Name: in vitro assay to study tumor-macrophage interaction
Uploaded: 2019-08-01T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Assay to Study Tumor-macrophage Interaction at JoVE.com

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...

Watch this Scientific Journal Video about In Vitro Assay to Study Tumor-macrophage Interaction at JoVE.com

In Vitro Assay to Study Tumor-macrophage Interaction

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

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

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...