Name: measurement of natural killer cell-mediated cytotoxicity and migration in the context of hepatic tumor cells
Uploaded: 2020-02-22T14:00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells at JoVE.com

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.

1. Preparation of Culture Medium for NK Cells and Hepatic Tumor Cells
<ol>
	<li>Use human natural killer cells (e.g., NK92MI) and human liver cancer cell line (e.g., SK-HEP-1).</li>
	<li>Prepare NK cell culture medium for NK92MI human NK cells by adding the following components to 500 mL of minimum essential medium Eagle alpha without ribonucleosides and deoxyribonucleosides to the indicated final concentrations: 0.02 mM folic acid (100 &#181;L of 100 mM folic acid), 0.2 mM myoinositol (500 &#181;L of 200 mM myoinositol), 0.1 mM &#946;-mercaptoethanol (3.5 &#181;L of 14.3 M &#946;-mercaptoethanol), 2 mM L-glutamine (5 mL of 200 mM L-glutamine), 1% penicillin/streptomycin (5 mL of 100x penicillin/streptomycin), 12.5% fetal bovine serum (FBS), and 12.5% horse serum. Mix and filter sterilize using a 0.22 &#181;m sterile filtration unit, and store at 4 &#176;C.</li>
	<li>Prepare culture medium for hepatic tumor cell line SK-HEP-1 by adding 10% FBS and 1% penicillin/streptomycin to high-glucose Dulbecco&#39;s modified Eagle medium (DMEM) containing 2 mM L-glutamine.</li>
</ol>
2. Colorimetric LDH Measurement-based NK Cell-mediated Cytotoxicity Assay
<ol>
	<li>Grow SK-HEP-1 cells to 70&#8722;80% confluency in a 100 mm cell culture Petri dish in 5% CO2 at 37 &#176;C in a CO2 incubator. Generate a single-cell suspension by first washing cells with 5 mL of 1x phosphate-buffered saline (PBS) followed by incubation with 1 mL of 0.25% trypsin-EDTA in 5% CO2 at 37 &#176;C in a CO2 incubator until a single cell suspension is generated. 
	NOTE: NK cell activity can be induced by stressed cancer cells due to changes in expression of NK cell-activating ligand in cancer cells. Therefore, healthy and sub-confluent cancer cells should be used for accurate results. It is also important to note that NK cell receptors and ligands can be sensitive to trypsinization26,27. Therefore, the trypsinization protocol should be carefully optimized and excessive trypsinization should be avoided.</li>
	<li>After trypsinization, add 10 mL of SK-HEP-1 culture medium and centrifuge at 160 x g for 3 min in a 15 mL sterile conical centrifuge tube. Wash the cell pellet with 5 mL of 1x PBS and resuspend in 5 mL of culture medium.</li>
	<li>In parallel, centrifuge the NK92MI cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube. Wash the cell pellet with 5 mL of 1x PBS and resuspend in 5 mL of NK92MI cell culture medium. 
	NOTE: NK92MI cells are grown in NK cell culture medium and obtained similarly as described in step 2.1.</li>
	<li>Count the SK-HEP-1 and NK92MI cells using a hemocytometer or any available automated cell counter.</li>
	<li>Add SK-HEP-1 cells (target cells) (1 x 104/100 &#181;L/well) and NK92MI cells (effector cells) (1 x 105/100 &#181;L/well) in the ratio of 1:10 target:effector and seed in triplicate wells in a 96 well plate.</li>
	<li>Incubate the 96 well plate at 37 &#176;C in an atmosphere of 95% air and 5% CO2 for 3 h. After incubation, centrifuge plate at 450 x g for 5 min at room temperature.</li>
	<li>Without disturbing the cell pellet, collect 100 &#181;L of supernatant from each well and transfer to a well in a new 96 well plate.</li>
	<li>Add 50 &#181;L of LDH substrate, mix well, and incubate the plate for 20 min at room temperature in the dark.</li>
	<li>Stop the reaction by adding 50 &#181;L of stop solution (50% dimethylformamide and 20% sodium dodecyl sulfate at pH 4.7). Immediately measure the absorbance of the plate at 490 nm and 680 nm using a plate reader.</li>
	<li>Subtract the absorbance at 680 nm from the absorbance at 490 nm. Calculate the percent (%) NK cell cytotoxicity using the formula below. 
	<img src="/files/ftp_upload/60714/60714eq1.jpg" /> 
	where LDH experimental (effector + target cells) is the absorbance of NK92MI cells and SK-HEP-1 cells, LDH effector cells is the absorbance of NK92MI cells alone, LDH spontaneous is the absorbance of SK-HEP-1 cells alone, and LDH maximal is the absorbance of SK-HEP-1 cells with lysis buffer. 
	NOTE: To reduce serum interference, always use the following controls: target cells alone (SK-HEP-1), effector cells alone (NK92MI), target cells with lysis buffer as a complete lysis control, target cell medium, NK92MI medium, as well as target cell medium and NK92MI medium in a 1:1 ratio.</li>
</ol>
3. Calcein AM Staining-based Microscopic Method for Measuring NK Cell-mediated Cytotoxicity
<ol>
	<li>Culture SK-HEP-1 cells to 70&#8722;80% confluency. Generate a single-cell suspension by first washing cells with 5 mL of 1x PBS followed by incubation with 1 mL of 0.25% trypsin-EDTA.</li>
	<li>Centrifuge SK-HEP-1 cells in a 1 mL sterile centrifuge tube at 160 x g for 3 min. Resuspend the pellet in 3 mL of serum-free DMEM.</li>
	<li>Add 1.5 &#181;L of calcein AM solution (10 mM) to SK-HEP-1 cells and incubate for 30 min at room temperature. Centrifuge calcein AM-labeled SK-HEP-1 cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube.</li>
	<li>Wash cells twice with 5 mL of 1x PBS to remove excess calcein AM dye.</li>
	<li>In parallel, centrifuge NK92MI cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube. Wash the cell pellet once with 5 mL of 1x PBS and resuspend in 5 mL of NK92MI cell medium.</li>
	<li>Count calcein AM-labeled SK-HEP-1 cells and NK92MI cells using a hemocytometer or an automated cell counter.</li>
	<li>Resuspend SK-HEP-1 cells in culture medium at 1 x 105 cells/mL and NK92MI cells at 1 x 106 cells/mL in NK cell medium.</li>
	<li>Plate calcein AM-labeled SK-HEP-1 cells (target cells) (1 x 104/100 &#181;L/well) with NK92MI cells (effector cells) (1 x 105/100 &#181;L/well) (1:10 target:effector ratio) per well in triplicate wells in a 96 well plate.</li>
	<li>Incubate the 96 well plate at 37 &#176;C in an atmosphere of 95% air and 5% CO2 for 4 h. After incubation, capture fluorescence images of the calcein AM-labeled cells using a fluorescence microscope at 10x magnification. Capture at least 10 different fields of each replicate for each treatment condition.</li>
	<li>Randomly select 10 images for each replicate and count calcein AM-positive labeled target cells incubated with or without NK92MI cells. Calculate % cytotoxicity using the formula below. 
	<img src="/files/ftp_upload/60714/60714eq2.jpg" /> 
	NOTE: As controls, use target cells without NK92MI cells and completely lysed target cells as complete lysis control. For complete lysis, incubate the cells in 0.5% Triton X-100 for 1 h (20 &#181;L of 5% Triton X-100 in 200 &#181;L of culture media).</li>
</ol>
4. NK Cell Migration Assay
<ol>
	<li>Grow NK92MI cells and centrifuge cells at 160 x g for 3 min in a 15 mL sterile centrifuge tube.</li>
	<li>Wash the cell pellet twice with 5 mL of 1x PBS and resuspend the cells in 3 mL of serum-free NK92MI cell medium. Count NK92MI cells using a hemocytometer or an automated cell counter.</li>
	<li>Plate NK92MI cells (2.5 x 105 cells/100 &#181;L/well) in the upper compartment of transwell permeable chamber (6.5 mm diameter insert and 5 &#181;m pore size).</li>
	<li>In the bottom chamber add 0.6 mL of serum-free medium containing material to be tested for NK cell chemoattractant properties (e.g., conditioned medium, chemokines, cytokines). 
	NOTE: When preparing conditioned medium, use reduced-serum medium without added serum to eliminate interference from serum proteins in the migration assay.</li>
	<li>Incubate the 24 well permeable chambers at 37 &#176;C for 4 h. After 4 h, collect the non-adherent and migrated NK92MI cells from the bottom chamber and transfer them to fluorescence-activated cell sorting (FACS) tubes for further analysis. 
	NOTE: The time of culture may vary depending on the type of target cells, as well as the amount and kinetics of chemokines produced by the target cells. Therefore, this time should be empirically determined for each cell type, chemokine, and experiment.</li>
	<li>Add predetermined number of counting beads for flow cytometry in a volume of 50 &#181;L to each tube containing migrated NK cells. Evaluate the volume of 300 &#181;L/well cell suspension using any flow cytometer capable of automated FACS-based cell counting. 
	NOTE: Mix or vortex-mix the counting beads for flow cytometry thoroughly each time before use to ensure that a constant number of beads is used to minimize experimental variability. Reverse pipetting is recommended with count beads to maintain accuracy. Use only NK cells and counting beads for flow cytometry as FACS analysis controls. The authors recommend reading at least 10,000 beads + NK cells combined, an amount that has worked well. However, this number may vary depending upon the experimental conditions. Therefore, the combined number of beads + NK cells should be empirically determined for each type of experiment. Additionally, it is important to perform experiments using biological triplicates to achieve statistically significant results and to account for variability between different cell counts.</li>
	<li>Calculate absolute number of migrated NK92MI cells using this formula: 
	<img src="/files/ftp_upload/60714/60714eq3.jpg" /> 
	where A = number of cells, B = number of beads, C = assigned bead count of the lot (number of counting beads for flow cytometry/50 &#181;L; in this example 49,500), and D = volume of sample (&#181;L). 
	NOTE: If 300 &#181;L of sample volume (migrated cells) is used for FACS analysis with 50 &#181;L of counting beads for flow cytometry, the absolute number of migrated cells = 1,700 cells /3,300 bead events x 49,500 beads/300 &#181;L = 84.975 cells/&#181;L. The calculation should be corrected if the sample is diluted or if a different volume of FACS counting beads are used.</li>
</ol>

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The authors have nothing to disclose.

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 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&#8722;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute counting beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M4256</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra Centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM dye</td>
			<td>Sigma-Aldrich</td>
			<td>17783</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050114</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>2530081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH cytotoxic assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>88953</td>
			<td></td>
		</tr>
		<tr>
			<td>myo-Inositol</td>
			<td>Sigma-Aldrich</td>
			<td>I5125</td>
			<td></td>
		</tr>
		<tr>
			<td>NK92MI cells</td>
			<td>ATCC</td>
			<td>CRL-2408</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>SKHEP-1 Cells</td>
			<td>ATCC</td>
			<td>HTB-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell permeable chambers</td>
			<td>Costar</td>
			<td>3241</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of natural killer cell-mediated cytotoxicity and migration in the context of hepatic tumor cells

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&#39; ability to eradicate dysfunctional target cells.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60714/60714fig1.jpg" /> 
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 &#177; SEM; ns, not significant; **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/60714/60714fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60714/60714fig2.jpg" /> 
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 &#956;m. (C) Percent of calcein AM-positive cells for the experiment presented in panel B. **p &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/60714/60714fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60714/60714fig3.jpg" /> 
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 &#177; SEM) is presented for the experiment shown in panel B. ***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/60714/60714fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells at JoVE.com

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

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 ...

measurement-natural-killer-cell-mediated-cytotoxicity-migration

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The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...