Name: in vitro assay to study tumor-macrophage interaction
Uploaded: 2019-08-01T14:00:00
Description: Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Grant Support: Z. An received support from Alex&#8217;s Lemonade Stand Foundation, American Brain Tumor Association, NIH T32CA108462 and Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation. W. Weiss was supported by NIH grants R01CA221969, R01NS091620, P50CA097257, U01CA217864, P30CA82103; the Samuel G. Waxman Cancer Research Foundation; and the Evelyn and Mattie Anderson Chair.

1. Medium Preparation
<ol>
	<li>Preparation of the serum-free stem cell medium
	<ol>
		<li>Thaw the 50x B27 supplement, the epidermal growth factor (EGF, 20 &#956;g/mL in 10 mM acetic acid with 0.1% BSA), and the fibroblast growth factor (FGF, 20 &#956;g/mL in 10 mM acetic acid with 0.1% BSA).</li>
		<li>Add 500 &#956;L of EGF (final concentration 20 ng/mL), 500 &#956;L of FGF (final concentration 20 ng/mL), 10 mL of 50x B27 to 500 mL of Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/...

<ol>
	<li>Liu, Y., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+function+of+tumor-associated+macrophages.">The origin and function of tumor-associated macrophages.</a> Cellular &amp; Molecular Immunology. 12 (1), 1-4 (2015).</li><li>Riabov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+tumor+associated+macrophages+in+tumor+angiogenesis+and+lymphangiogenesis.">Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis.</a> Frontiers in Physiology. 5, (2014).</li><li>Coussens, L. M., Zitvogel, L., Palucka, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+tumor-promoting+chronic+inflammation:+a+magic+bullet.">Neutralizing tumor-promoting chronic inflammation: a magic bullet.</a> Science. 339 (6117), 286-291 (2013).</li><li>Tsukamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Blockade+of+IL6+and+PD-1/PD-L1+Signaling+Abrogates+Mutual+Regulation+of+Their+Immunosuppressive+Effects+in+the+Tumor+Microenvironment.">Combined Blockade of IL6 and PD-1/PD-L1 Signaling Abrogates Mutual Regulation of Their Immunosuppressive Effects in the Tumor Microenvironment.</a> Cancer Research. 78 (17), 5011-5022 (2018).</li><li>Borgoni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+tumor-associated+macrophages+switches+the+epigenetic+profile+of+pancreatic+cancer+infiltrating+T+cells+and+restores+their+anti-tumor+phenotype.">Depletion of tumor-associated macrophages switches the epigenetic profile of pancreatic cancer infiltrating T cells and restores their anti-tumor phenotype.</a> Oncoimmunology. 7 (2), e1393596 (2018).</li><li>Argyle, D., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Macrophage-Recruiting+Chemokines+as+a+Novel+Therapeutic+Strategy+to+Prevent+the+Progression+of+Solid+Tumors.">Targeting Macrophage-Recruiting Chemokines as a Novel Therapeutic Strategy to Prevent the Progression of Solid Tumors.</a> Frontiers in Immunology. 9, 2629 (2018).</li><li>An, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+cooperates+with+EGFRvIII+to+recruit+macrophages+in+glioblastoma.">EGFR cooperates with EGFRvIII to recruit macrophages in glioblastoma.</a> Cancer Research. , (2018).</li><li>Fan, Q. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+phosphorylates+tumor-derived+EGFRvIII+driving+STAT3/5+and+progression+in+glioblastoma.">EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma.</a> Cancer Cell. 24 (4), 438-449 (2013).</li><li>Jacquelot, N., Duong, C. P. M., Belz, G. T., Zitvogel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Chemokines+and+Chemokine+Receptors+in+Melanoma+and+Other+Cancers.">Targeting Chemokines and Chemokine Receptors in Melanoma and Other Cancers.</a> Frontiers in Immunology. 9, 2480 (2018).</li><li>Arimont, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Chemokine+Receptor-Ligand+Interactions.">Structural Analysis of Chemokine Receptor-Ligand Interactions.</a> Journal of Medicinal Chemistry. 60 (12), 4735-4779 (2017).</li><li>Turner, M. D., Nedjai, B., Hurst, T., Pennington, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+chemokines:+At+the+crossroads+of+cell+signalling+and+inflammatory+disease.">Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease.</a> Biochimica et Biophysica Acta. 1843 (11), 2563-2582 (2014).</li><li>Sokol, C. L., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+system+in+innate+immunity.">The chemokine system in innate immunity.</a> Cold Spring Harbor Perspectives in Biology. 7 (5), (2015).</li><li>Pathria, P., Louis, T. L., Varner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Tumor-Associated+Macrophages+in+Cancer.">Targeting Tumor-Associated Macrophages in Cancer.</a> Trends in Immunology. 40 (4), 310-327 (2019).</li><li>Mantovani, A., Marchesi, F., Malesci, A., Laghi, L., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-associated+macrophages+as+treatment+targets+in+oncology.">Tumour-associated macrophages as treatment targets in oncology.</a> Nature Reviews Clinical Oncology. 14 (7), 399-416 (2017).</li></ol>

In this protocol, there are several key steps: 1) selection of the Transwell insert. For the MV-4-11 cell line, 5 &#956;m Transwell inserts work well. However, for other cell lines such as the commonly used monocyte cell line THP-1, a different pore size might work better. 2) As different cell lines grow at different speeds, it is important to adjust the volume of the conditioned media according to cell numbers. For this purpose, cell-free media incubated under the same experimental conditions can be used to dilute the c...

Macrophages are immune cells with high phenotypic and functional heterogeneity1. They play important roles in the host defense systems, tissue repair, development and tumor progression1. TAMs are macrophages in the microenvironment of solid tumors. Certain TAMs can promote tumor growth through inhibiting T cell-mediated cytotoxic activity, modulating the tumor microenvironment (TME), promoting angiogenesis, invasion and metastasis2,3,4,5. TAMs are among the most abundant cell types in the TME and higher number of TAMs generally correlates with worse patient survival in many types of solid tumors6. The distinct genetic signatures of the tumor cells affect their ability to recruit macrophages. In GBM, an aggressive brain tumor with no cure, macrophages can represent up to a half of the tumor mass7. Co-amplification of the epidermal growth factor receptor (EGFR) and its truncation mutant EGFRvIII is frequently observed in GBM, which confers tumor growth advantages8. Cells co-expressing EGFR and EGFRvIII attract more macrophages compared to cells expressing EGFR or EGFRvIII singly7.
Chemokines are a family of small cytokines that play significant roles in regulating immune composition in the TME6,9. Human cells express more than 50 cytokines10. Immune infiltration in the tumors is largely realized by the interaction between cytokines and cytokine receptors11. Each type of immune cells expresses distinct chemokine receptors/chemokines and can be recruited by cells secreting specific chemokines/chemokine receptors12. Cancer cells can increase expression of certain chemokines to recruit immune cells such as TAMs, regulatory T cells and myeloid-derived suppressor cells (MDSCs)6. Blockade of specific chemokine secreted by the tumors can be a promising way in inhibiting infiltration of immune cells into the tumor mass.
Here, we describe a protocol that allows in vitro evaluation of tumor-macrophage interaction, using conditioned media from the tumor cells containing chemokines and macrophage cell lines.

<table>
	<tbody>
		<tr>
			<td>0.1 &#956;m filtration cup</td>
			<td>Thermo fisher</td>
			<td>566-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m filter unit</td>
			<td>Millipore</td>
			<td>SLHA033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Olympus plastics</td>
			<td>12-104</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL sterile centrifuge tubes</td>
			<td>Olympus plastics</td>
			<td>28-103</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipette tip</td>
			<td>ART molecular bioproducts</td>
			<td>2779-RI</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL aspirating pipet</td>
			<td>Falcon</td>
			<td>357558</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Millipore</td>
			<td>ECM507</td>
			<td>Part of ECM507, or can be purchased separately</td>
		</tr>
		<tr>
			<td>4x lysis buffer</td>
			<td>Millipore</td>
			<td>ECM507</td>
			<td>Part of ECM507, or can be purchased separately</td>
		</tr>
		<tr>
			<td>5 &#956;m Transwell insert</td>
			<td>Millipore</td>
			<td>ECM507</td>
			<td>Part of ECM507, or can be purchased separately</td>
		</tr>
		<tr>
			<td>75cm2 flask</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative cell technologies</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>CyQuant Dye</td>
			<td>Millipore</td>
			<td>ECM507</td>
			<td>Part of ECM507, or can be purchased separately</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Biorad</td>
			<td>1450021</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assay to study tumor-macrophage interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

The results are usually showed via bar graphs (example shown in Figure 1). Samples with high 480/520 values indicate that the conditioned media has high capacity to recruit macrophages. Depending on experimental need, additional controls can be included. For example, one can use neutralizing antibodies to treat the conditioned media to abolish the macrophage chemotaxis, and perform the same assay. One can also add extra chemokines (i.e., CCL2) to the conditio...

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Tumor-associated macrophages play important roles in the tumor microenvironment. Understand how different genetic mutations in the tumor cells affect macrophage recruitment is critical for designing personalized treatment for cancer patients. This assay provides an easy and robust way to assess the interaction between tumor cells and macrophages in vitro.This assay can be modified to assess the interactions between the tumor cells and other immune cells in vitro. To begin this procedure, warm up the serum-free stem cell medium by placing it into a water bath at 37 degrees Celsius for 20 minutes. Spray the tissue culture hood surface with 70%ethanol and wipe down the sprayed surface with paper towels.Next spray the medium and cell detachment solution bottles with 70%ethanol and wipe them down with paper towels. Transfer the cleaned bottles into the tissue culture hood. Retrieve the tumor cell and transfer it into the tissue culture hood.Aspirate the medium and wash the cells with 10 milliliters of PBS. Then add two milliliters of cell detachment solution to the flask. Set the flask down in the hood, checking every minute to see if the tumor cells have rounded up.Once the tumor cells round up, gently tap the side of the flask to help the cells detach. After this, pipette eight milliliters of serum-free cell medium into the flask and pipette up and down three times to mix. Transfer this cell suspension to a 15 milliliter centrifuge tube and centrifuge at 200 times g and at four degrees Celsius for five minutes.Aspirate the supernatant. Then wash the cells in 10 milliliters of PBS, making sure to pipette up and down three to five times to mix. Centrifuge the cells at 200 times g and at four degrees Celsius for five minutes.Aspirate the supernatant, and resuspend the cells in 10 milliliters of serum-free medium. Pipette up and down three to five times to mix. Mix 10 microliters of this cell suspension with 10 microliters of Trypan Blue solution.Next pipette 10 microliters of the Trypan Blue and cell mixture into a counting slide, and use an automatic cell counter to quantify the living cell number. Use serum-free cell medium to adjust the cell density to 2.5 times 10 to the fifth cells per milliliter. Then seed two milliliters of the cells into each well of a six-well culture plate.At the same time, prepare six wells with only two milliliters of serum-free stem cell medium. After this, incubate the six-well plates at 37 degrees Celsius with 5%carbon dioxide for 24 hours. First spray the tissue culture hood surface with 70%ethanol and wipe down the sprayed surface with paper towels.Next spray the medium and cell detachment solution bottles with 70%ethanol and wipe them down with paper towels. Transfer the cleaned bottles into the tissue culture hood. After this, retrieve the six-well plates from the incubator.Carefully pipette the conditioned media into 15 milliliter sterile centrifuge tubes, and place the tubes on ice. Add 0.5 milliliters of cell detachment solution into each well of the six-well plates, and check every minute if the tumor cells have rounded up. Once the tumor cells round up, gently tap the side of the plate to help detach the cells.Next pipette 2.5 milliliters of tumor cell maintenance medium into the well and pipette up and down three times to mix. Transfer the cells into a 15 milliliter sterile centrifuge tube. Mix 10 microliters of cells with 10 microliters of Trypan Blue solution, and transfer 10 microliters of this mixture into the counting slide.Use an automatic cell counter to quantify the number of living cells, and use the serum-free stem cell medium that was incubated at 37 degrees Celsius overnight to adjust the volume of the conditioned media according to the cell number. Filter the conditioned media through 0.45 micrometer filters and place the conditioned media on ice. To begin, warm up the IMDM medium in a water bath at 37 degrees Celsius for 20 minutes.Clean the tissue culture hood with 70%ethanol and clean the bottle of medium and transport it into the hood as previously described. Next transfer the flask of MV-4-11 cells into the hood. Pipette 10 milliliters of cells into a 15 milliliter centrifuge tube.Use Trypan Blue and an automatic cell counter to quantify the number of living cells as previously described. Then centrifuge the cells at 200 times g and at four degrees Celsius for five minutes. Aspirate the supernatant and wash the cells with 10 milliliters of PBS.Centrifuge again at 200 times g and at four degrees Celsius for five minutes. Aspirate the supernatant, and resuspend the cells in IMDM medium at a final cell density of one million cells per milliliter. First bring the necessary materials to room temperature.Add 250 microliters of the prepared MV-4-11 cells to each insert. Next add 400 microliters of either the conditioned medium or medium only to the lower chambers of the 24-well plate, making sure to add the samples in triplicate. Incubate at 37 degrees Celsius with 5%carbon dioxide for four hours.Then gently tap the insert on the inner wall of the same well and discard the insert. Gently pipette the cells in the wells up and down three times to mix. Transfer 225 microliters of this cell suspension into the wells of a black-walled 96-well plate suitable for fluorescent measurement.After this, dilute the CyQUANT dye with 4x lysis buffer at a ratio of one to 75. Vortex briefly and spin down the solution. Transfer 75 microliters of this solution to each well of the 96-well plate and incubate at room temperature for 15 minutes.Using a fluorescence plate reader, read the fluorescence and analyze the data as outlined in the text protocol. In this study, an in vitro assay is used to study tumor macrophage interaction. Samples with high fluorescence values indicate that the conditioned media has a high capacity to recruit macrophages.Depending on experimental need, additional controls can be included. For example, one can use neutralizing antibodies to treat the conditioned media to abolish the macrophage chemotaxis and perform the same way. One can also add extra chemokines to the conditioned media, which serve as a positive control.It's important to control cell number differences for this assay because different cell types can grow at different speeds. In vivo confirmation of the in vitro results are critical. One can inject tumor cells into mice and analyze the tumors with flow cytometry, immunostaining, and CyTOF to confirm the results.

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