NK Cell Assays
Immunotherapy is now a frontline treatment in multiple cancers; as we learn how to harness the power of the immune system, the list of treatment indications continues to grow.
- One mechanism of natural killer cell-mediated tumor lysis is via antibody-dependent cytotoxicity (ADCC) where opsonizing antibodies bind to tumor cell antigens and engage with activating Fc receptors expressed by NK cells triggering their cytotoxic activity.
- NK cells can also be triggered to kill target cells by a combination of signals through their positive and negative receptors, such as recognition of down-regulated MHC class I or up-regulation of stress ligands.
- Recently PD-1/PD-L1 blockade has been shown to enhance NK cell anti-tumor responses, highlighting an alternative mechanism of action for immune checkpoint blockade (ICB) which was historically thought to be solely T cell-driven.
NK Cells in Drug Development
Drug development which specifically targets natural killer cells function is comparatively underexplored compared to T cell-directed therapies where we’ve seen huge breakthroughs. However, NK cells are an attractive target for therapies, due to their cytotoxic function and potential tumor killing activity which, unlike T cells, is not MHC restricted. Scientists are now turning their attention towards a better understanding of how we can therapeutically enhance the potent cytolytic function of NK cells in order to eliminate tumor growth.
To best support your immuno-oncology programs, we’ve developed a multifaceted suite of primary human NK cell assays to determine the ability of novel therapeutics to modulate NK cell activation, proliferation and tumor cell killing.
Natural Killer Cell Based Assays
Figure 1: Diagram of NK Cell Assays used to evaluate immunotherapies.
Charles River’s NK Cell Assays include:
- NK phenotyping and activation flow cytometry assays
- NK cell cytokine production by intracellular flow cytometry or Luminex
- NK cell proliferation assays
- IncuCyte® tumor killing assay (2D) ADCC dependent and independent
- IncuCyte® spheroid killing assay (3D) ADCC dependent and independent
- Flow cytometric tumor killing assay
NK Cell Phenotyping Assays
Natural killer cells can be isolated from human peripheral blood mononuclear cells (PBMCs) by magnetic sorting and flow cytometric analysis, with purified cells having a CD3-, CD56+, CD16+/- phenotype (Figure 1). Purified NK cells can then be used in downstream functional assays such as proliferation analysis and tumor cell killing.
Example Data: Flow cytometry analysis of NK cells
Figure 2: In NK cell assays, natural killer cells were purified from human PBMC by magnetic cell sorting and stained with a panel of antibodies for flow cytometry analysis. NK cells were identified as CD3- CD56+ and CD16+/-.
NK Cell Proliferation Assays
Purified cell cultures enable direct assessment of the activity of a therapeutic on a single cell type without the complication of interplay between different cell types found in heterogeneous cell cultures.
Natural killer cells are a heterogeneous population; the CD56bright population are specialized in their ability to produce large amounts of IFNγ whilst the CD56dim population are better killers. The ability of NK cell targeting agents to enhance NK cell proliferation and effector function may also be determined in FACS-based assays (shown here by dilution of cell proliferation dye (CPD)).
Example Data: FACs-based data from NK Cell Proliferation Assay
Figure 3: Flow cytometry-based data demonstrating the NK cells proliferation (left graph) and effector function (right graph in response to stimulation). NK cell effector mechanisms which may be evaluated include intracellular IFNγ (shown) granzyme B, perforin and degranulation as measured by LAMP-1 (CD107a) at the cell surface.
NK Cell Tumor Killing Assays
Natural killer cells can be cultured with novel therapeutics, antibodies (e.g., Cetuximab) or activating cytokines (such as IL-12/IL-15) and tested for their ability to kill tumor targets in FACS or IncuCyte (R)-based assays (Figure 3). Where IncuCyte (R)-based NK cell assays are employed, the number of viable tumor cells over time are kinetically assessed and Caspase 3/7-dependent tumor cell apoptosis is concurrently determined by co-localization of the caspase signal to tumor cells.
Example Data: Natural killer cell-mediated tumor killing in 2-D format
Figure 4: Data demonstrating enhancement of ADCC killing over time with Cetuximab (IgG1 targeting EGFR) in an Natural Killer cell based assay and the effect of activating NK cells with a combination of IL-12 and IL-15 on the ability of NK cells to kill tumor targets.
3D Tumor Killing Assays
NK cell-mediated tumor killing can also be assessed in a 3D ‘spheroid-like’ format using either whole PBMC or purified NK cells. The ability of novel therapeutics to potentiate NK cell killing can then be assessed by quantifying the spheroid area over time. Positive control agents which enhance tumor killing have been validated in this assay via either ADCC (Trastuzumab) or non-ADCC killing mechanisms.
Kinetic analysis of NK meditated target cell killing. Purified NK cells (phase bright cells) were co-cultured with nuclear RFP expressing target cells (red) in the presence of activating cytokines IL-12/IL-15 and a caspase reporter dye. Images were acquired every 2h for 72h by IncuCyte®. Analysis software was used to identify target cells dying by apoptosis (yellow circles).
Our team can help you determine which tumor killing assays or other NK assay can best support your program.
Frequently Asked Questions (FAQs) about Natural Killer Cells
Why should I use NK cell assays?
Natural killer cells' ability to kill tumor cells can be harnessed for drug discovery. For example, PD1-PD-L1 blockade has been shown to elicit a robust NK cell anti-tumor response in animal models, suggesting a role for NK cells in more ‘traditional’ checkpoint therapies which were thought to be solely CD8+ T cell-driven. NK cell assays and tumor killing assays can be used to test your drugs ability to target and destroy cancer cells.
Should I test my therapeutic in PBMC or NK cell assays?
Charles River are able to screen cell lines for target expression for you by flow cytometry, western blot or qPCR.
Is it better to test our therapeutics in a 2D or 3D NK cell assay?
This depends on your target and therapeutic approach. 2D natural killer cell based 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 PDX and in vivo PDX testing or syngeneic mouse models. Charles River’s immunologists can advise on the best assay.
How do we know our target is expressed on the tumor cell lines you commonly use?
This depends on your target and what you are hoping to see. Flow-based assays are often far more rapid than those run using the IncuCyte, which can run up to 3-5 days. You can discover additional mechanistic information using flow cytometry and assign function to specific NK cell subsets, which is not possible using IncuCyte. However Incucyte is far more high-throughput and gives a highly quantitative and visual representation of the ability of your therapeutic to modulate tumour killing. Charles River can work with you to select the best assay for your program.
Is it better to test our therapeutics in flow-based or IncuCyte®-based killing assays?
Flow-based assays are ideal for suspension tumor target cells while the IncuCyte® platform is best suited to adherent tumor cell lines. Flow cytometry-based assays enable additional mechanistic information via inclusion of surface markers and intracellular cytokine staining, allowing us to assign function to specific Natural Killer Cells subsets, which is not possible using live imaging. For example, expression of activation markers (such as CD25 and CD69) or effector molecules (e.g., IFNγ and Granzyme B) by phenotypically distinct Natural Killer Cells subsets can be quantified alongside therapeutic targets or potential inhibitory receptors like PD-1. Live 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. We can work with you to select the best NK cell assay for your program.