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/F12) and 5 mL of 100x Penicillin/Streptomycin (Pen/Strep). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 &#956;m filtration cup. Mark the bottle and store at 4 &#176;C. 
		NOTE: The medium is good to use for up to a month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the MV-4-11 culture medium: Add 50 mL of fetal bovine serum (FBS) to 500 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 &#956;m filtration cup. Mark the bottle and store at 4 &#176;C. 
	NOTE: The medium is good to use for up to a month at 4 &#176;C.</li>
	<li>Prepare the tumor cell maintenance medium: Add 50 mL of FBS to 500 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 &#956;m filtration cup. Mark the bottle and store at 4 &#176;C. 
	NOTE: The medium is good to use for up to a month at 4 &#176;C.</li>
</ol>
2. Cell Preparation
Day 1:
<ol>
	<li>Thawing tumor cells</li>
	<li>Warm up the tumor cell maintenance medium (as described in step 1.3) at a 37 &#176;C water bath for 20 min.</li>
	<li>Take the frozen U87, U87:EGFR, U87:EGFRvIII and U87:EGFR+EGFRvIII cells from the liquid nitrogen tank. Thaw the cells at the 37 &#176;C water bath for 2 min. 
	CAUTION: Be careful when taking the cells from the liquid nitrogen tank. Wear gloves with thermal protection and safety goggles as needed.</li>
	<li>Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels.</li>
	<li>Spray the cryogenic vials containing cells and the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.</li>
	<li>Pipette 5 mL of tumor cell maintenance medium into a 15 mL sterile centrifuge tube. Invert the tube containing tumor cells 4-6 times to mix. Pipette all the cells into the 15 mL centrifuge tube. Invert the centrifuge tube 4-6 times to mix.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
	<li>Wash the cells with 10 mL of phosphate buffered saline (PBS). Centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
	<li>Resuspend the cells in 12 mL of tumor cell maintenance medium (as described in step 1.3). Pipette up and down 3-5 times to mix. Transfer the cells into a T75 cell culture flask.</li>
	<li>Grow the cells in a 37 &#176;C incubator with 5% CO2. Put the tumor cell maintenance medium back to the 4 &#176;C fridge.</li>
</ol>
Day 2:
<ol start="2">
	<li>Check the cells under the microscope. Change the medium if needed. Cells should grow in a monolayer in culture.</li>
</ol>
Day 3:
<ol start="3">
	<li>Thawing MV-4-11 cells
	<ol>
		<li>Warm up the MV-4-11 culture medium (as described in step 1.2) at a 37 &#176;C water bath for 20 min. 
		NOTE: MV-4-11 can be changed to primary macrophages or other macrophage cell lines, depending on the need of the specific study.</li>
		<li>Take the frozen MV-4-11 cells from the liquid nitrogen tank. Thaw the cells at a 37 &#176;C water bath for 2 min. 
		CAUTION: Be careful when taking the cells from the liquid nitrogen tank. Wear gloves with thermal protection and safety goggles as needed.</li>
		<li>Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels.</li>
		<li>Spray the cryogenic vials containing cells and the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.</li>
		<li>Pipette 5 mL of MV-4-11 culture medium (as described in step 1.2) into a 15 mL sterile centrifuge tube. Invert the tube containing tumor cells 4-6 times to mix. Pipette all the cells into the 15 mL centrifuge tube. Invert the centrifuge tube 4-6 times to mix.</li>
		<li>Centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
		<li>Wash the cells in 10 mL of PBS and centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
		<li>Resuspend the cells in 12 mL of MV-4-11 culture medium (as described in step 1.2). Gently pipette up and down 3-5 times to mix. Transfer the cells into a T75 cell culture flask.</li>
		<li>Grow the cells in a 37 &#176;C incubator with 5% CO2.</li>
	</ol>
	</li>
	<li>Splitting tumor cells
	<ol>
		<li>Warm up the serum-free stem cell medium (as described in step 1.1) at a 37 &#176;C water bath for 20 min.</li>
		<li>Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the medium and cell detachment solution bottles with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood. 
		NOTE: Do not pre-warm the accutase cell detachment solution.</li>
		<li>Transfer the tumor cell culture from the incubator into the tissue culture hood. Aspirate the medium, add 2 mL of cell detachment solution into the flask. 
		NOTE: Rinsing with PBS before adding accutase is optional here.</li>
		<li>Set the flask in the hood. Check if the tumor cells round up every minute. Once the tumor cells round up, gently tap the side of the flask to help the cells detach from the flask.</li>
		<li>Pipette 8 mL of serum-free stem cell medium into the flask, pipette up and down 3 times to mix, and transfer the cells into a 15 mL sterile centrifuge tube. Centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
		<li>Wash the cells in 10 mL of PBS. Pipette up and down 3-5 times to mix. Centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant. 
		NOTE: This step is optional.</li>
		<li>Resuspend the cells in 10 mL of serum-free stem cell medium (as described in step 1.1). Pipette up and down 3-5 times to mix. Take 10 &#956;L of the cells and mix with 10 &#956;L of Trypan Blue solution. Pipette 10 &#956;L of the mixture into the counting slide, quantify living cell number using an automatic cell counter.</li>
		<li>Adjust the cell density to 2.5 x 105 cells/mL with serum-free stem cell medium (as described in step 1.1), and seed 2 mL of the cells into each well of a 6-well cell-culture plate. For each type of cells, seed 3 wells (triplicate sample). At the same time, prepare 6 wells with 2 mL of serum-free stem cell medium only (no cells). 
		NOTE: These samples will be used: 1) as negative controls and 2) for adjusting conditioned medium volume based on cell numbers.</li>
		<li>Put the 6-well plates in a 37 &#176;C incubator with 5% CO2. Allow the cells to grow for 24 h.</li>
	</ol>
	</li>
</ol>
3. In Vitro Macrophage Attraction Assay
<ol>
	<li>Preparation of conditioned media
	<ol>
		<li>Warm up the tumor cell maintenance medium (as described in step 1.3) at a 37 &#176;C water bath for 20 min.</li>
		<li>Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the bottles of the medium and the cell detachment solution with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.</li>
		<li>Take the 6-well plates out from the incubator. Carefully pipette the conditioned media into 15 mL sterile centrifuge tubes. Put the tubes on ice. Pipette the media in the &#8220;media only&#8221; well into a separate 15 mL sterile centrifuge tube. 
		NOTE: Before this step, make sure to check under the microscope to see if the cells attach. If not, one can use coated plates at step 2.4.8 to allow better attachment or include the floating cells in the counting step.</li>
		<li>Add 0.5 mL of cell detachment solution into each well of the 6-well plate. Check if the tumor cells round up every minute. Once the tumor cells round up, gently tap the side of the plate to help the cells detach.</li>
		<li>Pipette 2.5 mL of tumor cell maintenance medium into the well, pipette up and down 3 times to mix and transfer the cells into a 15 mL sterile centrifuge tube. Take 10 &#956;L of the cells and mix with 10 &#956;L of Trypan Blue solution. Pipette 10 &#956;L of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter.</li>
		<li>Adjust the volume of the conditioned media according to cell number using the serum free stem cell medium (37 &#176;C incubation overnight). For example, if Well A has 3x as many live cells as the well with the least number of cells, dilute its conditioned media 1:3. This step is used to control difference caused by cell proliferation rate. 
		NOTE: The reason to use serum free stem cell medium with 37 &#176;C incubation overnight is to control the possible change in the media with prolonged 37 &#176;C exposure.</li>
		<li>Filter the conditioned media through 0.45 &#956;m filters. Put the conditioned media on ice. 
		NOTE: This step removes cells and cell debris in the conditioned media.</li>
	</ol>
	</li>
	<li>Preparation of MV-4-11 cells
	<ol>
		<li>Warm up the IMDM medium (without FBS) at a 37 &#176;C water bath for 20 min.</li>
		<li>Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Spray the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.</li>
		<li>Take the flask of MV-4-11 cells into the tissue culture hood. Pipette 10 mL of cells into a 15 mL sterile centrifuge tube. Take 10 &#956;L of cells and mix with 10 &#956;L of Trypan Blue solution. Pipette 10 &#956;L of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter. Centrifuge the cells at 200 x g for 5min at 4 &#176;C. Aspirate the supernatant.</li>
		<li>Wash the cells with 10 mL of PBS and centrifuge the cells at 200 x g for 5 min at 4 &#176;C. Aspirate the supernatant.</li>
		<li>Resuspend the cells in IMDM medium (without FBS) for a final cell density of 1x106 cells/mL. 
		NOTE: The cell number of macrophages used here can be adjusted.</li>
	</ol>
	</li>
	<li>Transwell assay 
	NOTE: For this step, use a cell migration assay kit or purchase the reagents and insert separately. For MV-4-11 cells, choose Transwell inserts with 5 &#956;m pore size. To detect macrophage migration, directly quantify the number of macrophages in the lower chamber or use colorimetric/fluorimetric methods. Here, we demonstrate the assay using the colorimetric method.
	<ol>
		<li>Bring the 24-well plate, the insert and the reagents to room temperature.</li>
		<li>Add 250 &#956;L of MV-4-11 cells prepared above into each insert.</li>
		<li>Add 400 &#956;L of conditioned medium/medium only (with overnight incubation at 37 &#176;C, these samples serve as the negative control) to the lower chambers of the 24-well plate. For each tumor line or control, use triplicate samples. Avoid bubbles between the insert and the conditioned medium. Bubbles will inhibit macrophages from migrating to the bottom wells.</li>
		<li>Incubate the plates in a 37 &#176;C incubator with 5% CO2 for 4 h. 
		NOTE: The incubation time can be adjusted. Cells migrating into the lower chambers can be seen under the microscope.</li>
		<li>Gently tap the insert on the inner wall of the same well and discard the insert. As MV-4-11 cells grow in suspension, cell detachment solution or trypsin treatment is not needed here.</li>
		<li>Gently pipette the cells in the wells up and down 3 times to mix. Transfer 225 &#956;L of cell suspension to a black-walled 96-well plate suitable for fluorescent measurement.</li>
		<li>Dilute the CyQuant dye 1:75 with 4x lysis buffer. Vortex briefly and spin down the solution. Add 75 &#956;L of solution to each well of the 96-well plate. Incubate 15 min at room temperature.</li>
		<li>Read fluorescence using the 480/520 nm filter set with a fluorescence plate reader.</li>
		<li>Analyze data by subtracting the blank and normalizing to the control tumor cell line.</li>
	</ol>
	</li>
</ol>

<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>

William A. Weiss is a co-founder of StemSynergy Therapeutics. Zhenyi An has nothing to disclose.

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 ...

in-vitro-assay-to-study-tumor-macrophage-interaction

Research

JoVE Journal

Cancer Research

7.4K Views.  University of California, San Francisco. This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Video: In Vitro Assay to Study Tumor-macrophage Interaction

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 understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

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

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

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

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

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

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

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

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

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

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 therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

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

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

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

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

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

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

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

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

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

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive 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 ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...