Cytotoxic T Cell-Based Tumor Killing Assays
Charles River have a range of immune mediated tumor killing assays including cytotoxic T cell assays and natural killer (NK) cell assays. These assays are analysed using flow cytometry and live cell imaging (IncuCyte); IncuCyte-based assays quantify a target number that can be multiplexed with target specific apoptotic readout, and can be tailored to the anticipated mechanism of action by selecting from a panel of validated target cell lines.
Immune Mediated Tumor Killing Assays
Figure 1: Diagram of Immune Mediated Tumor Killing Assays used to evaluate oncology immunotherapies.
Our panel of immune-mediated tumor killing assays include:
- IncuCyte® tumor killing assays (2D)
- Macrophage-dependent ADCC assays
- IncuCyte® spheroid tumor killing assays (3D)/CDC assays
- Flow cytometry-based killing assays
- Natural killer (NK) cell tumor killing assays
Watch the video below to learn more about our assays and the value for your drug discovery.
Director of Science, Rhiannon Jenkinson, discusses how the use of tumor killing assays can aid oncology drug development.
Rhiannon Jenkins (00:07): The aim of many immune oncology therapies, is either to reinvigorate the immune response, or to initiate a novel, unique immune response against the tumor, which ultimately leads to tumor cell killing. Rhiannon Jenkins (00:21): When we're thinking about which cell populations are responsible for tumor cell killing, these are mostly T cell populations, so CD8 cytotoxic T cells, and natural killer or NK cells. Rhiannon Jenkins (00:33): To assess tumor cell killing, we use an NK cell-based platform, and we measure two different parameters. So we're co-culturing a tumor cell line, with PBMCs isolated from healthy human donors. Rhiannon Jenkins (00:47): And then we can read out tumor cell numbers, and the percentage of apoptotic tumor cells within these cultures. So this gives us information on whether the immune cells within that PBMC subset, are driving tumor killing over time. Rhiannon Jenkins (01:03): One of the different flavors of assays that we run, is one where we assess ADCC. So in that particular assay, we add an antibody into the tumor cell and PBMC co-culture. Rhiannon Jenkins (01:17): The antibody coats the tumor cells in a specific manner, and then that allows us to assess the amount of ADCC going on within those co-cultures. Rhiannon Jenkins (01:29): We run the tumor killing essays in two different flavors, either in a 2D format, where the PBMCs are added on top of tumor cells, which culture on a plastic, or we can run it in a spheroid format, where the tumor cells form an organoid or a ball of cells. Rhiannon Jenkins (01:47): And then we can monitor tumor cell killing in that context as well. And that particular assay setup is useful when we're thinking about antibody-dependent cellular cytotoxicity, or ADCC. Rhiannon Jenkins (02:00): And that's a very useful setting for understanding that process, and whether your therapeutic is enhancing killing in that context. Rhiannon Jenkins (02:08): We can define the subsets that are responsible within the PBMC mix for killing the tumor cells, using two different strategies. Rhiannon Jenkins (02:16): We can either deplete those subsets from the total PBMC mix, or we can take the approach where we purify the different subsets out, and add them on top of the tumor cells, and monitor tumor cell killing. Rhiannon Jenkins (02:28): On which assay set up you choose, it's very much dictated by what your therapeutic is, and your anticipated mode of action. Rhiannon Jenkins (02:37): These assays give you critical information on the mode of action of your drug, whether you're hitting your target for the expected outcomes, so are you enhancing tumor cell killing using relevant cell biology systems, before you move into efficacy models?
IncuCyte tumor killing assays (2D)
Cytotoxic T cells can be cultured with activating antibodies such as anti-CD3 and tested for their ability to kill tumor targets in an IncuCyte-based assay. The ability of novel therapeutics to potentiate cytotoxic T cell killing can then be assessed by quantifying the change in the number of viable tumor cells over time. Positive control agents which enhance tumor killing have been validated in this assay. Caspase 3/7-dependent tumor cell apoptosis can also be determined by co-localization of the caspase signal to NucLight Red positive tumor cells. The target cell population can be adapted to suit the disease indication or mechanism of action.
Data: Tumor killing assays (2D)
Figure 2: PBMC - mediated tumor killing in 2D format, and example image, demonstrating killing over time with checkpoint inhibitor treatment with or without depletion of CD8+ cytotoxic T cells. Alt tag: PBMC - mediated tumor killing assay in 2D format, demonstrating killing over time with checkpoint inhibitor treatment with or without depletion of CD8+ cytotoxic T cells.
IncuCyte spheroid immune mediated PDX tumor killing assays (3D)/CDC assays
Our team can also run immune cell killing assays with PDX (patient-derived xenograft) material; in vitro 3D assays incorporate live cell imaging to predict in vivo responses to matched PDX material. Screening PDX models in vitro prior to in vivo humanized tumor killing models enables stratification and prioritization of the wide range of in vivo PDX models available.
Data: Tumor killing assay (3D)
Figure 3: PBMC - mediated tumor killing in 3D format, and example images, demonstrating killing over time with or without IL-2 stimulation. Supernatants were harvested and levels of IFNg analysed as a surrogate marker to effector cell activation.
The combination of in vitro screening with in vivo trial design options will aid your drug development and increase throughput.
Have a question or need advice on which assay is right for you?
Frequently Asked Questions (FAQs) About Tumor Killing Assays
Can I test my therapeutics in both PBMC and purified cell assays?
Charles River offers both PBMC and purified cell assays. For example, purified CD8 T cells or monocytes can be co-cultured with target cells alone or in combination. These types of studies can help to refine the cellular target and understand which cell types work together to mediate tumor cell killing.
Which target cell lines are validated for tumor killing assays?
Charles River has an extensive list of 2D and 3D validated tumor cell targets from a range of tissue types including lung, ovarian, pancreatic and intestines. Where a specific line is required, cells are transduced with a nuclear RFP and a pilot experiment is run to determine the optimal seeding densities ahead of efficacy/mechanistic testing. This process has also been successfully applied to PDX cell lines after identifying relevant gene expression using our cancer model database.
Can I use murine lines to predict in vivo efficacy in tumor models?
Murine tumor cell lines are compatible with nuclear RFP transduction and we see variation in the extent of killing depending on the strain of murine PBMC used in co-culture. Murine tumor killing assays are useful to refine test conditions ahead of limited and expensive in vivo studies.
How do I know my target is expressed on the tumor cell lines you commonly use?
Our team can screen cell lines for your target expression by flow cytometry, western blot or qPCR. Target cells can also be induced to express a target, for example, IFNγ stimulates upregulation of PD-L1 by many tumor lines. Alternatively, genetic manipulation can force expression or deletion of required proteins.
Is it better to test our therapeutics in 2D or 3D PBMC cell tumor killing assays?
This depends on your target and therapeutic approach. 2D IncuCyte assays enable target and apoptotic readouts while 3D formats better model the in vivo tumor structure. If successful, testing could then progress to in-vitro/in-vivo PDX testing or syngeneic mouse models. Our team of immunologists can advise the best assay for your purpose.
Is it better to test our therapeutics in flow-based or IncuCyte-based assays?
Flow-based assays are ideal for suspension tumor target cells while the IncuCyte platform is best suited to adherent tumor cell lines. Flow cytometric based assays enable additional mechanistic information via inclusion of surface markers and intracellular cytokine staining, providing the ability to assign function to specific PMBC cell subsets, which is not possible using the IncuCyte. For example, expression of activation markers (such as CD25 and CD69) or effector molecules (e.g., IFNγ and Granzyme B) by phenotypically distinct PBMC subsets can be quantified alongside therapeutic targets or potential inhibitory receptors like PD-1. IncuCyte imaging is adaptable to higher-throughput screening and gives a highly quantitative and visual representation of the ability of your therapeutic to modulate tumor killing. Charles River can work with you to select the best assay for your program.