Analysis of acute lymphoblastic leukemia drug sensitivity by changes in impedance via stromal cell adherence.

The bone marrow is a frequent location of primary relapse after conventional cytotoxic drug treatment of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Because stromal cells have a major role in promoting chemotherapy resistance, they should be included to more realistically model in vitro drug treatment. Here we validated a novel application of the xCELLigence system as a continuous co-culture to assess long-term effects of drug treatment on BCP-ALL cells. We found that bone marrow OP9 stromal cells adhere to the electrodes but are progressively displaced by dividing patient-derived BCP-ALL cells, resulting in reduction of impedance over time. Death of BCP-ALL cells due to drug treatment results in re-adherence of the stromal cells to the electrodes, increasing impedance. Importantly, vincristine inhibited proliferation of sensitive BCP-ALL cells in a dose-dependent manner, correlating with increased impedance. This system was able to discriminate sensitivity of two relapsed Philadelphia chromosome (Ph) positive ALLs to four different targeted kinase inhibitors. Moreover, differences in sensitivity of two CRLF2-drivenBCP-ALL cell lines to ruxolitinib were also seen. These results show that impedance can be used as a novel approach to monitor drug treatment and sensitivity of primary BCP-ALL cells in the presence of protective microenvironmental cells.

In vitro immunotherapy potency assays using real-time cell analysis.

A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes.

The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs) and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP) kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl), an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs). The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.

High throughput physiological screening of iPSC-derived cardiomyocytes for drug development.

Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

High Throughput Measurement of Ca++ Dynamics in Human Stem Cell-Derived Cardiomyocytes by Kinetic Image Cytometery: A Cardiac Risk Assessment Characterization Using a Large Panel of Cardioactive and Inactive Compounds.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

DUSP3/VHR is a pro-angiogenic atypical dual-specificity phosphatase.

BACKGROUND: DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies showed that DUSP3 is a negative regulator of ERK and JNK pathways in several cell lines. On the other hand, DUSP3 is implicated in human cancer. It has been alternatively described as having tumor suppressive and oncogenic properties. Thus, the available data suggest that DUSP3 plays complex and contradictory roles in tumorigenesis that could be cell type-dependent. Since most of these studies were performed using recombinant proteins or in cell-transfection based assays, the physiological function of DUSP3 has remained elusive. RESULTS: Using immunohistochemistry on human cervical sections, we observed a strong expression of DUSP3 in endothelial cells (EC) suggesting a contribution for this phosphatase to EC functions. DUSP3 downregulation, using RNA interference, in human EC reduced significantly in vitro tube formation on Matrigel and spheroid angiogenic sprouting. However, this defect was not associated with an altered phosphorylation of the documented in vitro DUSP3 substrates, ERK1/2, JNK1/2 and EGFR but was associated with an increased PKC phosphorylation. To investigate the physiological function of DUSP3, we generated Dusp3-deficient mice by homologous recombination. The obtained DUSP3-/- mice were healthy, fertile, with no spontaneous phenotype and no vascular defect. However, DUSP3 deficiency prevented neo-vascularization of transplanted b-FGF containing Matrigel and LLC xenograft tumors as evidenced by hemoglobin (Hb) and FITC-dextran quantifications. Furthermore, we found that DUSP3 is required for b-FGF-induced microvessel outgrowth in the aortic ring assay. CONCLUSIONS: All together, our data identify DUSP3 as a new important player in angiogenesis.

High throughput measurement of Ca²âº dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry.

Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca²âº indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca²âº](i) at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca²âº dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.

Multidentate small-molecule inhibitors of vaccinia H1-related (VHR) phosphatase decrease proliferation of cervix cancer cells.

Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.

Cell-type specific and cytoplasmic targeting of PEGylated carbon nanotube-based nanoassemblies.

In this paper we report the fabrication of a multivalent, cell-type specific and cytoplasmic delivery system based on single-walled carbon nanotubes. The latter were functionalized through adsorption of phospholipids terminated by biotinylated PEG chains functionalized with fluorochrome-coupled neutravidin, and subsequently with antibodies (anti-CD3epsilon and anti-CD28) for T cell receptor post-signaling endocytosis and a synthetic fusogenic polymer for disruption of lysosomal compartments. The biomimetic nanoassemblies were composed by PEGylated individual/very small bundles of carbon nanotubes having an average length and a standard deviation of 176 nm and 77 nm, respectively. The nanoassemblies were stably dispersed under physiological conditions, visible by conventional optical and confocal microscopy and specifically targeted to T cells both in vitro and in living animals. The addition of a fusogenic polymer to the nanoassemblies did not affect the cellular uptake and allowed the release into the cytosol of the targeted cells both in vitro and in the animals. The present manuscript is the first report about the cytoplasmic delivery of carbon nanotubes in a specific cell type in intact animals and paves the way for their use as in vivo intracellular delivery systems.

SKAP55 modulates T cell antigen receptor-induced activation of the Ras-Erk-AP1 pathway by binding RasGRP1.

Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is an adapter protein with an N-terminal region, a pleckstrin homology domain, a linker with tyrosine phosphorylation sites, and a C-terminal Src homology 3 domain. We report that overexpression of SKAP55 disrupts signaling from the TCR to the Ras-Erk-AP-1 pathway and transcription of the IL-2 gene in primary human T cells and in Jurkat T leukemia cells. In contrast, moderate overexpression of SKAP55 increased TCR-dependent AP-1 transcriptional activity, suggesting that high-level SKAP55 overexpression interfered with the assembly of functional signaling complexes required for TCR coupling to the Ras pathway. In support of this view, knock-down of SKAP55 by RNA interference resulted in decreased reporter gene activation and decreased ERK phosphorylation. In contrast, TCR-induced NF-kappaB activation was not affected. Since constitutively active forms of Ras or Raf-1 overcame the inhibitory effects of SKAP55 overexpression, we searched for a mechanism upstream of Ras and found that SKAP55 co-immunoprecipitated with the Ras activator RasGRP1. The binding of RasGRP1 to SKAP55 required the C-terminus of SKAP55 and was enhanced by tyrosine phosphorylation of SKAP55. These results suggest that SKAP55 modulates signal transduction from the TCR to Ras by binding to RasGRP1.

Cutting edge: selective tyrosine dephosphorylation of interferon-activated nuclear STAT5 by the VHR phosphatase.

Cytokine-induced tyrosine phosphorylation of the transcription factor STAT5 is required for its transcriptional activity. In this article we show that the small dual-specificity phosphatase VHR selectively dephosphorylates IFN-alpha- and beta-activated, tyrosine-phosphorylated STAT5, leading to the subsequent inhibition of STAT5 function. Phosphorylation of VHR at Tyr(138) was required for its phosphatase activity toward STAT5. In addition, the Src homology 2 domain of STAT5 was required for the effective dephosphorylation of STAT5 by VHR. The tyrosine kinase Tyk2, which mediates the phosphorylation of STAT5, was also responsible for the phosphorylation of VHR at Tyr(138).

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes.

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

Luminescent silica nanobeads: characterization and evaluation as efficient cytoplasmatic transporters for T-lymphocytes.

We report the fabrication and characterization of neutravidin-conjugated silica nanobeads doped with a ruthenium-complex luminophore and functionalized with antihuman CD3, antihuman CD28, and an acid-sensitive polymer. We observed that the nanobeads were readily delivered into Jurkat T leukemia cells by endocytosis, transported into lysosomes and subsequently into the cytoplasm as revealed by pH-sensitive luminescence. Since signs of cytotoxicity were not observed, the reported nanobeads could be an excellent and nontoxic building block for efficient intracellular transporters.

Identification and characterization of DUSP27, a novel dual-specific protein phosphatase.

A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4-14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism.

Regulation of MAP kinases by the VHR dual-specific phosphatase: implications for cell growth and differentiation.

Although it is well established that a transient activation of the mitogen-activated protein kinases Erk and Jnk is a crucial step in most growth promoting signaling pathways, it has also been demonstrated that a prolonged activation of these kinases can induce differentiation, cell cycle arrest, and cell senescence. We recently found that the expression of the 21-kDa human Vaccinia H1-related (VHR) dual-specific phosphatase fluctuates during cell cycle progression and affects Erk and Jnk activity in a cell cycle-dependent manner. Cells lacking VHR arrested at the G(1)/S and G(2)/M transitions of the cell cycle and exhibited senescence phenotypes. Cells lacking VHR upregulated p21(Cip/Waf1) and downregulated many genes for cell cycle regulators, DNA replication, transcription, and mRNA processing. In the absence of VHR, the serum-induced activation of Jnk and Erk was further elevated and was required for the G(1)/S and G(2)/M blocks, which were attenuated upon Jnk and Erk inhibition. Collectively, VHR provides a long sought layer in the regulation of Jnk and Erk during cell cycle progression thereby contributing to cell cycle arrest, differentiation or senescence.

Full-length single-walled carbon nanotubes decorated with streptavidin-conjugated quantum dots as multivalent intracellular fluorescent nanoprobes.

We report the formation of a supramolecular luminescent nanoassembly composed of individual or small ropes of full-length, single-walled carbon nanotubes decorated with streptavidin-conjugated quantum dots. The supramolecular luminescent nanoassembly was stably dispersed under physiological conditions and was readily visible by both optical and confocal fluorescent microscopies. Jurkat T leukemia cells were able to internalize the nanoassembly by multivalent CD3 receptor-mediated endocytosis (adsorption by cell). Once internalized by cells, the nanoassembly was found to be transported to lysosomes. These properties should make this supramolecular luminescent nanoassembly an excellent building block for the construction of intracellular polyvalent nanoprobes, mimicking natural viral delivery entities with enhanced loading capacity compared to small molecules.

Loss of the VHR dual-specific phosphatase causes cell-cycle arrest and senescence.

Protein tyrosine phosphatases regulate important processes in eukaryotic cells and have critical functions in many human diseases including diabetes to cancer. Here, we report that the human Vaccinia H1-related (VHR) dual-specific protein tyrosine phosphatase regulates cell-cycle progression and is itself modulated during the cell cycle. Using RNA interference (RNAi), we demonstrate that cells lacking VHR arrest at the G1-S and G2-M transitions of the cell cycle and show the initial signs of senescence, such as flattening, spreading, appearance of autophagosomes, beta-galactosidase staining and decreased telomerase activity. In agreement with this notion, cells lacking VHR were found to upregulate p21(Cip-Waf1), whereas they downregulated the expression of genes for cell-cycle regulators, DNA replication, transcription and mRNA processing. Loss of VHR also caused a several-fold increase in serum-induced activation of its substrates, the mitogen-activated protein (MAP) kinases Jnk and Erk. VHR-induced cell-cycle arrest was dependent on this hyperactivation of Jnk and Erk, and was reversed by Jnk and Erk inhibition or knock-down. We conclude that VHR is required for cell-cycle progression as it modulates MAP kinase activation in a cell-cycle phase-dependent manner.

Adsorption of streptavidin onto single-walled carbon nanotubes: application in fluorescent supramolecular nanoassemblies.

We report the solubilization of full-length single-walled carbon nanotubes into a physiological buffer by sonication in presence of streptavidin. Transmission electron microscopy showed that the resultant dispersion was enriched of individual/small ropes of nanotubes. By the analysis of the crystal structure of tetrameric streptavidin and of the tryptophan emission of adsorbed proteins we hypothesized that proteins adsorbed onto SWNT sidewalls through their amine functionalities. Our results suggested using streptavidin as an interlinker between carbon nanotubes and semiconducting nanocrystals. We fabricated a supramolecular luminescent nanoassembly composed of individual or small ropes of full-length single-walled carbon nanotubes decorated with streptavidin-conjugated quantum dots. The luminescent nanoassembly was stably dispersed under physiological conditions and was readily visible by optical fluorescent microscopies.

High mobility group A1 is a molecular target for MYCN in human neuroblastoma.

High mobility group A1 (HMGA1) is an architectural transcription factor and a putative protoncogene. Deregulation of its expression has been shown in most human cancers. We have previously shown that the expression of the HMGA family members is deregulated in neuroblastoma cell lines and primary tumors. On retinoic acid (RA) treatment of MYCN-amplified neuroblastoma cell lines, HMGA1 decreases with a kinetics that strictly follows MYCN repression. In addition, MYCN constitutive expression abolishes HMGA1 repression by RA. Here we explored the possibility that HMGA1 expression might be sustained by MYCN in amplified cells. Indeed, MYCN transfection induced HMGA1 expression in several neuroblastoma cell lines. HMGA1 expression increased in a transgene dose-dependent fashion in neuroblastoma-like tumors of MYCN transgenic mice. In addition, it was significantly more expressed in MYCN-amplified compared with MYCN single-copy primary human neuroblastomas. MYCN cotransfection activated a promoter/luciferase reporter containing a 1,600 bp region surrounding the first three transcription start sites of the human HMGA1 and eight imperfect E-boxes. By heterodimerizing with its partner MAX, MYCN could bind to multiple DNA fragments within the 1,600 bp. Either 5' or 3' deletion variants of the 1,600 bp promoter/luciferase reporter strongly decreased luciferase activity, suggesting that, more than a single site, the cooperative function of multiple cis-acting elements mediates direct HMGA1 transactivation by MYCN. Finally, HMGA1 repression by RNA interference reduced neuroblastoma cell proliferation, indicating that HMGA1 is a novel MYCN target gene relevant for neuroblastoma tumorigenesis.