Cited by 105Immune Cell Function and InteractionCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

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In vitro immunotherapy potency assays using real-time cell analysis

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Cerignoli, Fabio, Abassi, Yama A., Lamarche, Brandon J., Guenther, Garret, Santa Ana, David, Guimet, Diana, Zhang, Wen, Zhang, Jing, Xi, Biao·2018·PLOS ONE

DOI 10.1371/journal.pone.0193498PMC PMC5834184

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Abstract

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.

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Fig 1 Working principle of the xCELLigence RTCA system and result from NK92 and TALL-104. (A) Working principle of xCELLigence impedance technology applied to immunotherapy monitoring. The xCELLigence RTCA label-free technology monitors cell number by changes in impedance measured through gold electrodes embedded in proprietary E-Plates. When seeded alone, target adherent cancer cells proliferation rate is registered as increase in the impedance-related Cell Index (CI) parameter over time. Effector non-adherent immune cells produce small baseline level signal due absence of tight surface adhesion over the gold electrodes. When immune cells are added to adherent target cells, their cytolytic activity causes the adherent cells to round up and detach, consequently reducing CI value. (B) Impedance monitoring is validated using NK92 as effector cells over nuclear red-labeled PC3 prostate cancer cells (left) or MCF7 breast cancer cells (right) as targets. When seeded alone, target cells adhere to the plate and proliferate, increasing the CI readout (blue lines). NK92 effector cells seeded alone caused only a small increase in the CI value over the initial background measurement (green lines). When added to target cells, NK92 cause cell cytolysis and subsequent progressive decrease in CI (red lines). Y-axis is the normalized cell index generated by the RTCA software and displayed in real time. X-axis is the time of cell culture and treatment time in hour. Mean values of the CI were plotted ± standard deviation. Time interval is 6 hours for all the figures unless indicated. (C) The same PC3 target cells are treated with a cytotoxic T cell line (Tall-104), showing the suitability of the technology to use different effector cell types. (D) Images taken at different time points after NK92 addition show correlation between CI drop (red line in the plot), reduction in target cells number (red nuclei PC3 in the images) and changes in cell morphology/adhesion in apoptotic cells (red nuclei in images enlargements).CC BY · PubMed Central

Fig 2 Parameters of % cytolysis and KT50 determined through impedance measurement. (A) Cell Index plot for nuclear-red labeled PC3 target cells treated with different E:T ratios of NK92 cytolytic cells. Samples have been internally normalized for the Cell Index value measured before NK92 addition (Normalized Cell Index plot). (B) The Cell Index plot is converted to a % Cytolysis plot by the xCELLigence Immunotherapy Software (xIMT). (C) % Cytolysis measured at 6 and 24 hours after NK92 addition for the different E:T ratios. One way ANOVA result indicates significant difference between individual treatment and control at 6 hours (light blue) and at 24 hours (red); (*** p< 0.001) and (**** p<0.0001). (D) 50% Killing Time (KT50) for the same E:T ratios in (C). ND: Not Detected.CC BY · PubMed Central

Fig 3 Correlation between % cytolysis determined through impedance measurement and flow cytometry. (A) Replica plates for the same experiment in Fig 2 has been collected and analyzed by Flow Cytometry. Nuclear red-gated PC3s show ratio and time dependent increase of early apoptotic (annexin V+, DAPI-; bottom right of each plot), and late apoptotic (annexin V+, DAPI+; upper right of each plot) cells. (B) Charts show the % apoptotic cells for the flow data. (C) Total apoptosis measured by flow cytometry is similar to the results of impedance analysis.CC BY · PubMed Central

Fig 4 Impedance assessment of BiTE-mediated cytotoxicity. (A) Normalized Cell Index plot for PC3 target cells incubated with PBMCs at different E:T ratios without BiTE. (B) Same E:T ratios as (A) but with 1 μg/ml anti-EpCAM/CD3 BiTE. (C) At E:T ratio of 10:1, different BiTE concentrations resulted in varied dynamic cytolysis of the target cells. (D) Same result from (C) showed as % cytolysis plot. (E) Example of BiTE concentration depended % cytolysis from E:T ratio 10:1 and 1.25:1. (F) KT50 comparison for result from (E). Significant analysis performed by one way ANOVA. (*** p< 0.001),); (** p< 0.01);); (* p< 0.05);); (NS Not Significant); (ND Not Detected).CC BY · PubMed Central

Fig 5 Adaptation of the xCELLigence killing assay to suspension B cells. (A). Illustration of B cell attachment through anti-CD40 antibody on the surface of the gold sensor embedded in the well. (B). Raji, 60,000 cells per well, were seeded on the E plate and NK92 cells were added to the well at different E:T ratios. Cytolysis and target cells only are used as positive and negative controls for cyutolysis. (C). Same data as (B) are plotted by xIMT software and displayed as % cytolysis. (D). Comparison of the % cytolysis of Raji cells at different E:T ratios measured by RTCA and flow cytometer at 4 hours (left, correlation coefficient R = 0.9967) and 24 hours (right, R = 0.9903).CC BY · PubMed Central

Fig 6 CAR-T mediated killing assay of tethered Raji B cells. (A) Raji cells were seeded at 40,000 cells per well on E plate. One day after seeding, effector CAR-T cells were added to the well at E:T ratio of 2:1. Mock CAR-T cells and NK92 were added for comparison. (B) Same data from (A) but displayed as % cytolysis.CC BY · PubMed Central

Fig 7 PC3 killing by PBMCs is enhanced by anti PD-1 blocking antibody. (A) Freshly isolated PMBCs shows increased PD-1 expression after SEB stimulation. (B) PC3 at 5,000 cells per well were seeded on E plate and treated with freshly isolated PBMCs two days after initial seeding in presence with increasing concentrations of the anti PD-1 blocking antibody. PC3 cells only and PC3 cells plus PBMCs and without antibody were included as controls. The insert show the dose dependent curve and EC50 calculated at t = 150 hours. (C) Same data from (B) but displays as % cytolysis.CC BY · PubMed Central

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“Conversely, a reduction in CI is indicative of target cell death [31].”
Background
Combination Blockade of the IL6R/STAT-3 Axis with TIGIT and Its Impact on the Functional Activity of NK Cells against Prostate Cancer Cells
Ochoa, S. G., Téllez‐Bañuelos, M. C., Méndez-Clemente, A. S., Bravo‐Cuéllar, A., Hernández‐Flores, G., Palafox-Mariscal, L. A., Haramati, J., Pedraza‐Brindis, E. J., Sánchez-Reyes, K., & Ortiz‐Lazareno, P. C. (2022) · Journal of Immunology Research · doi:10.1155/2022/1810804 · Cited by 16
“3.4 (Agilent Technologies Inc., Santa Clara, CA, USA) [28].”
Influential
CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK CellsPreprint
Kararoudi, M. N., Likhite, S. B., Elmas, E., Yamamoto, K., Schwartz, M., Sorathia, K., Pereira, M. D. S. F., Devin, R. D., Lyberger, J. M., Behbehani, G. K., et al. (2021) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2021.03.17.435886 · Cited by 15
Comparative efficacy and safety of PSCA CAR-engineered Vδ1 γδ T cells for immunotherapy of pancreatic cancer
Li, Z., Liu, N., Shah, Z., Jin, L., Tian, L., Sun, X., Zhang, J., Li, Z., Caligiuri, M. A., & Yu, J. (2025) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2025-011890 · Cited by 16
Self-citation
A Novel Approach for Quantifying the Pharmacological Activity of T-Cell Engagers Utilizing In Vitro Time Course Experiments and Streamlined Data Analysis
Vyver, A. J. V. D., Eigenmann, M. J., Ovacik, M., Pohl, C., Herter, S., Weinzierl, T., Fauti, T., Klein, C., Lehr, T., Bacac, M., et al. (2021) · The AAPS Journal · doi:10.1208/s12248-021-00637-2 · Cited by 13
“Here, we highlight the utility of real-time imag - ing systems such as incuCyte or real-time cellular imped - ance like xCELLigence ( 34 ) that generate richer datasets and are less labor-and time-intensive than analogous work - flows that use flow cytometry ( 35 ) in early drug discovery.”
Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products
Logun, M. T., Colonna, M. B., Mueller, K. P., Ventarapragada, D., Rodier, R., Tondepu, C., Piscopo, N. J., Das, A., Chvatal, S. A., Hayes, H. B., et al. (2023) · Cytotherapy · doi:10.1016/j.jcyt.2023.01.008 · Cited by 14
“While impedance is extremely sensitive to adherent cells, immune effector cells are non-adherent and do not directly affect impedance signal [23].”
Background
Identification of a Promiscuous Epitope Peptide Derived From HSP70
Matsui, H., Hazama, S., Tamada, K., Udaka, K., Irie, A., Nishimura, Y., Miyakawa, T., Doi, S., Nakajima, M., Kanekiyo, S., et al. (2019) · Journal of Immunotherapy · doi:10.1097/cji.0000000000000274 · Cited by 13
A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B
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Optimization and Validation of CAR Transduction into Human Primary NK Cells Using CRISPR and AAVPreprint
Kararoudi, M. N., Likhite, S. B., Elmas, E., Yamamoto, K., Schwartz, M., Sorathia, K., Pereira, M. D. S. F., Devin, R. D., Lyberger, J. M., Behbehani, G. K., et al. (2021) · SSRN Electronic Journal · doi:10.2139/ssrn.3869896 · Cited by 13
Charting the Cannabis plant chemical space with computational metabolomics
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“However, since this study was based on crude index analysis to show real-time cytotoxic activities of the extracts over time (Kho et al., 2015; Cerignoli et al., 2018).”
Background
Phase I/IIa Trial in Advanced Pancreatic Ductal Adenocarcinoma Treated with Cytotoxic Drug-Packaged, EGFR-Targeted Nanocells and Glycolipid-Packaged Nanocells
Ganju, V., Marx, G. M., Pattison, S. T., Amaro-Mugridge, N. B., Zhao, J., Williams, B. R. G., MacDiarmid, J. A., & Brahmbhatt, H. N. (2023) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-23-1821 · Cited by 12
Lentivirus Manufacturing Process for Primary T‐Cell Biofactory Production
Radhakrishnan, H., Javitz, H. S., & Bhatnagar, P. (2020) · Advanced Biosystems · doi:10.1002/adbi.201900288 · Cited by 12
MAST: a hybrid Multi-Agent Spatio-Temporal model of tumor microenvironment informed using a data-driven approach
Cesaro, G., Milia, M., Baruzzo, G., Finco, G., Morandini, F., Lazzarini, A., Alotto, P., de Miranda, N. F. C. C., Trajanoski, Z., Finotello, F., et al. (2022) · Bioinformatics Advances · doi:10.1093/bioadv/vbac092 · Cited by 11
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Xi, B., Berahovich, R., Zhou, H. F., Xu, S., Wei, Y., Guan, J., Harto, H., Guan, J., Wu, L., Ana, D. S., et al. (2019) · Journal of Visualized Experiments · doi:10.3791/59033 · Cited by 11
Self-citation
Healing Field: Using Alternating Electric Fields to Prevent Cytokine Storm by Suppressing Clonal Expansion of the Activated Lymphocytes in the Blood Sample of the COVID-19 Patients
Abadijoo, H., Khayamian, M. A., Faramarzpour, M., Ghaderinia, M., Simaee, H., Shalileh, S., Yazdanparast, S. M., Ghabraie, B., Makarem, J., Sarrami‐Forooshani, R., et al. (2022) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2022.850571 · Cited by 11
“Due to the HLA mismatch between the MDA-MB-231 cells and the donor WBCs, it is supposed that the cytotoxic T cells (CTLs), as well as natural killer cells (NK cells), are activated and invade the cells (Figure 3L) (Martínez-Lostao et al., 2015; Shenouda et al., 2017; Cerignoli et al., 2018).”
Background
Dual‐Sensing Nanoreporter for Dynamic and High‐Throughput Monitoring of Immune Checkpoint Inhibitor Responses in Tumor‐Derived Organoids
Nguyen, A., Ramesh, A., Fish, A., & Kulkarni, A. A. (2024) · Advanced Functional Materials · doi:10.1002/adfm.202400393 · Cited by 10
A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation
Lee, H. W., O'Reilly, C., Beckett, A., Currier, D. G., Chen, T., & DeRenzo, C. C. (2024) · Journal of Experimental & Clinical Cancer Research · doi:10.1186/s13046-024-03022-x · Cited by 11
“Percent cytotoxicity was calculated using RTCA Software Pro immuno-therapy module (Agilent) [23].”
Method
A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes
Jia, Y., Reboulet, J., Gillet, B., Hughes, S., Forcet, C., Tribollet, V., Hajj Sleiman, N., Kundlacz, C., Vanacker, J.-M., Bleicher, F., et al. (2023) · Cells · doi:10.3390/cells12010200 · Cited by 10
Distinct functions of CAR-T cells possessing a dectin-1 intracellular signaling domain
Liang, X., Huang, Y., Li, D., Yang, X., Jiang, L., Zhou, W., Su, J., Chen, N., & Wang, W. (2021) · Gene Therapy · doi:10.1038/s41434-021-00257-7 · Cited by 10
“xCELLigence RTCA SP) as previously described [23].”
A Novel Age-Related Circular RNA Circ-ATXN2 Inhibits Proliferation, Promotes Cell Death and Adipogenesis in Rat Adipose Tissue-Derived Stromal Cells
Song, X., He, N., Xing, Y., Jin, X., Li, Y., Liu, S., Gao, Z.-Y., Guo, C., Wang, J., Huang, Y., et al. (2021) · Frontiers in Genetics · doi:10.3389/fgene.2021.761926 · Cited by 9
Enhanced Antitumor Effect of the Combination of Bacille Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer-On-a-Chip
Choi, S. Y., Kim, M., Kang, S. J., Choi, Y. W., Maeng, S., Kim, S.-H., & Chang, I. H. (2023) · BioChip Journal · doi:10.1007/s13206-023-00125-z · Cited by 8
“Current potency assays involve the direct measurement of the cytotoxic effects of effector cells or the indirect assessment of cytokine release [6].”
Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell
Haeseleer, F., Eichholz, K., Tareen, S. U., Iwamoto, N., Roederer, M., Kirchhoff, F., Park, H., Okoye, A. A., & Corey, L. (2020) · AIDS Research and Human Retroviruses · doi:10.1089/aid.2020.0163 · Cited by 8
“Impedancebased assays are another option to quantify the cytolytic activity of immune cells in real time but require target cells to be attached to the electrodes and are thus less suitable to assess killing of nonadherent target cells such as primary CD4 T cells.(23) The preparation of target cells is a critical fact…”
Release Assays and Potency Assays for CAR T-Cell Interventions
Dias, J., Cadiñanos-Garai, A., & Roddie, C. (2023) · Advances in experimental medicine and biology · doi:10.1007/978-3-031-30040-0_8 · Cited by 10
Expediting in vitro characterization of mRNA-based gene therapies via high-content fluorescent imaging
Vigil, T. N., Zhang-Hulsey, D., Santos, J. L., & Hussmann, G. P. (2021) · Analytical Biochemistry · doi:10.1016/j.ab.2021.114259 · Cited by 7
A protocol for quantifying lymphocyte-mediated cytotoxicity using an impedance-based real-time cell analyzer
Kanemaru, H., Mizukami, Y., Kaneko, A., Kajihara, I., & Fukushima, S. (2022) · STAR Protocols · doi:10.1016/j.xpro.2022.101128 · Cited by 7
“If you use nonadherent cells as targets, such as leukemia-derived cell lines, consult previously reported approaches to tether target cells to the plate using a specific antibody, such as an anti-CD40 antibody for Raji cells (Cerignoli et al., 2018).”
Background
LRRC15-CAR T Cells for the Treatment of Osteosarcoma
O'Reilly, C., Tuladhar, S., Nguyen, P. T., Sheppard, H., Koo, S. C., Chockley, P. J., Houke, H., Santiago, T., Tian, L., Jeschke, J., et al. (2026) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-24-2335 · Cited by 6
EF1α, rather than CMV promoter, is suitable for luciferase tag expression in target cells for in vitro cytotoxicity assays of CAR-T cells
Hou, R., Zhang, Z., Li, H., He, W., Wang, X., Zhao, X., Li, S., Guan, Z., Liu, D., Zheng, J.-N., et al. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101537 · Cited by 5
“42,43 46 In Luc-based cytotoxicity assays, target cell viability is inferred from Luc activity; however, under certain assay conditions, Luc enzymatic activity may persist post lysis, leading to discrepancies between luminescence signals and actual cell death levels.”
In vitro assays to evaluate CAR-T cell cytotoxicity
Cao, M., Carlson, R. D., Staudt, R. E., & Snook, A. E. (2023) · Methods in cell biology · doi:10.1016/bs.mcb.2023.05.009 · Cited by 4
Co-SLD suppressed the growth of oral squamous cell carcinoma via disrupting mitochondrial function
Li, S., Li, G., Zhang, T., Li, J., Zhao, Q., Zhang, B., Wang, R., Zhou, R., Si, J., Gan, L., et al. (2019) · Artificial Cells Nanomedicine and Biotechnology · doi:10.1080/21691401.2019.1608218 · Cited by 4
“KT50 (the amount of time it takes to kill 50% of the target tumour cells) was selected as a quantitative parameter for evaluating the efficient killing kinetic of Co-SLD [68].”
BackgroundMethod
Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13
Moetlhoa, B., Naicker, L., Hayeshi, R. K., Grobler, A., Mokoena, N. B., & Mawadza, C. (2020) · Access Microbiology · doi:10.1099/acmi.0.000191 · Cited by 4
Evaluating CD8+ T Cell Responses In Vitro
Lemonnier, F. A. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-9450-2_15 · Cited by 3
Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism
Pereira, M. D. S. F., Sorathia, K., Sezgin, Y., Thakkar, A., Maguire, C. T., Collins, P. L., Mundy-Bosse, B. L., Lee, D. A., & Kararoudi, M. N. (2023) · Cancers · doi:10.3390/cancers15030705 · Cited by 3
“Briefly, the xCELLigence SP instrument (Agilent) was utilized to record cellular impedance following the previously described [21,22].”
Method
A New Approach to In Vivo Transformation of Killer T Cells
Rodriguez, N., Hofmann, C., Yang, O. O., & Gelbart, W. M. (2025) · Journal of Molecular Biology · doi:10.1016/j.jmb.2025.169369 · Cited by 4
High-Throughput Direct Cell Counting Method for Immuno-Oncology Functional Assays Using Image Cytometry
Chan, L. L. (2020) · Methods in pharmacology and toxicology · doi:10.1007/978-1-0716-0171-6_2 · Cited by 3
Potency Assays: The ‘Bugaboo’ of Stem Cell Therapy
Torrents, S., Grau-Vorster, M., & Vives, J. (2023) · Advances in experimental medicine and biology · doi:10.1007/978-3-031-30040-0_3 · Cited by 2
Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method
Talib, F. N. A. A., Marzuki, M., & Hoe, S. L. L. (2023) · Heliyon · doi:10.1016/j.heliyon.2023.e17480 · Cited by 2
A Proximity-Dependent Biosensor System for Visualizing Cell-Cell Interactions Induced by Therapeutic Antibodies
Tang, Y., Liao, X., & Cao, Y. (2022) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2023.12.008 · Cited by 2
Impact of eggshell membrane on metabolism and cell adhesion in oxidatively stressed canine chondrocytes
Vozár, J., Hudáková, N., Nosálová, N., Huniadi, M., Marcinčáková, D., Horňák, S., Horňáková, Ľ., Majerová, P., & Cizkova, D. (2025) · Frontiers in Veterinary Science · doi:10.3389/fvets.2024.1517349 · Cited by 2
“On the other hand, RTCA monitors real-time changes in cell adhesion, proliferation, and morphology (36).”
In Vitro Cell Impedance Assay to Examine Antigen-Specific T-Cell-Mediated Melanoma Cell Killing to Support Cancer Immunotherapy Drug Discovery
Stirling, E. R., & Soto-Pantoja, D. R. (2022) · Methods in molecular biology · doi:10.1007/978-1-0716-1896-7_8 · Cited by 2
NK Cell Effector Functions and Bystander Tumor Cell Killing in Immunovirotherapy
Floerchinger, A., & Engeland, C. E. (2022) · Methods in molecular biology · doi:10.1007/978-1-0716-2441-8_12 · Cited by 2
In Vitro Functional Assay Using Real-Time Cell Analysis for Assessing Cancer Immunotherapeutic Agents
Xi, B., Ye, P., Golubovskaya, V., & Abassi, Y. A. (2020) · Methods in pharmacology and toxicology · doi:10.1007/978-1-0716-0171-6_3 · Cited by 2
Self-citation
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Xi, B., Berahovich, R., Zhou, H., Xu, S., Wei, Y., Guan, J., Harto, H., Guan, J., Wu, L., Ana, D. S., et al. (2019) · Journal of Visualized Experiments · doi:10.3791/59033-v · Cited by 2
Self-citation
Genetically encoded biosensor of apoptosis and necrosis reveals the dynamics and biphasic cytotoxicity by Anti-EGFR CAR-T cells
Rather, A. A., Halikar, A. M., Sachdeva, R., Tiwari, S. K., & Santhoshkumar, T. R. (2026) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2026.118471 · Cited by 1
Membrane-proximal binding of PSMA facilitates synapse formation with CAR and enhances antitumor activity of PSMA CAR-T cells against prostate cancer
Li, J., Li, D., Huang, Y., Zhou, W., Li, J., Wang, F., Zhang, Y., Yan, F., Gao, H., Gu, X., et al. (2026) · Biomedicine & Pharmacotherapy · doi:10.1016/j.biopha.2026.119110 · Cited by 1
From production to bedside: A tiered QC framework for regulatory alignment and analytical comparability in decentralized CGT
Nahum, S., Doshi, D., Kosnik, C., Jeffries, E., Nishida, K. S., Caplan, V., & von der Leyen, H. E. (2026) · Regenerative Therapy · doi:10.1016/j.reth.2026.101112 · Cited by 1
“…analysis for mycoplasma detection, replication-competent virus (RCV) testing, vector copy number (VCN) quantification [100 – 102], and transgene confirmation, image-based functional assays for potency [103] and impedance-based functional assays for real-time cell activity monitoring [104].”
Engineering Organ-on-a-Chip Systems for Cancer Immunotherapy: Strategies and Assay Integration
Wang, J., & Wang, Z. (2026) · Bioengineering · doi:10.3390/bioengineering13050492 · Cited by 1
“The resulting killing curves correlated closely with endpoint measurements from image-based assays and flow cytometry (Figure 6a) [81].”
Influential
A review of organoid–immune co-culture platforms to model the immune microenvironment of hepatocellular carcinoma and guide immunotherapy
Liu, W.-W., Xie, X.-S., & Yang, X.-J. (2026) · Journal of Translational Medicine · doi:10.1186/s12967-026-08278-9 · Cited by 1
“Tumor cell killing is commonly quantified using real-time impedance assays (e.g., xCELLigence), lactate dehydrogenase (LDH) release assays, or ATP-based viability assays [47, 48].”
Two-dimensional dynamic evaluation of natural killer cell-mediated lysis of adherent target cells
Frazao, A., Rethacker, L., & Caignard, A. (2019) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/bs.mie.2019.07.033 · Cited by 1
A high-content screen identified ingenol-3-angelate as an enhancer of B7-H3-CAR T cell activity by increasing B7-H3 protein expression on the target cell surface via PKCα activationPreprint
Lee, H. W., O'Reilly, C., Beckett, A., Currier, D. G., Chen, T., & DeRenzo, C. C. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.05.26.542130 · Cited by 1
Anti-cancer activity and immunoadjuvant properties of edible bird's nest extracts on human breast cancer cell line
Maruthai, M. Y. S. (2019) · UTM Institutional Repository
Data from Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy
Abou‐El‐Enein, M., Elsallab, M., Feldman, S. A., Fesnak, A. D., Heslop, H. E., Marks, P. W., Till, B. G., Bauer, G., & Savoldo, B. (2023) · doi:10.1158/2643-3230.c.6550449
Pooled genetic screens identify breast cancer risk genes involved in evasion from T cell-mediated killingPreprint
Shi, W., Luo, Y., Burrows, J. M., Black, D. M., Civitarese, A., Perlaza-Jiménez, L., Zhang, P., Manning, M., Tuano, N., Rentería, M. E., et al. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.05.10.593465
“…MCF-7, SKBR3, MDA-MB-231 and the immortalised normal breast cell line B80-T5 24 were cultured in 96 well E-Plates (ACEA Biosciences) for 24 hours followed by co-culturing with HLA-A2-HER2-restricted T cells on the xCELLigence RTCA instrument (ACEA Biosciences) until the cytolysis plateaus 25,26 .”
Method
Directed evolution and modular integration of a high-affinity ICOS-L variant for potent T cell–mediated tumor elimination
Ha, J. Y., Song, T. W., Jebamani, P., Lee, S., & Jung, S. T. (2025) · Journal of Biological Engineering · doi:10.1186/s13036-025-00536-6
“T cell-mediated tumor cell lysis was assessed by monitoring the cell index and % cytolysis using the xCELLigence real-time cell analysis (RTCA) instrument (Agilent, Santa Clara, CA, USA) [32].”
Development of an In Vitro Electrical Impedance Model to Assess Cytotoxicity and Antifungal Efficacy of Aspergillus fumigatus
Kakuno, S., Shibata, W., Kawamoto, K., Imoto, W., Yamada, K., Niki, M., Kaneko, Y., Umeyama, T., Miyazaki, Y., & Kakeya, H. (2025) · Medical Mycology Journal · doi:10.3314/mmj.25-00003
Additional file 1 of A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation
Lee, H. W., O'Reilly, C., Beckett, A. N., Currier, D. G., Chen, T., & DeRenzo, C. C. (2024) · Figshare · doi:10.6084/m9.figshare.25522554
Serial Killing Assay Using Longitudinal Impedance-Based Tumor Cell Viability Measurement - A Useful Method to Assess T Cell Performance
Tschaidse, T., Trefny, M. P., Carlini, E., Andreu-Sanz, D., Michaelides, S., Nguyen, T., & Kobold, S. (2025) · Journal of Visualized Experiments · doi:10.3791/69623
From Bench to Bedside: Innovations and Challenges in CAR-T Cell Therapy Manufacturing and Regulation
Talukdar, J., Baishya, D., Karmakar, S., Ghosh, A., Seth, T., Kumar, U., & Ghatak, A. (2026) · Springer eBooks · doi:10.1007/978-981-95-3628-3_8
Dynamics of natural killer cell function upon recurrent stimulation
One, J., Narayan, J., Cichocki, F. M., Hu, W., & Azarin, S. M. (2026) · Biotechnology Progress · doi:10.1002/btpr.70100
“This could be due to the use of a high effector to target cell ratio (10:1), which could mask subtle differences in killing efficacy, 33 particularly for cell lines that exhibit faster rates of killing by NK cells.”
Coverslip Hypoxia High-Content Screening (CH-HCS): A 2D Imaging Platform for Spatially Resolved Analysis of CAR-T Function Under Oxygen GradientsPreprint
Murarolli, J., Capuz, G. B. C. B., Arrojo, M. L., Mizukami, A., Caruso, S. R., Orellana, M. D., & Panepucci, R. A. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.01.30.702833

Authors: Cerignoli, F., Abassi, Y. A., Lamarche, B. J., Guenther, G., Santa Ana, D., Guimet, D., Zhang, W., Zhang, J., & Xi, B.
Affiliations: ACEA Biosciences (United States) (US)
Corresponding author: Xi, Biao
ORCID: Zhang, Wen
Zhang, Jing
Xi, Biao
Venue: PLOS ONE
Publisher: Public Library of Science (PLoS)
Published: 2018-03-02
Volume: 13
Issue: 3
Pages: e0193498
ISSN: 1932-6203 (ISSN-L)
DOI: 10.1371/journal.pone.0193498
PMC: PMC5834184
OpenAlex: W2791857742
Cited by: 105
References: 47
Citations by year: 2026: 13 · 2025: 9 · 2024: 6 · 2023: 13 · 2022: 15 · 2021: 19 · 2020: 11 · 2019: 17 · 2018: 2
FWCI: 5.27
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
creativecommons.org/licenses/by/4.0/ (vor)
Locations: PLoS ONE (publishedVersion, cc-by)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Figshare (submittedVersion, cc-by)
Topics: Life Sciences › Immunology and Microbiology › Immunology › Immune Cell Function and Interaction
Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords: cancer immunotherapy, potency assay, real-time cell monitoring, xCELLigence, tumor cell lysis, chimeric antigen receptor T cells, natural killer cells, bispecific T-cell engagers, EpCAM, gold microelectrodes, liquid tumors
MeSH terms: Epithelial Cell Adhesion Molecule, Antibodies, Biological Assay, Cytological Techniques, Humans, Immunotherapy, Leukocytes, Mononuclear, Apoptosis, Cell Line, Tumor, Programmed Cell Death 1 Receptor, MCF-7 Cells

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In vitro immunotherapy potency assays using real-time cell analysis

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