Axion Biosystems (United States)

Abstract Introduction: Harnessing the potent cytotoxicity of chimeric antigen receptor T (CAR-T) cells has revolutionized cancer immunotherapy. A key factor influencing CAR-T cell efficacy is target antigen density on cancer cells. Human epidermal growth factor receptor 2 (HER2), overexpressed in various cancers like ovarian and lung carcinomas, is a promising target for CAR-T cell therapy. Traditional cytotoxicity assays generally provide only static endpoint measurements and often rely on 2D cell culture models, which fail to capture the structural complexity of tumors in vivo—a factor crucial to CAR-T cell efficacy. Live-cell imaging offers a solution by enabling non-invasive, real-time monitoring of cytotoxicity. Therefore, this study aims to explore the dynamics of CAR-T cell-mediated cytotoxicity against cancer cells with varying HER2 expression levels, comparing outcomes between 2D and 3D cell culture models using the Omni platform. Methods: Monolayers and spheroids of SKOV3 (ovarian carcinoma) and A549 (lung adenocarcinoma) cells, tagged with GFP, were exposed to HER2 CAR-T cells at different Effector : Target (E:T) ratios. Using the Omni platform, hourly brightfield and fluorescent images were captured over 72 hours assess cancer cell growth and cytotoxicity and quantify the cytolysis of the fluorescent target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent intensity of treated wells to no treatment control wells. Results: The study demonstrated a dose-dependent cytotoxic effect of HER2 CAR-T cells that correlated with increasing amounts of CAR-T cells. Comparing SKOV3 cells with A549 cells showed higher sensitivity and earlier response for all E:T ratios. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells, while it demonstrated 40.3% cytolysis of A549 cells. In comparing the cytotoxic effect of HER2 CAR-T cells on SKOV3 spheroids versus monolayers, the 3D spheroid model exhibited a more rapid and efficient dose-dependent response. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells in spheroid, while it demonstrated 61.1% cytolysis of SKOV3 cells in monolayer. Conclusion: This study underscores the importance of target antigen density in determining CAR-T cell efficacy, as SKOV3 cells, with higher HER2 expression, were more prone to CAR-T cell killing compared to A549 cells. Additionally, it also highlights the critical role of the assay model type, 2D versus 3D, in accurately assessing cytotoxicity. The Omni platform provided dynamic, real-time insights into cell viability and cytotoxicity, highlighting its potential to optimize immunotherapy strategies. Citation Format: Skylar Dewees, Nathalie Opdam - van de Laar, Stacie Chvatal, Danielle Califano, Ben Streeter. Exploring CAR-T cell killing dynamics through live-cell fluorescence microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3908.

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer that has no effective treatments and a prognosis of only 12-18 months. Immune effector T cells are a promising therapy due to their innate cytotoxicity. In addition, engineering chimeric antigen receptors (CAR) to target tumor-associated or neo-antigens can lend high specificity. The ability to assess the efficacy and potency of such T cell therapies in vitro at high throughputs is vital for the preclinical development of these promising therapies. Cellular impedance assays offer a sensitive, non-destructive, and label-free method to continuously monitor cancer cell proliferation and immune cell-mediated cytotoxicity in real-time, revealing not only the potency but also the kinetics of T cell killing. Here, we used co-culture to compare the cytolytic potency and kinetics of naïve activated T Cells and targeted GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells. Patient-derived N08 GSCs were plated at 50k cells per well on a PEI and laminin-coated CytoView-Z 96-well plates, and their impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). After 48 hours, human naïve activated T Cells (ImmunoCult CD3/CD28 activation media) or GD2 CAR-T cells were added at varying effector:target ratios ranging from 0.1:1 to 10:1. Impedance and cytolysis were subsequently monitored for up to 7 days. The addition of activated T Cells or GD2 CAR-T Cells resulted in cell swelling, followed by a decrease in impedance consistent with T cell-mediated lysis of GSCs. GD-2 CAR-T Cells resulted in a significantly higher percent cytolysis of GSCs compared to naïve activated T Cells after 7 days of exposure. In addition, the kill time 50, defined as the time to reach 50% cytolysis of target cells, was shorter for GD-2 CAR-T Cells compared to naïve activated T Cells. Cytotoxic function was validated with subsequent flow cytometry and cytokine analysis. After 7 days in co-culture, GD2 CAR-T cells exhibited markers of chronic activation, including greater CD8 expression than CD4, upregulation of CD69 (33% of cells), and induction of GrB (70%). Initial exhaustion was suggested by expression of PD1 (80% cells) and LAG3 (35%), but not TIM3. Overall, both naïve activated T-Cells and GD2 CAR-T Cells were effective for cytolysis of GSCs, with CAR-T exhibiting more efficient killing. CAR-T Cells engineered to target the GD2 antigen exhibited stronger potency and faster kinetics, suggesting greater clinical potential against glioblastoma. Citation Format: Heather Brant Hayes, Meghan T. Logun, Stacie A. Chvatal, Katie Mueller, Nicole Piscopo, Amritava Das, Krishanu Saha, Daniel C. Millard, Lohitash Karumbaiah. Kinetics and potency of T cell and CAR-T cell mediated cytolysis of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1552.

Recent in vitro cardiac safety studies demonstrate the ability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to detect electrophysiologic effects of drugs. However, variability contributed by unique approaches, procedures, cell lines, and reagents across laboratories makes comparisons of results difficult, leading to uncertainty about the role of hiPSC-CMs in defining proarrhythmic risk in drug discovery and regulatory submissions. A blinded pilot study was conducted to evaluate the electrophysiologic effects of 8 well-characterized drugs on 4 cardiomyocyte lines using a standardized protocol across 3 microelectrode array platforms (18 individual studies). Drugs were selected to define assay sensitivity of prominent repolarizing currents (E-4031 for IKr, JNJ303 for IKs) and depolarizing currents (nifedipine for ICaL, mexiletine for INa) as well as drugs affecting multichannel block (flecainide, moxifloxacin, quinidine, and ranolazine). Inclusion criteria for final analysis was based on demonstrated sensitivity to IKr block (20% prolongation with E-4031) and L-type calcium current block (20% shortening with nifedipine). Despite differences in baseline characteristics across cardiomyocyte lines, multiple sites, and instrument platforms, 10 of 18 studies demonstrated adequate sensitivity to IKr block with E-4031 and ICaL block with nifedipine for inclusion in the final analysis. Concentration-dependent effects on repolarization were observed with this qualified data set consistent with known ionic mechanisms of single and multichannel blocking drugs. hiPSC-CMs can detect repolarization effects elicited by single and multichannel blocking drugs after defining pharmacologic sensitivity to IKr and ICaL block, supporting further validation efforts using hiPSC-CMs for cardiac safety studies.

Abstract Here we present an open-source R package ‘meaRtools’ that provides a platform for analyzing neuronal networks recorded on Microelectrode Arrays (MEAs). Cultured neuronal networks monitored with MEAs are now being widely used to characterize in vitro models of neurological disorders and to evaluate pharmaceutical compounds. meaRtools provides core algorithms for MEA spike train analysis, feature extraction, statistical analysis and plotting of multiple MEA recordings with multiple genotypes and treatments. meaRtools functionality covers novel solutions for spike train analysis, including algorithms to assess electrode cross-correlation using the spike train tiling coefficient (STTC), mutual information, synchronized bursts and entropy within cultured wells. Also integrated is a solution to account for bursts variability originating from mixed-cell neuronal cultures. The package provides a statistical platform built specifically for MEA data that can combine multiple MEA recordings and compare extracted features between different genetic models or treatments. We demonstrate the utilization of meaRtools to successfully identify epilepsy-like phenotypes in neuronal networks from Celf4 knockout mice. The package is freely available under the GPL license (GPL>=3) and is updated frequently on the CRAN web-server repository. The package, along with full documentation can be downloaded from: https://cran.r-project.org/web/packages/meaRtools/.Author summaryCultured neuronal networks are widely used to study and characterize neuronal network activity. Among the many uses of neuronal cultures are the capabilities to evaluate neurotoxicity and the effects of pharmacological compounds on cellular physiology. Multi-well microelectrode arrays (MEAs) can collect high-throughput data from multiple neuronal cultures simultaneously, and thereby make possible hypotheses-driven inquiries into neurobiology and neuropharmacology. The analysis of MEA-derived information presents many computational challenges. High frequency data recorded simultaneously from hundreds of electrodes can be difficult to handle. The need to compare network activity across various drug treatments or genotypes recorded on the same plate from experiments lasting several weeks presents another challenge. These challenges inspired us to develop meaRtools; an MEA data analysis package that contains new methods to characterize network activity patterns, which are illustrated here using examples from a genetic mouse model of epilepsy. Among the highlights of meaRtools are novel algorithms designed to characterize neuronal activity dynamics and network properties such as bursting and synchronization, options to combine multiple recordings and use a robust statistical framework to draw appropriate statistical inferences, and finally data visualizations and plots. In summary, meaRtools provides a platform for the analyses of singular and longitudinal MEA experiments.

Microelectrode array (MEA) technology is a neurophysiological method that allows for the spontaneous measure of activity in neural cultures and determination of drug and chemical effects thereon. Recent introduction of multi-well MEA (mwMEA) formats have dramatically increased the throughput of this technology, allowing more efficient compound screening. Rapid characterization of compounds for neuroactivity or neurotoxicity hazard evaluation following acute, chronic, or developmental exposures ideally would also consider compound effects on cell health, and to do so in the same well requires a multiplexed approach. Procedures describing the multiplexed method to acute and developmental screening are described in this chapter.

Optogenetic techniques enable precise excitation and inhibition of firing in specified neuronal populations and artifact-free recording of firing activity. Several studies have suggested that optical stimulation provides the precision and dynamic range requisite for closed-loop neuronal control, but no approach yet permits feedback control of neuronal firing. Here we present the ‘optoclamp’, a feedback control technology that provides continuous, real-time adjustments of bidirectional optical stimulation in order to lock spiking activity at specified targets over timescales ranging from seconds to days. We demonstrate how this system can be used to decouple neuronal firing levels from ongoing changes in network excitability due to multi-hour periods of glutamatergic or GABAergic neurotransmission blockade in vitro as well as impinging vibrissal sensory drive in vivo. This technology enables continuous, precise optical control of firing in neuronal populations in order to disentangle causally related variables of circuit activation in a physiologically and ethologically relevant manner.DOI: http://dx.doi.org/10.7554/eLife.07192.001