Tumor killing assays
T Cell–Mediated Tumor Killing Assays
Does your candidate enhance immune cell-mediated tumor killing, by activating T cells, sensitizing tumor cells or redirecting cytotoxicity toward a defined target?
We co-culture human tumor cells with primary immune cells and monitor tumor-cell growth and apoptosis in real time, alongside immune cell activation and effector function readouts. The assay can be tailored to checkpoint inhibitors, immune agonists, tumor-sensitizing compounds and T-cell engagers, depending on the mechanism under investigation.
A flexible functional platform to characterize T cell-dependent tumor killing across therapeutic modalities and mechanisms.
T cell killing assay format and readouts
- Format
- 96- or 384-well plates; dose-response
- Model
- >100 human tumor cell lines co-cultured with human primary immune cells, including PBMCs or purified T cell populations.
- Core readouts
- Tumor cell count and apoptosis by live-cell imaging (Incucyte), together with soluble markers of immune activation (e.g. IFN-γ / TNF-α / IL-6) selected according to the biological question.
- Reference molecules
- Relevant reference therapies or controls selected according to modality and mechanism of action.
- On request
- Flow cytometry immunophenotyping, cytokine / secretome profiling, proteomics, transcriptomics
72h post-treatment
Untreated
Tumor-target inhibition sensitizes breast MDA-MB-231 tumor cells to immune cell-mediated killing. Breast cancer MDA-MB-231 cells were treated with a tumor target inhibitor, and subsequently co-cultured with activated PBMCs. Real-time monitoring of tumor cell count & apoptosis - as functional tumor cell response surrogates - showed increased immune-mediated tumor cell killing following target inhibition.
In our assay models, we connect the functional phenotype to its underlying biology. We go beyond tumor cell death by combining functional and mechanistic readouts to understand how the anti-tumor immune response happens and which cellular mechanisms are associated with it.
Imane Nafia, PhD
Chief Scientific Officer, Explicyte
Assay principle
How the T cell killing assay works
1
Set up the co-culture
Co-culture human tumor cells with primary PBMCs, T cells, or CD8 T cells at an optimized effector-to-target ratio. The candidate is applied to tumor cells, immune cells, or the complete co-culture according to its mechanism of action and experimental design.
2
Monitor tumor cell killing and immune activation
Track tumor-cell count and apoptosis over 3–5 days by live-cell imaging, while measuring selected soluble markers of immune activation in the coculture supernatant.
3
Characterize the underlying mechanism
Representative tumor-killing data
Checkpoint blockade enhances immune-mediated tumor killing
PD-L1 blockade enhances immune cell-mediated killing of breast MDA-MB-231 tumor cells.
MDA-MB-231 tumor cells were cultured either alone (ctrl) or with PBMCs in the presence and absence of PD-L1 inhibitor (Atezo). Supernatants were collected at 72h post-co-culture and analyzed by HTRF for TNF-α and IL-6 release as soluble markers of the immune response (A). Tumor cell count was also kinetically monitored and analyzed (B). While tumor cell exposure to activated immune cells is sufficient to induce an immune cell response against MDA-MB-231 tumor cells, Atezolizumab further enhanced immune cell-mediated tumor killing.
A. Atezo promotes immune cell response
B. Atezo promotes anti-tumor immune cell-mediated activity
PDL1 blockade enhances immune cell-mediated killing of A549 lung cancer cells
Lung cancer A549 cells were seeded and then co-cultured – at an appropriate E:T ratio – with activated hPBMCs, in the presence and absence of Atezo. (A) Tumor cell apoptosis was monitored over ~5 days by mean of a caspase 3/7 fluorescent probe and a NucRED probe expression, respectively, as a surrogate measure of immune cell killing activity towards tumor cells, and quantified in kinetics (left) or as AUC (right). (B) Tumor cell count was monitored as a surrogate of tumor cell proliferation, and quantified in kinetics (left) or as AUC (right).
CD8 T cell-mediated killing of A375 melanoma cells increases with anti-CD3 stimulation
Melanoma A375 tumor cells were seeded and 48h later were co-cultured with activated CD8 cells, in the presence and absence of increasing concentrations of anti-CD3 antibody. (A) Tumor cell apoptosis was monitored and quantified over a period of ~3 days, by mean of a caspase 3/7 fluorescent probe and a NucRED probe expression, respectively, as surrogate measures of immune cell killing activity towards tumor cells. (B) IFNg released levels in the co-culture supernatants were quantified by mean of a specific HTRF detection kit. Data are expressed as means ± SEM.
T-cell engager case study
T-cell engagers redirect cytotoxicity toward target-expressing tumor cells
Making a difference as a preclinical CRO in oncology
Why work with Explicyte
Proven experience
>10 years in preclinical immuno-oncology, deep expertise in T-cell killing assays, over 100 in vitro campaigns delivered.
Built around your program
A PhD-level study director designs the study around your target, tumor model, and modality, from experimental plan to final-report discussion.
From functional response to mechanism
Immunophenotyping, cytokine and secretome analysis, proteomics, and single-cell or spatial transcriptomics can be integrated to characterize the cellular and molecular responses associated with tumor killing.
Reports you can act on
Progress reports let you adjust the plan as the study runs; final reports arrive with ready-to-publish data that holds up in due diligence.
Paul Marteau, PharmD (study director), Imane Nafia, PhD (CSO), Loïc Cerf, MSc (COO), Alban Bessede, PhD (founder, CEO), Jean-Philippe Guégan, PhD (CTO)
Contact our team
Discuss your tumor killing study
Tell us your target, the modality you're testing (checkpoint, sensitizer, or T-cell engager), and the tumor indication — we'll scope the right model, effector cells, readouts, and controls, and send a fit-for-purpose proposal.
Frequently asked questions
Answers about T-cell killing assays
What is a T cell–mediated tumor killing assay?
An in vitro co-culture assay that measures how primary human immune cells affect tumor-cell growth and survival over time. Tumor-cell count and apoptosis are monitored by live-cell imaging, alongside selected immune-response readouts. Purified T-cell or CD8 T-cell formats can be used when a more focused assessment of T-cell-mediated activity is required.
What can I test with it?
Any modality whose mechanism runs through T-cell-mediated killing — immune checkpoint inhibitors, T-cell agonists and co-stimulators, tumor-sensitizing agents, and bispecific / multispecific T-cell engagers — alone or in combination, including with standard-of-care checkpoint antibodies. If your candidate is meant to make T cells kill tumor cells, the assay can be designed around it.
What effector cells and tumor models do you use?
Primary human PBMCs, purified T cells, or CD8 T cells, co-cultured with human tumor cell lines from a library of 100+ (breast, lung, melanoma, small-cell lung cancer, and others) at an optimized effector-to-target ratio.
What are the main readouts?
Core readouts include tumor-cell count and caspase-3/7-dependent apoptosis monitored kinetically over approximately 3–5 days. Tumor-cell count reflects net tumor growth, while caspase-3/7 provides a complementary measure of apoptosis. Soluble markers such as IFN-γ, TNF-α or IL-6 are selected according to the immune cell format and study biology. Optional readouts include flow-cytometry immunophenotyping, cytokine and cytotoxic-effector profiling, proteomics and transcriptomics.
How do you show a T-cell engager is specific?
The dose-response kill is run on a target-positive tumor line alongside a target-negative control line (both confirmed by flow cytometry). Target-dependent activity is demonstrated by comparing killing in target-positive and target-negative models, with and without immune effector cells — as shown in our tarlatamab (DLL3) case study.
What controls and reference standards are available?
Benchmark standards include anti-PD-1 (nivolumab, pembrolizumab) and anti-PD-L1 (atezolizumab, avelumab), with anti-CD3 as an activation reference. Sensitization studies compare treated versus untreated tumor cells; engager studies use a target-negative control line.
Which indications and cell lines can you cover?
We work from a library of 100+ human tumor cell lines spanning solid and hematological indications, so most targets and indications can be covered — and we can source or bring in a specific line on request. Lines used in the examples on this page include MDA-MB-231 (breast), A549 & SHP77 (lung), and A375 (melanoma).
What is the format and timeline?
96- or 384-well plates in dose-response format, with kinetic imaging reads over roughly 3–5 days. Timeline depends on the number of conditions, cell lines, and add-on readouts — a study director scopes it with you.
