq analysis pipeline (Brayer et al., Cancer Discov 2016; Brown et al., PLos One 2017), we developed molecular profiles for 126 salivary gland tumors with accompanying clinical data–68 adenoid cystic carcinoma (ACC), 23 acinic cell carcinoma (Acinic), 5 basal cell adenoma (BCA), 20 basal cell adenocarcinoma (BCAC), and 10 secretory carcinoma (SC)–and have identified distinct gene expression patterns for each neoplasm. Additionally, a detailed analysis within each cancer type has revealed heterogeneous gene expression patterns, with altered signaling pathways, suggesting molecular subtypes exist. In both ACC and Acinic samples we identified unique molecular signatures found in patients with poor overall survival. Transcripts within each molecular signature that are independent of gender, age, and cancer stage, but correlated with clinical outcome data, are being identified for use as diagnostic or prognostic biomarkers and for use in drug discovery assays. Preliminary results to identify biomarkers for these neoplasms will also be presented. Citation Format: Kathryn J. Brayer, Yoshitsugu Mitani, Adel El-Naggar, Scott A. Ness. Differential expression analysis and biomarker identification in five salivary gland carcinoma types [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 1511.

Abstract 749: Antibody internalization assays for cancer drug discovery

Abstract Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used as anti-cancer therapeutics. A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmaco-dynamic profile. Quantifying and comparing the internalization rates of different Ab drug candidates and production batches is therefore a critical step in the biopharmaceutical selection and optimization process. Here, we describe novel and enabling cell-based Ab internalization assays that are turnkey, medium throughput and geared toward industrial biologics discovery. Internalization measurements are made over time on 96-well microplates using live-cell analysis (IncuCyte S3) and a fluorescent pH-sensitive dye coupled antibody-binding fragment (FabFluor) that binds the test mAb Fc region using a single step, no wash labeling protocol. An increase in fluorescence signal is observed as the mAb complex is internalized into the acidic lysosome. To validate this approach trastuzumab (Herceptin, Her-2) or rituximab (Rituxan, CD20) were mixed with the hFabFluor reagent (1: 3 molar ratio, 15min), serially diluted in complete media (1:2) and added to pre-plated BT-474 or Raji cells (no wash). Cell images (10-20x) were taken and automatically analysed for fluorescence area every 30min for up to 48h. Both trastuzumab (BT-474) and rituximab (Raji) caused clear time- and concentration-dependent internalization (EC50 values 2.1 and 2.6nM, respectively). The fluorescence signal was punctate, outside of the nucleus and strongly co-localized with a lysosomal marker (LysoSensor). In line with known marker expression profiles, specific internalization of mAbs to CD45, CD71 and CD3, but not CD20, was observed in Jurkat T-lymphocytes and CD45, CD71 and CD20, but not CD3 in Raji B cells. As a proof of concept for screening and direct comparison of test mAbs, 6 commercially available CD71 (transferrin receptor) Abs were labeled with mFabFluor reagent, serially diluted (1:2, 4.6-10000ng mL-1) and added to HT1080 osteosarcoma cells. 3 of the Abs produced a large internalization signal with detection less than 50ng mL-1, whilst the other 3 were internalized weakly with signal only visible at higher concentrations. A mean Z' value of 0.82 was calculated from control wells indicating a microplate assay with high precision and robustness. Taken together these data support the validation of a simple, integrated and quantitative solution for directly studying internalization of mAbs into cells which can easily be scaled to compare multiple Abs in parallel. This method enables mAb and ADC internalization measurements to be implemented at early stages of the biologics discovery process and will prove valuable in efficacy, safety and pharmacokinetic optimization. Citation Format: Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. Antibody internalization assays for cancer drug discovery [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 749.

Phenotypic Assays in Drug Discovery: a resurgence

Phenotypic Assays in Drug Discovery: a resurgence

Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells.

The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor's function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays.

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor's function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor's function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay's readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors

Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Fluorescence lifetime based assays in drug discovery

Fluorescence lifetime based assays in drug discovery

SIRT1 activator isolated from artificial gastric juice incubate of total saponins in stems and leaves of Panax ginseng

Panax ginseng as a traditional Chinese medicine has been extensively used for the treatment of many diseases, especially in prolonging life and anti-tumor. Dammarane-type triterpenoids from P. ginseng have diverse beneficial effects and their chemical structures can be modified in the gastrointestinal tract after oral administration. In this paper, the dammarane-type triterpenoids were isolated from artificial gastric juice incubate of total saponins in the stems and leaves of P. ginseng through column chromatographic methods and their chemical structures were determined based on spectral data. Two new dammarane-type triterpenoids named ginsenotransmetins B (1) and C (2), along with twenty-nine known compounds (3–31), were obtained. All 31 compounds isolated were investigated for their activities of SIRT1 using SIRT1 fluorometric drug discovery assay kit. Among them, compounds 11, 17, 18, 20, 23, 24, 28, and 29, which were found to be potential as SIRT1 activators, exhibited significant stimulation of SIRT1 activity. The results showed that these compounds may be considered to be a useful medicinal resource for prolonging life and anti-tumor. In addition, the results were helpful to explain the longevity effect of ginseng from the new field of view.

Effectiveness of Curcumin for Treating Cancer During Chemotherapy

Abstract Oncologists tend to avoid the use of supplements during chemotherapy. To understand the safety issue, a literature review was conducted to evaluate safety and possible benefits of taking curcumin during chemotherapy. Curcumin was chosen because of its wide use in the public and because of the availability of clinical trials utilizing curcumin during chemotherapy. Curcumin is considered a “pan-assay interference compound,” creating false leads in drug discovery assays. The pharmacodynamics of curcumin presents many challenges for a therapeutic agent, including poor bioavailability and rapid metabolism and excretion. The proposed molecular targets for curcumin include inhibition of nuclear factor kappa B and inhibition of cyclooxygenase-2. Clinical trials investigating the efficacy of curcumin treatment for cancer have been conducted in patients with colorectal cancer (CRC), pancreatic cancer, breast cancer, prostate cancer, multiple myeloma, lung cancer, and head and neck cancer. Outcomes revealed...

The Generation of Three-Dimensional Head and Neck Cancer Models for Drug Discovery in 384-Well Ultra-Low Attachment Microplates.

The poor success rate of cancer drug discovery has prompted efforts to develop more physiologically relevant cellular models for early preclinical cancer lead discovery assays. For solid tumors, this would dictate the implementation of three-dimensional (3D) tumor models that more accurately recapitulate human solid tumor architecture and biology. A number of anchorage-dependent and anchorage-independent in vitro 3D cancer models have been developed together with homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth, and viability. However, several significant technical challenges have restricted the implementation of some 3D models in HTS. We describe a method that uses 384-well U-bottomed ultra-low attachment (ULA) microplates to produce head and neck tumor spheroids for cancer drug discovery assays. The production of multicellular head and neck cancer spheroids in 384-well ULA-plates occurs in situ, does not impose an inordinate tissue culture burden for HTS, is readily compatible with automation and homogeneous assay detection methods, and produces high-quality uniform-sized spheroids that can be utilized in cancer drug cytotoxicity assays within days rather than weeks.

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

Sturge-Weber Syndrome (SWS) is a neurocutaneous disease with clinical manifestations including ocular (glaucoma), cutaneous (port-wine birthmark), neurologic (seizures), and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against its progression. Development of such platforms of bioassay can bring along the implementation of high-throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of molecular targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholinesterase, alkaline phosphatase, GABAergic receptors, Hypoxia-Inducible Factor (HIF)-1α and 2α are suggested.

A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine

N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, making them attractive therapeutic targets. Overexpression of functional NMDAR in non-neuronal cells results in cell death by excitotoxicity, hindering the development of cell-based assays for NMDAR drug discovery. Here we report a plate-based, high-throughput approach to study NMDAR function. Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. This allows to investigate the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor’s sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.

Elucidating Substrate Promiscuity within the FabI Enzyme Family.

The rapidly growing appreciation of enzymes' catalytic and substrate promiscuity may lead to their expanded use in the fields of chemical synthesis and industrial biotechnology. Here, we explore the substrate promiscuity of enoyl-acyl carrier protein reductases (commonly known as FabI) and how that promiscuity is a function of inherent reactivity and the geometric demands of the enzyme's active site. We demonstrate that these enzymes catalyze the reduction of a wide range of substrates, particularly α,β-unsaturated aldehydes. In addition, we demonstrate that a combination of quantum mechanical hydride affinity calculations and molecular docking can be used to rapidly categorize compounds that FabI can use as substrates. The results here provide new insight into the determinants of catalysis for FabI and set the stage for the development of a new assay for drug discovery, organic synthesis, and novel biocatalysts.

Abstract 5929: A novel image cytometric analysis method for T-cell-mediated cytotoxicity of 3D tumor spheroids

Abstract Cell-mediated cytotoxicity assays have been frequently performed to characterize cancer cytotoxic potential of immune cells, antibodies, and drug compounds. Traditionally, these assays are performed using release assays such as Cr51 (radioactivity), Calcein (fluorescence), or LDH (enzymatic). However, release assays have limitations such as the handling of hazardous material, the indirect measurement of cell death leading to an under estimation of cytotoxicity, the requirement for a large volume of cell sample, and the inability to visually confirm, image ad track the assay kinetically. In the recent years, Celigo image cytometry has been used to perform high-throughput cell-mediated cytotoxicity assays using a direct cell counting method where cancer cells (Target) are stained with Calcein AM. Upon the addition of effector immune cells with the fluorescently stained Target cells in the presence or absence of antibody or drug compounds, the Celigo image cytometer is used to capture bright-field and fluorescent images and analyze the change in Target cell count over time to determine the cytotoxicity percentage. In general, these assays are performed in a 2D cultures. The 2D assays for cancer drug discovery have had some difficulty in identifying more qualified drug candidates for clinical testing, thus there has been an increase in interest of performing cytotoxicity assays in 3D tumor models. Traditional 3D spheroid analysis methods require the use of standard microscopy, which is time-consuming and subjective. Celigo imaging cytometer has also been used to rapidly analyze drug effects on 3D tumor spheroids. In this work, we demonstrate a novel method of analyzing T cell-mediated cytotoxicity on 3D tumor spheroids in the presence of absence of ImmTAC molecules, which can promote higher T cell killing. In this experiment, MDA-MB-453 GFP expressing breast cancer cells are used to form tumor spheroids in an ultra-low attachment plate. The spheroids are then treated with primary T cells at 1:10 and 1:50 E:T ratios, as well as 10, 1, 0.1, 0.01, and 0.001 nM ImmTAC. The results showed a dose response effect of T cell killing with the addition of ImmTAC molecules by measuring spheroid size in GFP fluorescence and viability using propidium iodide. The image cytometry method was able to monitor changes in spheroid size and fluorescence over time and rapidly quantify T cell killing, which was as high as 75%. The captured bright-field and fluorescent images also clearly showed the cytotoxicity effect from the combination of T cells and ImmTAC. The ability to screen cytotoxic effects of immune cells, antibody, and drug compounds on 3D tumor spheroids can provide an alternative tumor model for identifying more qualified cancer drug candidates for drug discovery campaigns. Citation Format: Leo Li-Ying Chan, Laure Humbert, Scott Cribbes. A novel image cytometric analysis method for T-cell-mediated cytotoxicity of 3D tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5929. doi:10.1158/1538-7445.AM2017-5929

MAPPING PROTEIN-PROTEIN INTERACTIONS BY PEPTIDE ARRAY FOR DISRUPTOR PEPTIDE DEVELOPMENT AND TRANSLATIONAL IMPACT!

Aberrant cellular signalling is central to the pathogenesis of a number of neurodegenerative disorders. Our group focuses in particular on the role of Wnt signalling in β-amyloid toxicity, and have demonstrated not only that aberrant Wnt activation, APP proteolysis and synapse loss are intimately linked, but have identified the key protein-protein interactions responsible. A fundamental aspect of our pathway-driven approach is the mapping and disruption of protein-protein interactions using peptide array technology. Peptide arrays are created from peptide libraries consisting of overlapping peptides spanning the entire sequence of the protein of interest and spotted onto cellulose coated slides. These peptide arrays can be overlaid with putative binding partners to identify potential binding regions or novel post-translational modification sites. Key amino acid residues can be rapidly identified by alanine/ single substitution scanning. Crucially, we can delineate the minimal “core” binding regions, which can be synthesised and used as cell permeable “disruptor peptides” capable of selectively blocking specific protein-protein interactions. Furthermore, disruptor peptides can be used as the basis of high throughput biochemical and cell based assays for drug discovery. We wish to showcase the power of this technology, using specific examples from our own work on Alzheimer's disease, and demonstrate its capabilities, which are readily amenable across a broad range of applications, to provide useful research tools in dissecting specific signalling events. Mapping and disrupting protein-protein interactions by peptide array has significant potential to provide translational outcomes for neurodegenerative disease.

Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.