Cancer remains one of the most prevalent and lethal diseases globally. Liquid tumor (hematological cancer) cell lines are vital in studying blood cancers and developing therapies. Immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has shown great promise, improving outcomes in hematological malignancies. Since 2017, the FDA has approved six CAR T-cell therapies for blood cancers, including lymphomas, leukemia, and multiple myeloma.

Advancing CAR T-cell therapy requires basic mechanistic research, bioengineering, and clinical trials to enhance T-cell biology and improve CAR T-cell proliferation and durability. CRISPR-Cas9 technology offers new opportunities for genome-wide screening to identify genes that can boost CAR T-cell resilience.

This application note describes how liquid tumor cells can be monitored and how effects of cytolysing reagents or effector cells such as CAR T-cells can be quantified using an impedance-based assay employing the AtlaZ platform.

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play crucial roles in various cellular processes, including cell growth and proliferation, differentiation, migration
or metabolism. They function by binding extracellular ligands, which triggers receptor dimerization and activation of their intracellular tyrosine kinase domains. This initiates downstream
signaling cascades that regulate key cellular functions (Figure 1). RTKs are highly relevant targets within drug discovery because they are frequently dysregulated in cancer and other diseases. Many successful targeted therapies inhibit RTKs (e.g. imatinib, erlotinib), showing that the receptors provide opportunities for developing selective inhibitors.

The interconnection between ageing, cancer development, and cellular growth suggests common origins despite distinct outward processes. The accumulation of cellular damage is widely acknowledged as a primary cause of ageing, potentially leading to abnormal cellular
advantages (aberrant properties of cells, enabling them to bypass normal growth controls) and cancer. Uncontrolled cellular overgrowth is implicated in age-related pathologies like atherosclerosis and inflammation. Impedance analysis of mammalian cells grown on planar  film electrodes provides a label-free, non-invasive and unbiased observation of cellular properties addressing the biological response to putative senescence inducers, drugs, toxins or stressors in general. Being label-free, automation and continuous monitoring of barrier function are among the most significant advantages offered by electrical techniques in comparison to macromolecular solute permeability studies.

The label-free AtlaZ impedance recording system enables acute and chronic assessment of cellular toxicity as well as senescence in a continuous fashion from living cells under physiological temperatures and without the confounding effects of dyes that may affect cell function. The system uses 96-well plates with 96 parallel sensors offering a time resolution of down to 1 s for impedance measurements, thus allowing investigation of fast effects like GPCR related morphology changes. A frequency spectrum can be recorded ranging from 100 Hz – 100 kHz.
In general, cell adhesion and proliferation assays such as immune-cell -mediated killing of cancer cells can be successfully performed.

This Application Note demonstrates the in vitro characterization of GPCR pharmacology, covering agonist and antagonist mode of action, dose-response relationships, and the involvement of signal transduction cascades. Experimentally, cells expressing H1R or Y4R are cultured on planar gold-film electrodes integrated into standard cell culture dishes.

Hepatotoxicity and drug-induced liver injury (DILI) are leading reasons for drug failure to market, leading to approximately 18% of market withdrawals of drugs in the last decade.
Additionally, only half of drugs with a potential to induce hepatotoxicity are actually identified during preclinical animal studies. This highlights the importance of generating more
advanced cell-based models and experimental strategies to enhance the predictivity of these assays. Current existing in vitro models employed to predict DILI mostly focus on
hepatocytes, though primary hepatocytes do not maintain their phenotype. iCell® Hepatocytes 2.0 (FUJIFILM Cellular Dynamics, FCDI) are human iPSC-derived cells with a
wide variety of basic and functional characteristics which make them amenable to applications such as compound mediated ADME-T and DILI toxicity. In addition to displaying
characteristic hepatocyte morphology, (i.e., polygonal shape, polynucleation and formation of bile canalicular channels), these cells also express liver cell markers, including albumin,
A1AT, and HNF4a, and exhibit basic and induced P450 functions, as observed in primary human hepatocytes. iCell® Hepatocytes 2.0 maintain morphology, marker expression,
and metabolic function in culture over a longer time frame compared to primary human hepatocytes, rendering these cells useful for investigations of acute and chronic DILI responses in a 2D culture system using impedance. Combining these cells with the planar gold-film electrodes on the impedance systems reveal alterations in confluency, cell contact (morphological shape) and conductivity of adherent cells, thereby providing a measure of toxicity. Dose-dependent harmful effects of drugs could be evaluated over time in a functional 2D cell-based model without the need for 3D spheroid formation.

Long-term exposure to cancer-related therapeutics has been linked to alterations of cardiac function in patients. The Stem Cell Working Group as part of the Health and Environmental
Science Institute (HESI) currently endeavors to gain further insight into chronic cardiotoxicity. The objective of the HESI study is to optimize non-clinical safety assessment strategies
of chronic cardiotoxicity by testing prolonged exposure of compounds on different cell-based assay systems using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), by investigating e.g. contractility or contractile force

The US Food and Drug Administration has approved a number of chimeric antigen receptor (CAR) T-cell therapies. Due to the nature of CAR T cells as “living drugs”, they display a unique toxicity profile. As CAR T-cell therapy is extending towards multiple diseases and being broadly employed in hematology and oncology, being able to reliably predict treatment efficacy and a quantification of responses are of high relevance. Furthermore, for continued breakthroughs, novel CAR designs are needed. This includes different antigenbinding domains such as antigen-ligand binding partners and variable lymphocyte receptors (1). Now, after amazing advances for treating blook cancers, CAR T cell therapy is showing promise for solid tumors.

In general, identifying T cells that kill cancer cells in vivo is critical to the development of successful cell therapies. The label-free AtlaZ immune cell killing assay can be used to measure rate of killing at Effector:Target (E:T) ratios to predict in vivo activity. In order to gain a deeper understanding of cancer cells, real-time and continuous monitoring is necessary to access kinetic and phenotypic information. Such monitoring captures also unique toxicity profiles of CAR T cells.

AtlaZ accelerates cellular research by enabling the investigation of a large variety of effects in cells over time. It offers label-free and real-time monitoring capabilities. It can simultaneously or independently record data from up to six 96-well plates.

The numerous different cell types in the human body are greatly specialized and often require a conjunctive action of a population of cells, for example in tissues. Cells are interconnected via cell junctions, multiprotein complexes found in the cell membrane of animal cells, and such cell junctions allow for a mechanical, chemical or electrical transmission of signals. These junctions can be subdivided into (I) tight junctions, (II) anchoring junctions or (III) gap junctions. Defects in cell–cell junctions give rise to a wide range of tissue abnormalities that disrupt homeostasis and are common in genetic abnormalities and cancers (1). The so-called tight junctions form the barrier in endothelial and epithelial cells. Classical transepithelial electrical resistance measurements are performed using microelectrodes, where trans- and para-cellular conductivities can be calculated (2).
Here the cells are grown on a porous filter membrane which is placed between two fluid compartments. Flux of solutes from one compartment to the other must pass the interfacial cell
layer then, and this is determined by the functional properties of the tight junctions.

Cancer remains one of the leading causes of death, with, according to the World Health Organisation (WHO), around 10 million people dying due to the disease in 2020 (1). Chemo and
radio-therapy are still the dominant treatment types, but advancing therapies such as immuno-therapy have emerged as tools to fight against the disease. In general, identifying T cells that kill cancer cells in vivo is critical to the development of successful cell therapies. The label-free AtlaZ immune cell killing assay can be used to measure rate of killing at Effector : Target (E:T) ratios to predict in vivo activity. In order to gain a deeper understanding of cancer cells, real-time and continuous monitoring is necessary to access kinetic and phenotypic information.

The platform used here, AtlaZ, is a quantitative live-cell analysis system and allows for cellular research on cell adhesion and proliferation, cytotoxicity, GPCR, morphology and
barrier function, label-free and in real-time. Recordings can be performed in up to six 96-well plates simultaneously or independently. Electrical impedance spectroscopy (2,3) as
the methodology behind the AtlaZ system, in combination with the throughput of 6 x 96-wells allows for a so far unmet quantity and richness of information which can be gained from cells.