Real-Time Monitoring Of CAR-T Cell Therapy Using A Bioelectronic Assay


By Lohitash Karumbaiah, Ph.D., Associate Professor, Regenerative Bioscience Center, University of Georgia

Glioblastoma accounts for nearly half of all cancers that affect the central nervous system,1 and yet there are no advanced therapies approved to treat this deadly disease. CAR-T cell therapies have shown promise with liquid tumors, but in developing CAR-T cell lines for solid tumors like gliomas, there are still many unknowns: researchers need to identify appropriate molecular targets, unlock critical molecular pathways involved in CAR-T cell function, and assess the potency of these potential treatments. To answer these questions, researchers need reliable, objective in vitro assays that monitor the dynamic interactions between immune cells and tumor cells in real time and reveal the success or failure of candidate cell lines quickly.

Can CAR-T Cell Therapy be an Answer to Glioblastoma?

As the most aggressive form of glioma,2 glioblastoma is the deadliest central nervous system cancer: the five-year survival rate for patients under 44 is only 22%, while less than 10% of patients over 44 survive for five years.3 Unfortunately, the only treatment options are surgery, chemotherapy, and radiation.1 An approved CAR-T cell therapy could dramatically improve outcomes for people with this fatal condition.

The US Food and Drug Administration has approved five CAR-T cell therapies for liquid tumors, all of which have shown tremendous success. For instance, in clinical trials, a CAR-T therapy for mantle cell lymphoma led to significant improvement in 87% of patients, while 62% of patients exhibited a complete response.4 Based on these successes, forecasters expect the CAR-T market to grow rapidly, reaching a valuation of $6.1 billion by 2030.5 One reason for this progress is increasing interest in CAR-T cells among academic researchers and biopharmaceutical companies. According to, more than 700 CAR-T cell therapy clinical trials are currently in planning or underway.6

But since CAR-T cell therapies are so new, scientists still have much to learn about their function, reliability, and potency. Their status as “living drugs” creates unique challenges in transforming them into a safe and effective therapy. This is because cell therapies operate via biological mechanisms that we do not fully understand.

It is critically important for researchers to gain an accurate understanding of a CAR-T cell line’s potency to dose the therapy correctly. If one overestimates potency in the laboratory, then biomanufacturers will underestimate the effective dosage and the treatment will not work.

Many Techniques Don’t Provide Enough Answers

Although scientists have been testing cell health for years, continuously monitoring tumor cell health in response to treatment with CAR-T cells can be burdensome. Researchers can use methods like flow cytometry to measure CAR-T cell activity, but the assay must be repeated at each time point and it fails to capture the cell-killing behavior of immune cells in real time. Since different antigen targets or manufacturing methods can lead to different potencies and kinetic profiles, it can be challenging to test the impact of all the variables and dosing regimens that affect CAR-T cell therapy safety and efficacy.

The most used method for testing immune cell behavior, the chromium release assay, is also an endpoint assay. Like any endpoint assay, without careful planning and some luck, researchers can easily miss critical events such as peak tumor cell killing, potential CAR-T cell exhaustion, and subtle kinetic changes between conditions. This technique also uses a hazardous radioactive isotope of chromium as an indicator and requires significant technical skill to run it safely.

Live-cell imaging offers real-time monitoring of cell viability and death, but this optical technique presents its own challenges. First, it requires the use of multiple dyes to distinguish between immune cells and tumor cells, and these dyes may impact the target biology in unknown ways. Furthermore, the analysis is often complex and requires scientists to make subjective decisions, which can introduce extra variability to the data. Live-cell imaging assays can also suffer from a spatial problem: immune cells are usually placed on top of target cells in a well and can interfere with proper imaging analysis.

Monitoring Immune Cell Potency Using a Bioelectronic Assay

In contrast to other methods, bioelectronic assays enable objective, real-time monitoring of CAR-T cells without the need for hazardous reagents or complex protocols. These assays rely on multi-well plates with multielectrode arrays (MEAs) embedded in the bottom. These MEAs can detect cell death by measuring electrical impedance across the well.

This assay measures the degree to which the cells in a well are opposing, or impeding, the electrical currents running between electrodes. When living cells attach to a well, they cause a rise in impedance. Conversely, as cells die, their membrane becomes unstable, or they detach, decreasing impedance. This provides a sensitive measurement of cell viability and death in real time. With standard microtiter formats up to 384 wells, researchers can use this assay to simultaneously compare multiple conditions. For instance, they can assess antigen targets, compare donors, rank transduction and other manufacturing methods, or determine effectiveness at various effector-to-target ratios. These capabilities enable researchers to quickly and comprehensively profile the potency of CAR-T products and focus on effective treatments sooner in the development process.

Currently, my team at the University of Georgia is using one such bioelectric assay to characterize CAR-T cell-mediated killing of glioblastoma.

Using a Bioelectronic Assay to Evaluate CAR-T Cell Potency Against Glioblastoma

One step in designing effective cell therapies for cancer is identifying appropriate molecular targets on tumor cells. The ideal target is expressed at high levels on tumor cells and nowhere else in the body, leading to a positive treatment outcome with few side effects. But finding a specific target is challenging, especially for brain tumors. GD2 is overexpressed in many cancers including glioma, making it a strong candidate as a target for glioblastoma therapy.7 To assess the potency of GD2-targeted CAR-T cells, my group at the University of Georgia, in collaboration with researchers from the University of Wisconsin-Madison and Axion Biosystems, assessed their cytotoxic activity using a bioelectronic assay.8 We cultured glioma stem cells collected from patients in a 96-well plate with embedded electrodes and monitored impedance within each well over a seven-day period.

As the glioma cells attached to the wells, impedance rose as expected. After 48 hours, we added GD2-targeted CAR-T cells or naive activated T cells to the glioma cultures at various effector-to-target (E:T) ratios. The GD2-targeted CAR-T cells showed significantly higher cell killing than naive T cells over 7 days, reducing impedance at all ratios. All the CAR-T cells killed more than 50% of glioma cells on average.

The bioelectronic assay was not only sensitive enough to measure cytolysis at even low E:T ratios; it also measured the different rates at which the conditions performed, all in one plate. Overall, the bioelectronic assay enabled us to gain more insights into GD2-targeting CAR-T cells as a potential therapeutic option for people with glioblastoma.

The Value of Bioelectronic Assays in Cancer Therapy Development

Given the importance of CAR-T therapy effectiveness to patients, it is critical that scientists accurately profile the potency before the treatment reaches the clinic. Reducing uncertainty about therapeutic potency will make both preclinical and clinical testing more efficient by enabling researchers to “fail fast” and arrive at a viable therapy sooner. Impedance assays facilitate this by providing researchers with a simpler, safer, more reliable tool for monitoring CAR-T cell activity in real time and in a label-free manner. This tool creates greater opportunity for researchers to develop novel immune cell products that are effective against cancer and to profile their activity more comprehensively, giving people with glioblastoma and other cancers a better chance of living healthy lives.

  1. Thakkar JP, Paruzzi PP, Prabhu VC, eds. Glioblastoma multiforme. Patients. Accessed September 8, 2021.

  2. Perlmutter Cancer Center. Types of Glioma & Astrocytoma, NYU Lagone Health. Available at:

  3. Survival rates for selected adult brain and spinal cord tumors. Early Detection, Diagnosis, and Staging. Accessed September 8, 2021.

  4. NCI Staff. CAR T-Cell THERAPY approved by FDA for Mantle cell lymphoma. Cancer Currents Blog. Published August 24, 2020. Accessed September 8, 2021.

  5. Research and Markets. Global CAR-T therapy market report 2020: market is expected to stabilize and reach $3,150 million in 2025 - COVID-19 impact and recovery forecast to 2030. PR Newswire. Published February 1, 2020. Accessed September 8, 2021.

  6. Search: car-t | Recruiting, Not yet recruiting, Active, not recruiting, Enrolling by invitation Studies. National Library of Medicine. Published September 8, 2021. Accessed September 8, 2021.

  7. Sujjtjoon J, Sayour E, Tsao S, Uiprasertkul M, Sanpakit K, Buaboonnam J, Yenchitsomarius P, Atchaneeyasakul L, Chang L, GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma – assessing tumor and T cell interaction, Trans Oncology, 14:100971 (2021) .

  8. Hayes HB,  Logun MT, Chvatal SA, Mueller K, Piscopo N, Das A, Saha K,  Millard D, Karumbaiah L, Abstract 1552: Kinetics and potency of T cell and CAR-T cell mediated cytolysis of glioma stem cells, Cancer Res, 81(13 Supplement): 1552 (2021).

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