High-throughput impedance-based assessment of cytotoxicity and senescence in cancer immunotherapy models

Abstract

Immunotherapy has become a transformative approach in cancer treatment, complementing traditional therapies. Key to its success is the ability to assess T cells that selectively eliminate cancer cells and to monitor the functional performance of engineered immune cells such as CAR T cells. This study evaluates the AtlaZ, a high-throughput impedance-based platform, for real-time, label-free monitoring of immune-mediated cytotoxicity and senescence-induced growth arrest across solid tumor and liquid tumor cell models. We utilized the AtlaZ system (Nanion Technologies), based on Electrical Impedance Spectroscopy (EIS), which enables monitoring of cell behavior across six 96-well plates. The platform operates over a frequency range of 0.1 kHz to 100 kHz, distinguishing between changes in proliferation, cytotoxicity, adhesion, and barrier integrity. We evaluated cytolytic activity in epithelial A549 lung adenocarcinoma cells, K562 leukemia cells, and SKOV-3 ovarian carcinoma cells. CAR T cell responses targeting the EGFR receptor were studied, along with prototypic immune cell-mediated cytolysis using CD8+ T cells and Natural Killer (NK) lymphocyte cells. Additionally, we investigated senescence-related growth arrest in murine RT2 β-cc and human HCT116 cells using established senescence-inducing protocols. Time-resolved impedance analysis captured progressive cytolysis of adherent A549 cells co-cultured with cytotoxic T lymphocytes, showing 37–59% killing over 27 h depending on effector-to-target ratios. In tethered suspension K562 leukemia cells, antibody-mediated cell adhesion enabled robust signal detection similar to adherent cells. Upon addition of CD8+ T cells and NK cells, impedance signals rapidly declined, indicating cytolysis. Full cytolysis was achieved in 7.8 h with CD8+ T cells and 4.6 h with NK cells. Importantly, for liquid tumor cell models, tethering reagents were selective for target cells, avoiding signal interference from effectors. In senescence studies, impedance monitoring successfully captured a transition from proliferative to growth-arrested states in both murine and human cancer cells, reflected by stable, plateaued impedance profiles over time. The AtlaZ system enables dynamic, label-free analysis of cytotoxicity and senescence across various liquid (suspension) or solid (adherent) cell models. Its ability to detect early cellular responses and differentiate between growth inhibition and cell death makes it a powerful tool for preclinical immunotherapy development and toxicological research.