Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.
Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells' ability to eradicate dysfunctional target cells.
The ability of the human body to identify non-self and eradicate foreign objects is key to human survival against pathogens and malignancies1. The human immune response plays the most important role in this process2,3,4. Based on key characteristics and functions, the human immune system can be broadly classified into two major functional groups: the adaptive immune system and the innate immune system. The adaptive immune system is typically specific to a given pathogen and has immunological memory and, thus, is long-lasting and responsive to future re-infection by the same pathogen5,6,7,8,9. In contrast, innate immunity is much broader in its target eradication and relatively nonspecific. Therefore, typically, the innate immune response serves as the first line of immunological defense10. Natural killer (NK) cells belong to the innate immune system and represent 10−15% of total circulating lymphocytes11. NK cells eradicate target cells via two major mechanisms. First, upon binding to target cells expressing activating ligands, NK cells release the membrane-disrupting protein perforin and serine protease granzymes through exocytosis, which jointly induce apoptosis in target cells12,13,14,15. Additionally, NK cells expressing FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) interact with target cells expressing death receptors (Fas/CD95), leading to caspase-dependent apoptosis16. Most importantly, NK cells do not require prestimulation, such as antigen presentation, to eradicate pathogen-infected or malignant cells; thus, they are usually in a ready-to-kill state17,18. In order to inhibit tumor development and progression and eradicate cancer cells, NK cells must migrate to the tumor site and, once in the tumor microenvironment, identify and attack the target cells.
In the past, NK-cell effector functions were mainly monitored by degranulation and cytotoxicity assays19,20,21. NK cell cytotoxicity can also be measured by 51chromium release assay22,23,24,25. However, this assay has some specific requirements, including the need for a gamma counter, and is radioactivity-based, which requires training in handling and disposal of radioactive materials and poses a level of risk to the user. Therefore, several new non-radioactivity-based assays have been developed and employed to study NK cell activity.
Here, we describe two such protocols, colorimetric lactic dehydrogenase (LDH) measurement-based NK cell-mediated cytotoxicity assay and calcein acetoxymethyl (AM) staining-based microscopic method to measure NK cell-mediated cancer cell eradication. These assays do not require the use of radioisotopes, are straightforward, sensitive, and reproducibly identify factors that modulate NK cell function. Additionally, because NK cell function cannot be fully evaluated without monitoring changes in NK cell migration, we also present a flow cytometry-based quantitative method to monitor NK cell migration.
1. Preparation of Culture Medium for NK Cells and Hepatic Tumor Cells
2. Colorimetric LDH Measurement-based NK Cell-mediated Cytotoxicity Assay
3. Calcein AM Staining-based Microscopic Method for Measuring NK Cell-mediated Cytotoxicity
4. NK Cell Migration Assay
NK cell cytotoxicity assays and NK cell migration assay were performed using the SK-HEP-1 hepatic tumor cell line as a model system. To measure NK cell cytotoxicity using the LDH assay, SK-HEP-1 cells expressing either a nonspecific (NS) shRNA or shRNA targeting activating transcription factor 4 (ATF4) were incubated with NK92MI cells in a 96 well plate for 3 h (Figure 1A). ATF4 has been previously shown to regulate NK cell cytotoxicity by upregulating the activating ligand ULBP128. LDH activity associated with NK cell-mediated target cell killing was measured colorimetrically, and percent cytotoxicity calculated using the formula described in protocol 1. ATF4 knockdown significantly reduced NK cell-mediated cytotoxicity compared to NK cells expressing NS shRNA (Figure 1B-D). We also measured NK cell cytotoxicity using a calcein AM staining-based assay. To this end, SK-HEP-1 cells expressing NS shRNA or ATF4-targeting shRNAs were labelled with calcein AM and incubated with NK92MI cells in 96 well plates for 4 h as illustrated in Figure 2A. After incubation, images of calcein AM-positive cells were captured by fluorescence microscopy using a FITC/GFP filter. Cells killed by NK cells are not detected by this approach because they no longer retain the calcein AM dye. As shown in Figure 2B,C, the number of calcein AM-positive SK-HEP-1 cells is decreased after co-culture with NK92MI cells compared to SK-HEP-1 cells grown without NK92MI cells. However, as expected, ATF4 knockdown via shRNA reduced NK cell-mediated killing of SK-HEP-1 cells, observed by a greater number of calcein AM-positive cells (Figure 2B,C). Therefore, both LDH-based and calcein AM assays showed consistent results and confirmed that ATF4 knockdown reduces NK-mediated cancer cell cytotoxicity. Either of these assays is sufficient to assess NK cell-mediated cytotoxicity; however, we recommend using both methods to increase both the stringency and confidence in the results.
We also present the results of a 24 well NK cell migration assay. NK92MI cells were resuspended in serum-free NK92MI medium in the upper chamber, and chemokine, CC motif, ligand 2 (CCL2) was added to the lower permeable chamber. NK cell migration was assayed as described in section 3 (Figure 3A). The number of migrated NK92MI cells was quantified by adding counting beads for flow cytometry and followed by FACS analysis. As shown in Figure 3B-C, there was a significant increase in the number of NK92MI cells that had migrated toward CCL2-containing medium compared to control medium.
Figure 1: Colorimetric LDH activity-based NK cell-mediated cytotoxicity assay. (A) Schematic depicting the key steps of colorimetric LDH activity-based NK cell cytotoxicity assay. (B,C) ATF4 expression was analyzed in SK-HEP-1 cells expressing either nonspecific (NS) shRNA or shRNAs targeting ATF4 by quantitative RT-PCR and western blotting. (B) Relative ATF4 mRNA level is shown after normalization to ACTINB level in SK-HEP-1 cells expressing NS shRNA or ATF4 shRNAs. (C) ATF4 and ACTINB protein levels in SK-HEP-1 cells expressing NS shRNA or ATF4 shRNAs. (D) NK cell-mediated cytotoxicity was analyzed in SK-HEP-1 cells expressing either NS shRNA or ATF4 shRNAs by the LDH method. Percent (%) NK cell-mediated cytotoxicity is shown. Data are presented as mean ± SEM; ns, not significant; **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
Figure 2: Calcein AM staining-based microscopic method for measuring NK cell-mediated cytotoxicity. (A) Schematic depicting the key steps of calcein AM staining-based microscopic method for measuring NK cell-mediated cytotoxicity. (B) NK cell-mediated cytotoxicity was analyzed in SK-HEP-1 cells expressing either NS shRNA or ATF4 shRNAs using the calcein AM method. Representative images are shown. Scale bar = 200 μm. (C) Percent of calcein AM-positive cells for the experiment presented in panel B. **p < 0.01. Please click here to view a larger version of this figure.
Figure 3: FACS-based quantitative NK cell migration assay. (A) Schematic depicting the key steps of FACS-based NK cell migration assay. (B,C) NK cell migration assay was carried out after adding 50 ng of CCL2 to the bottom chamber of the 24 well plate. (B) Representative NK cell migration dot plots for control or CCL2-treated culture medium are shown. (C) NK cell migration data (mean ± SEM) is presented for the experiment shown in panel B. ***p < 0.001. Please click here to view a larger version of this figure.
The cytotoxicity and migration methods described here can be used to evaluate NK cell cytotoxicity to cancer cells and NK cell-mediated immune evasion mechanisms in malignant tumors, as well as to identify therapeutic agents that will enhance NK cell activity/function. The protocols are simple, sensitive, reproducible, and preferable alternatives to classical radioactivity-based 51chromium release assay. The protocols have been specifically designed to be readily adaptable for use in most laboratories, with straightforward colorimetric, microscopic, or FACS-based readouts that are easy to interpret, allowing researchers to reach reliable conclusions. All are scalable for high-throughput screening-based approaches. Although the protocols are presented in the context of a single hepatic tumor cell line, they can be easily adapted easily to other cancer cell types and/or other non-cancer target cells.
Whereas all of the methods presented are robust and reproducible, inter-experimental variation could occur with different batches of cancer cells and NK cells. To ensure that results accurately support the conclusions of the experiments, it is advisable to repeat the experiment at least two times using biological triplicates.
A limitation of this method is that the growth rate of NK92MI cells can be slow. Therefore, for experiments with 50 or more samples, sufficient numbers of NK92MI should be grown beforehand to prevent delays. Also, all of the controls described in the protocols must be implemented to avoid spurious and nonreproducible results. Another limitation of the NK cytotoxicity assay is that the NK to cancer cell ratio, as well as the incubation time, must be optimized for each target cell. For example, we have tested various ratios of cancer cells to NK cells (1:5, 1:10, 1:20, 1:40, and 1:80) for the hepatic cancer cell lines, as well as multiple incubation times (2, 3, 4, 5, and 6 h). Based on our results we observed most consistent results with 1:10 and 1:20 ratios of cancer cells to NK cells and incubation for 3 h.
In addition, cancer cells derived from solid tumors will attach and grow on the surface of the culture plate, whereas NK cells grow in suspension. If the incubation time is longer than 3 h, it is advisable to use ultralow attachment 96 well plates, which will improve consistency and reproducibility between experiments and biological replicates. It is also important to note that NK cell-induced cytotoxicity assays can also be performed using primary NK cells isolated from peripheral blood mononuclear cells (PBMCs). However, there are several limitations with such experiments. First, these experiments cannot be as easily scaled as with NK92MI cells. Second, batch-to-batch variation of cytotoxic activity of NK cells isolated from PMBCs can be problematic in terms of reproducibility and interpretation of the results. Similarly, other human NK cell lines are described in the literature and could be used in these types of experiments, including NKL cells29; however, unlike NK-92MI cells, NKL cells are IL-2 dependent and not commercially available.
When considering the calcein AM experiments described, it is important to note that calcein AM remains in apoptotic bodies after cell death30. Therefore, the quantitation of calcein AM staining should be carefully performed, as it may lead to an underestimation of NK cytotoxicity.
Similar to NK cell-mediated cytotoxicity, modulation of NK cell migration by cytokines and other chemoattractants plays an important role in the regulation of NK cell function. The NK cell migration assay described here provides a simple platform to evaluate NK cell migration in the context of a stimulus such as a chemokine or chemoattractant. This assay can be used to evaluate agents that can either promote or interfere with NK cell migration – thus, identifying enhancers and repressors of NK cell migration. This method can also be useful to study the NK cell migration regulatory capacity as a result of a genetic/epigenetic alterations (upregulation or downregulation) or arising due to drug treatment.
To accurately measure NK cell migration, there are few critical steps that should be followed. For all of the experiments using conditioned medium, it is important that equivalent conditioned medium be used from both control and treatment conditions to obtain accurate measurements. The time of incubation will be varied with purified chemoattractants and conditioned medium. Finally, transwell migration assays are well established and considered an excellent method to assess NK cell migration; however, the homogeneous monocultures employed in the assays lack the complex physiology of tissues or even 3D cultures that might more accurately mimic the tumor microenvironment.
Thus, although there are some limitations to the NK cell cytotoxicity assays and NK migration assay presented in this article, these assays are applicable to a wide range of immunological studies and thus provide important and reliable methods to assess NK cell function and NK cell modulatory immune therapeutics.
The authors have nothing to disclose.
We gratefully acknowledge grants from the National Institutes of Health: R01CA195077-01A1 (NW), R01CA200919-01 (NW), and 1R01 CA218008-01A1 (NW). We also acknowledge Department of Defense funding W81XWH1910480 and W81XWH-18-1-0069 to NW.
Absolute counting beads | Thermo Fisher Scientific | C36950 | |
Alpha-MEM | Sigma-Aldrich | M4256 | |
Amicon ultra Centrifugal filters | Millipore | UFC900324 | |
Calcein AM dye | Sigma-Aldrich | 17783 | |
DMEM | Gibco | 11965-092 | |
Fetal Bovine Serum | Gibco | 26140079 | |
Folic Acid | Sigma-Aldrich | F8758 | |
Horse Serum | Gibco | 16050114 | |
L-Glutamine (200 mM) | Gibco | 2530081 | |
LDH cytotoxic assay Kit | Thermo Fisher Scientific | 88953 | |
myo-Inositol | Sigma-Aldrich | I5125 | |
NK92MI cells | ATCC | CRL-2408 | |
Opti-MEM | Gibco | 31985070 | |
Phosphate Buffer Saline (PBS) | Sigma-Aldrich | P4417 | |
SKHEP-1 Cells | ATCC | HTB-52 | |
Transwell permeable chambers | Costar | 3241 | |
Trypsin EDTA solution | Gibco | 25200056 |