NK Cell Assays
There is significant interest in harnessing Natural Killer Cell’s ability to kill tumor cells and one approach is to generate allogeneic NK cell therapies which are applicable to multiple cancers. 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.
As part of our immuno-oncology offering, Charles River have developed a range of primary human NK cell immuno-oncology assays to determine the ability of your novel therapeutics to modulate NK cell activation, proliferation and tumor cell killing.
Charles River’s Natural Killer Cell Assays include:
- IncuCyte tumour killing assay (2D) ADCC
- IncuCyte spheroid killing assay (3D) ADCC
- NK cell proliferation assays
- NK effector function flow cytometry assays
Tumor Killing Assays
Natural Killer Cells can be cultured with activating cytokines such as IL-12 and IL-15 and tested for their ability to kill tumor targets in a FACS-based or Incucyte based assay, depending on your requirements. NK cell-mediated tumor killing can also be assessed in a 2D format or a 3D ‘spheroid-like’ format (Figure 2) using either whole PBMC or purified NK cells. For more information on Charles River’s tumor killing assays visit our web page.
Figure 1: Natural Killer cell-mediated tumor killing in 3D/spheroid format, and example data demonstrating enhancement of killing over time with Trastuzumab.
The ability of novel therapeutics to potentiate NK cell killing can then be assessed by counting the number of viable tumor cells or spheroid area over time. Positive control agents which enhance tumor killing have been validated in this assay via either ADCC (Trastuzumab) or cell-mediated (TLR7 agonists) killing mechanisms. Caspase 3/7-dependent tumour cell apoptosis can also be determined by co-localisation of the caspase signal to NuclightRed positive tumor cells.
Natural Killer Cell Proliferation Assays and Effector Function Assays
There are circumstances where it may be more relevant to focus on the NK cells, as well as tumor cells, in a functional assay and a flow cytometry panel may be used to assess a range of NK-based readouts in co-cultures with tumour cells. NK cells are heterogeneous in nature and the CD56bright population are specialised in their ability to produce large amounts of IFNg whilst the CD56dim population are better killers. The ability of NK targeting agents to enhance NK cell proliferation and effector function may also be determined in FACS-based assays.
Figure 2: FACs-based assays data demonstrating the ability of NK targeting therapies to enhance NK cell proliferation (left graph) and effector function (right 2 graphs). NK cell effector mechanisms which may be evaluated include intracellular granzyme B, perforin or IFNγ and membrane associated LAMP-1 (CD107a).
Natural Killer Cell Assay FAQs
Can we test our therapeutics in both PBMC and NK cell killing assays?
Charles River offer both PBMC and NK cell killing assays, both work equally well in this format and using whole PBMC may be beneficially if bystander cell involvement is required.
How do we know our target is expressed on the tumor cell lines you commonly use?
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 2D or 3D assays?
This depends on your target but we would recommend 3D for ADCC and starting with 2D for cell-mediated mechanisms. If successful, the latter may then progress into 3D testing. Charles River’s immunologists can advise on the best assay.
Is it better to test our therapeutics in flow-based or Incucyte-based killing assays?
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.