If you’ve worked in drug development, you already know that the road from discovery to clinical success is long, expensive, and rarely linear. One of the most pivotal checkpoints in that journey is cytotoxicity testing.
Whether you’re developing a small molecule, a biologic, or a cell therapy like CAR‑T, the ability to accurately assess how a candidate interacts with healthy cells is non-negotiable. Cytotoxicity assays are often the first meaningful gatekeeper for ruling out poorly tolerated or dangerous compounds; getting this right early can save an extraordinary amount of time and resources later.
Cytotoxicity Assays: What They Tell Us and Why It Matters
Cytotoxicity assays aim to answer a straightforward question: Does this compound kill cells it shouldn’t? But the simplicity of that question belies its importance. Identifying safety liabilities before they move into complex preclinical models, let alone the clinic, is crucial. Just as importantly, these assays help flag non-viable candidates fast, allowing developers to fail smart and early rather than incurring large costs and time delays.
The most basic colorimetric assays (e.g., MTT, LDH, ATP-based) are useful screens, but they fall short when therapies get more nuanced — especially when cell types are heterogeneous, or when you need to separate cytotoxicity from immune activation, apoptosis, or sublethal stress. That’s where flow cytometry steps in.
Why Flow Cytometry Is a Game-Changer for Cytotoxicity Testing
Unlike bulk assays that average signals across populations, flow cytometry analyzes individual cells. This means you don’t just get a measure of overall viability — you can identify which cells are dying, how they’re dying, and whether they’re also undergoing changes like immune activation, cell cycle arrest, or cytokine release.
Using fluorophore-labeled antibodies and dyes, like annexin V, propidium iodide, caspase substrates, or mitochondrial potential markers, we can dissect cell fate in multiplex. In the setting of CAR‑T therapy or ADC development, this can be the difference between spotting an early safety issue or missing a signal entirely.
From Insight to Intervention: Flow Cytometry and Safety Profiling
A key application of high‑content flow cytometry is in detecting early signs of on‑target, off‑tumor toxicity in engineered cell therapies, well before these signals appear in gross viability assays or animal models.
In a recent study, Yang et al. (2024) developed a panel of mesothelin-targeting CAR T cells with varying antigen-binding affinities and assessed them in vitro and in vivo for both efficacy and safety. Using a luciferase-based cytotoxicity assay, they observed that high-affinity CARs triggered unintended toxicity even against mesothelin-low target cells, including in mouse models where off‑tumor effects were lethal. Flow cytometry was used throughout to monitor CAR T cell phenotype, expansion, and memory subset distribution, key indicators of T cell behavior and persistence (Yang et al., JCI Insight, 2024).
However, the study also highlights an opportunity: while luciferase readouts confirmed cytotoxicity, complementary flow cytometry–based co-culture assays could have directly quantified activation markers on CAR T cells during engagement with mesothelin‑low healthy targets. This would have provided mechanistic insights into the activation thresholds and signaling states driving off‑tumor effects and offered a richer, cell‑by‑cell view of therapeutic window limitations.
By performing flow cytometry-based cytotoxicity assays, future studies could move beyond binary live/dead readouts to more predictive, translationally relevant safety profiles. The Yang study makes clear that affinity tuning alone can shift safety outcomes, but it also reinforces the value of flow‑based functional profiling in identifying those risks earlier and more precisely.
A concrete example of using flow cytometry to measure CAR T–mediated cytotoxicity is provided in Kiesgen et al. (2021). In this Nature Protocols–review article, researchers evaluated multiple cytotoxicity platforms, including flow cytometry–based assays that used live/dead staining (e.g. 7‑AAD and Annexin V) to quantify target cell death in co‑culture with CAR T cells. They demonstrated that this method permits simultaneous analysis of target viability and cellular behavior, offering a high-throughput, single-cell resolution view of CAR T potency and functionality.
Together, these examples demonstrate how fit‑for‑purpose, flow‑based cytotoxicity platforms, especially when combined with antigen expression profiles of healthy cell types, can sensitively detect off‑target activation or killing and turn ambiguous safety signals into actionable mechanistic insights, guiding safer CAR T‑cell construct design.
Supporting Smarter Development
At KCAS Bio we’ve had the opportunity to partner with biotechs and pharmas across a range of therapeutic areas. We’ve learned that success often hinges on how early and how thoroughly teams interrogate their candidate’s cytotoxicity — not just on tumor cells, but on healthy, primary human cells as well.
With the right panel design, you can combine readouts like viability, apoptosis, T-cell activation, and even cytokine expression in a single flow run. Whether you’re screening candidates, optimizing constructs, or addressing reviewer feedback on a regulatory package, this kind of assay provides the kind of high-resolution data that stands up to scrutiny.
And when timelines are tight, which they always are, getting definitive, interpretable data from cytotoxicity assays can make the difference between doubling down on a promising lead or pivoting to save time and budget.
Final Thoughts
Cytotoxicity assays are more than a checkbox, they’re a window into how a therapy behaves in the real world. And in our experience, flow cytometry makes that window wider, clearer, and more actionable.
If you’re working on a program where the safety margins are tight, whether it’s a CAR-T, an antibody-drug conjugate, or a first-in-class biologic, it’s worth investing in the tools that can reveal issues early and guide you toward a safer, more effective final product.
At KCAS Bio, we see the difference every day, and we’re always happy to talk through assay design with partners looking to raise the bar on safety screening.
References
- Yang et al., 2024
Yang, Qian, et al. “Affinity-Tuned Mesothelin CAR T Cells Demonstrate Enhanced Targeting Specificity and Reduced Off-Tumor Toxicity.” JCI Insight, vol. 9, no. 22, 2024, https://doi.org/10.1172/jci.insight.186268. - Kiesgen et al., 2021
Kiesgen, Samantha, et al. “Comparative Analyses of CAR T Cell Cytotoxicity Protocols.” Nature Protocols, vol. 16, no. 2, 2021, pp. 689–719, https://doi.org/10.1038/s41596-020-00467-0. - DePriest et al., 2021
DePriest, Andrew D., et al. “An Overview of Multiplexed Analyses of CAR T-cell Therapies: Insights and Potential.” Expert Review of Proteomics, vol. 18, no. 3, 2021, pp. 173–189, https://doi.org/10.1080/14789450.2021.1885390.