Label-free single cell kinetics of the invasion of spheroidal colon cancer cells through 3D Matrigel.

This article reports label-free, real-time, and single-cell quantification of the invasion of spheroidal colon cancer cells through three-dimensional (3D) Matrigel using a resonant waveguide grating (RWG) imager. This imager employs a time-resolved swept wavelength interrogation scheme to monitor cell invasion and adhesion with a temporal resolution up to 3 s and a spatial resolution of 12 µm. As the model system, spheroids of human colorectal adenocarcinoma HT-29 cells are generated by culturing the cells in 96-well round-bottom ultralow attachment plates. 3D Matrigel is formed by its gelation in 384-well RWG biosensor microplates. The invasion and adhesion of spheroidal HT29 cells is initiated by placing individual spheroids onto the Matrigel-coated biosensors. The time series RWG images are obtained and used to extract the optical signatures arising from the adhesion after the cells are dissociated from the spheroids and invade through the 3D Matrigel. Compound profiling shows that epidermal growth factor accelerates cancer cell invasion, while vandetanib, a multitarget kinase inhibitor, dose-dependently inhibits invasion. This study demonstrates that the label-free imager can monitor in real-time the invasion of spheroidal cancer cells through 3D matrices.

Innovative fixed bed bioreactor platform: Enabling linearly scalable adherent cell biomanufacturing with real-time biomass prediction from nutrient consumption.

Scalable single-use adherent cell-based biomanufacturing platforms are essential for unlocking the full potential of cell and gene therapies. The primary objective of this study is to design and develop a novel fixed bed bioreactor platform tailored specifically for scaling up adherent cell culture. The bioreactor comprises a packed bed of vertically stacked woven polyethylene terephthalate mesh discs, sandwiched between two-fluid guide plates. Leveraging computational fluid dynamics modeling, we optimized bioreactor design to achieve uniform flow with minimal shear stress. Residence time distribution measurements demonstrated excellent flow uniformity with plug flow characteristics. Periodic media sampling coupled with offline analysis revealed minimal gradients of crucial metabolites (glucose, glutamine, lactate, and ammonia) across the bioreactor during cell growth. Furthermore, the bioreactor platform demonstrated high performance in automated cell harvesting, with ≈96% efficiency and ≈98% viability. It also exhibited linear scalability in both operational parameters and performance for cell culture and adeno-associated virus vector production. We developed mathematical models based on oxygen uptake rates to accurately predict cell growth curves and estimate biomass in real-time. This study demonstrates the effectiveness of the developed fixed-bed bioreactor platform in enabling scalable adherent cell-based biomanufacturing with high productivity and process control.

GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis.

G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy and cancerous cells. However, little is known about the response of cytosolic metabolic pathways to GPCR-mediated signaling. Here we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. Through a library screen of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gα(i)-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.

Resonant waveguide grating biosensor for whole-cell GPCR assays.

Current drug discovery campaigns for G protein-coupled receptors (GPCRs) heavily rely on assay technologies that use artificial cell systems tailored to a point-of-contact readout and as a consequence are mostly pathway biased. Recently, we have developed label-free optical biosensor cellular assays that are capable of examining systems cell biology of endogenous receptors and systems cell pharmacology of GPCR ligands in both physiologically and disease relevant environments. We have shown that these biosensor assays enable high-throughput screening of pathway-biased ligands acting on endogenous beta(2)-adrenergic receptor in cells. These biosensor cellular assays hold the potential to reduce attrition rates in drug discovery and development process.

Label-free optical biosensor for ligand-directed functional selectivity acting on beta(2) adrenoceptor in living cells.

Recent realization of ligand-directed functional selectivity demands high-resolution tools for studying receptor biology and ligand pharmacology. Here we use label-free optical biosensor to examine the dynamic mass redistribution of human epidermoid A431 cells in response to diverse beta(2)-adrenoceptor ligands. Multi-parameter analysis reveals distinct patterns in activation and signaling of the receptor induced by different agonists. Sequential and co-stimulation assays categorize various ligands for their ability to modulate signaling induced by catechol, a structural component of catecholamines. This study documents multiple ligand-specific states of the beta(2)-adrenoceptor and highlights the power of the biosensor assays for screening pathway-biased ligands.

Cellular functions of cholesterol probed with optical biosensors.

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.

Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells.

BACKGROUND: Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown. RESULTS: Here, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR1) and 2 (PAR2) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR1 and PAR2 agonists, but neither PAR3 nor PAR4 agonists, trigger dose-dependent Ca2+ mobilization as well as Gq-type DMR signals. Both Ca2+ flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR1 and PAR2 exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca2+ flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities. CONCLUSION: Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR1 and PAR2 in A431.

Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells.

INTRODUCTION: Screening drugs against G protein-coupled receptors (GPCRs) - the single largest family of drug targets in the human genome - is still a major effort in pharmaceutical and biotech industries. Conventional cell-based assays generally measure a single cellular event, such as the generation of a second messenger or the relocation of a specific protein target. However, manipulation or engineering of cells is often a prerequisite for these technologies to achieve desired sensitivities. The present study is focused on the use of non-invasive and manipulation-free optical biosensors for assaying endogenous GPCRs in adherent cells. METHODS: Resonant waveguide grating (RWG) biosensor was applied to manifest ligand-induced dynamic mass redistribution (DMR) within the bottom portion of adherent cell layer. The DMR signatures mediated through the activation of several endogenous GPCRs in cells were characterized. Endogenous receptor panning was examined at cell system level by using a panel of agonists known to activate many GPCRs, and also at family receptor level by determining the efficacies of a set of family-specific agonists. RESULTS: Three major types of optical signatures were identified; each was correlated with the activation of a class of GPCRs, depending on the G protein with which the receptor is coupled (i.e., G(q), G(s) and G(i)). The characteristics of DMR signals, mostly the amplitude and kinetics of a DMR event, were dependent on the doses of agonists and the expression levels of endogenous receptors. All three classes of endogenous receptors were found in human epidermoid carcinoma A431 cells. Interestingly, the dose-dependent switching from one type of DMR signal to another was observed for several GPCR agonists examined. A small panel of P2Y receptor agonists exhibited distinct efficacies in three cell lines examined. DISCUSSIONS: The RWG biosensors were applicable to study the activation of endogenous GPCRs. Like second messengers or gene expression, the DMR signals obtained could be considered as novel and quantifiable physiological responses of living cells mediated through GPCRs and used for studying receptor biology.

Intensity interrogation near cutoff resonance for label-free cellular profiling.

We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

Probing cytoskeleton modulation by optical biosensors.

This paper reported the use of resonant waveguide grating biosensors for studying the cytoskeleton structure in cells. This was achieved by measuring the changes in mass within the bottom portion of cells upon exposure to saponin in the absence and presence of cytoskeleton modulators. Treatment of Chinese hamster ovary cells with saponin led to a dose-dependent and dynamic mass changes. When a higher concentration of saponin (> 60 microg/ml) was used, a net loss in mass was observed. This is probably resulted from the diffusion of soluble intracellular materials away from the bottom portion of cells after pore formation in the cell plasma membranes by saponin. The pretreatment of cells with actin disruption agents, cytochalasin B and latrunculin A, led to significantly increased loss in cell mass induced by either 75 or 125 microg/ml saponin. These results suggested that optical biosensors provide an attractive means to study the cytoskeleton structure and screen modulators that affect the cytoskeleton structure.

Label-free functional selectivity assays.

G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the ß2-adrenergic receptor (ß2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of ß2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the ß2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

Divergent label-free cell phenotypic pharmacology of ligands at the overexpressed ß2-adrenergic receptors.

We present subclone sensitive cell phenotypic pharmacology of ligands at the ß2-adrenergic receptor (ß2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged ß2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled ß2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

A label-free optical biosensor with microfluidics identifies an intracellular signalling wave mediated through the ß(2)-adrenergic receptor.

The canonical model of G protein-coupled receptor (GPCR) signalling states that it is solely initiated at the cell surface. In recent years, a handful of evidence has started emerging from high-resolution molecular assays that the internalized receptors can mediate the third wave of signalling, besides G protein- and ß-arrestin-mediated signalling both initiating at the cell surface. However, little is known about the functional consequences of distinct waves of GPCR signalling, in particular, at the whole cell system level. We here report the development of label-free biosensor antagonist reverse assays and their use to differentiate the signalling waves of an endogenous ß2-adrenergic receptor (ß2-AR) in A431 cells. Results showed that the persistent agonist treatment activated the ß2-ARs, leading to a long-term sustained dynamic mass redistribution (DMR) signal, a whole cell phenotypic response. Under the persistent treatment scheme in microplates, a panel of known ß-blockers all dose-dependently and completely reversed the DMR signal of epinephrine at a relatively low dose (10 nM), except for sotalol which partially reversed the DMR. Under the perfusion conditions with microfluidics, the subsequent perfusion with sotalol only reversed the DMR induced by epinephrine or isoproterenol at 10 nM, but not at 10 µM. Furthermore, the degree of the DMR reversion by sotalol was found to be in an opposite relation with the duration of the initial agonist treatment. Together, these results suggest that the hydrophilic antagonist sotalol is constrained outside the cells throughout the assays, and the early signalling wave initiated at the cell surface dominates the DMR induced by epinephrine or isoproterenol at relatively low doses, while a secondary and late signalling wave is initiated once the receptors are internalized and contributes partially to the long-term sustainability of the DMR of epinephrine or isoproterenol at high doses.

Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists.

We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango ß-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism.

Dynamic mass redistribution assay decodes differentiation of a neural progenitor stem cell.

Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.

Label-free integrative pharmacology on-target of drugs at the ß(2)-adrenergic receptor.

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the ß(2)-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

Agonist-directed desensitization of the ß2-adrenergic receptor.

The ß(2)-adrenergic receptor (ß(2)AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the ß(2)AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective ß(2)AR desensitization at the whole cell level.

Discovery of 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as G protein-coupled receptor 35 (GPR35) agonists.

Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC(50) of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango ß-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.

Resonant waveguide grating imager for live cell sensing.

We report on a resonant waveguide grating imager for high throughput screening using live cells. This imager can generate a snapshot image of all biosensors in a 384-well microtiter plate with a time resolution of ∼3 s and a spatial resolution of 80 µm. This imager is well tolerant to variability in plate configurations and cell confluency. The resonant wavelength and its shifts induced by cell responses at each pixel correlate well with cell confluency. Data filtration protocol can be used to improve assay quality for partially confluent cells.

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics.

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.