The authors would like to acknowledge Jacqueline Rosenberg for proofreading the manuscript. This work was supported by the US Department of Health and Human Services/National Institutes of Health with a grant to YA DeClerck (grant 5R01 CA 129377) and the TJ Martell Foundation. MH Kubala is the recipient of a Research Career Development Fellowship award of The Saban Research Institute at Children's Hospital Los Angeles.

The protocol section that uses human monocytes obtained from healthy volunteers follows the guidelines of the Children&#39;s Hospital Los Angeles Human Research Ethics Committee and has been approved by the Institutional Review Board under the Human Material Protocol number: CCI 08-00208.
1. Preparation of Cancer Cell Lines with Tetracycline-regulated shRNA Expression
<ol>
	<li>Cloning of shRNA PAI-1 into Tet-pLKO-puro vector8,9
	<ol>
		<li>Design shRNA oligomers that contain AgeI and EcoRI cleavage site at the 5&#39; end, the sense sequence, a 6 nucleotide loop, and the antisense sequence. 
		NOTE: Typical oligonucleotides are designed as follows: Forward oligo: 5&#39; CCGG - 19 - 21 bp sense - CTCGAG - 19 - 21 bp antisense - TTTTT, Reverse oligo: 5&#39; AATTAAAAA- 19-21 bp sense - CTCGAG - 19 - 21 bp antisense 3&#39;. Detailed protocol for the oligomer design can be found in 8. In this study 3 shRNA sequences against PAI-1 were tested for the strongest silencing effect: shRNA 132, shRNA 2, shRNA 333, and scrambled33 are included in Table 1.</li>
		<li>Anneal the shRNA oligomers and scrambled shRNA oligomers.
		<ol>
			<li>Reconstitute oligonucleotides to 100 &#181;M in water and mix 1 &#181;L Forward oligonucleotide, 1 &#181;L Reverse oligonucleotide, and 8 &#181;L H2O.</li>
			<li>To anneal the oligonucleotides, mix the following: 1 &#181;L oligonucleotide mixture, 5 &#181;L buffer 10 mM Tris(hydroxymethyl)aminomethane (Tris) pH 7.5, 50 mM Sodium Chloride (NaCl), 10 mM Magnesium Chloride (MgCl2), 1 mM dithioerythritol (DTE), and 44 &#181;L H2O.</li>
			<li>Incubate the mixture in a polymerase chain reaction (PCR) thermal cycler at 95 &#176;C for 5 min, switch off the instrument, and wait until room temperature (RT) is reached.</li>
		</ol>
		</li>
		<li>Precipitate annealed oligonucleotides by adding 5 &#181;L 3 M Sodium Acetate pH 5.2 to 50 &#181;L annealed oligonucleotides.
		<ol>
			<li>Add 100 &#181;L cold 100% ethanol (EtOH) and incubate for 30 min at -80 &#176;C. Centrifuge in a benchtop centrifuge at maximum speed for 30 min at 4 &#176;C.</li>
			<li>Remove supernatant, add 500 &#181;L cold 70% EtOH, centrifuge for 30 min, remove supernatant, and dissolve pellet in 20 &#181;L H2O.</li>
			<li>Assess the concentration of annealed oligonucleotides by spectrophotometry, measuring the absorbance at 260 nm. Dilute oligonucleotides to the final concentration of 1 ng/&#181;L.</li>
		</ol>
		</li>
		<li>Prepare Luria Bertani (LB) medium and LB agar plates.
		<ol>
			<li>For LB medium, dissolve 10 g tryptone, 5 g yeast extract, 10 g NaCl in 1 L of water. Adjust pH of the medium to pH 7.0 using 1 N NaOH and autoclave.</li>
			<li>For LB agar plates, add 15 g/L of agar into LB medium. Autoclave and cool down to approximately 50 &#176;C. Add antibiotic and pour the plates.</li>
		</ol>
		</li>
		<li>Streak bacteria transduced with Tet-pLKO-puro vector (see the Table of Materials) on LB agar plate supplemented with 100 &#181;g/mL ampicillin and incubate overnight (O/N) at 37 &#176;C.</li>
		<li>Inoculate 100 mL LB medium supplemented with 100 &#181;g/mL ampicillin (LB/ampicillin) with 1 colony from the plate and grow O/N with shaking at 37 &#176;C.</li>
		<li>IsolateTet-pLKO-puro from 100 mL bacterial culture using a plasmid isolation kit.</li>
		<li>Prepare the restriction reaction for the Tet-pLKO-puro vector with EcoRI and AgeI restriction enzymes as follows: 1 &#181;L EcoRI, 1 &#181;L AgeI, 5 &#181;L 10x buffer, 1 &#181;L plasmid DNA (1 &#181;g), 42 &#181;L H2O. Incubate 1 h at 37 &#176;C. To obtain enough of cleaved vector, prepare up to 5 x 50 &#181;L reactions.</li>
		<li>Use 1% agarose gel to purify cleaved vector from the agarose gel using a DNA purification kit. To increase the yield of DNA, minimize the agarose to DNA ratio in the gel by using an agarose gel comb for large wells and by carefully removing most of the agarose from the band using a scalpel.</li>
		<li>Prepare the ligation reaction mix as follows: 1 ng annealed oligonucleotides, 50 ng cleaved vector, 2 &#181;L 10x ligase buffer, 1 &#181;L ligase, and water up to 20 &#181;L. Ligate the vector with annealed shRNA oligonucleotides at 16 &#176;C O/N.
		<ol>
			<li>Prepare in addition, a negative control reaction without oligonucleotides to account for uncleaved, partially cleaved, and self-ligated vector that will result in false positive colonies.</li>
		</ol>
		</li>
		<li>Using standard transformation techniques, transform ligation mixtures into chemically competent bacterial cells designed to prevent homologous recombination of lentiviral vectors containing direct repeats.</li>
		<li>Grow bacteria on plates with ampicillin O/N at 37 &#176;C. 
		NOTE: If growth of satellite colonies occurs, repeat the transformation and grow bacteria at 30 &#176;C to slow down the growth and thus prevent satellite colonies.</li>
		<li>To verify successful ligation, pick between 10 - 20 single colonies, inoculating 2 mL LB/ampicillin cultures and an ampicillin plate as a stock plate (to be used as a positive clone later). Grow liquid cultures with shaking O/N at 37 &#176;C. Incubate the plate O/N at 37 &#176;C.</li>
		<li>Seal the plate with parafilm and store at 6 &#176;C.</li>
		<li>Isolate plasmid DNA from liquid cultures using a plasmid isolation kit.</li>
		<li>Perform restriction analysis of plasmid DNA isolated from ampicillin-resistant colonies using XhoI enzyme. For each clone, prepare the following reaction mix: 0.5 &#181;L XhoI, 2.5 &#181;L 10x buffer, 1 &#181;L plasmid DNA (0.5 &#181;g), and 21 &#181;L H2O. Incubate for 1 h at 37 &#176;C. Analyze the result of the restriction reaction by running the reaction on a 1% agarose gel. 
		NOTE: Restriction analysis of the WT vector cleaved by XhoI will generate 3 fragments: 8,447, 1,800, and 200 bp. The stuffer sequence in Tet-pLKO-puro vector of 1,800 bp is not present in positive clones containing shRNA-PAI-1 and the XhoI digest will result in DNA fragments of the size: 8,447, 190, and 130 bp. As shown in Figure 1, the 2,000 bp fragment is not found in DNA isolated from ampicillin resistant colonies number: 3, 8, and 11. Depending on the design of oligonucleotides, the length and number of obtained fragments in DNA from positive colonies may vary.</li>
		<li>Verify the proper insertion of the shRNA sequences into the Tet-pLKO-puro vector by sequencing8,9.</li>
		<li>Inoculate 100 mL LB/Ampicillin culture with the colony containing the correct clone and grow O/N at 37 &#176;C.</li>
		<li>Isolate plasmid DNA using a plasmid isolation kit.</li>
	</ol>
	</li>
	<li>Generation of lentivirus particles.
	<ol>
		<li>Coat a 15 cm tissue culture dish with Poly-L-Lysine by covering the surface of the dish with sterile water solution of 0.01% Poly-L-Lysine and incubating at RT for 15 min. Remove the solution by aspiration and air-dry the dishes.</li>
		<li>Seed 5 x 106 Human Embryonic Kidney 293 cells (HEK293) cells in the Poly-L-Lysine-coated 15 cm dish, and culture in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) medium supplemented with 10% FBS and 1% Penicillin/Streptomycin mix (Pen/Strep) at 37 &#176;C, 5% CO2 until they reach 70% confluency.</li>
		<li>To produce viral particles, transfect HEK293 cells with 25 &#181;g pLKO-Tet-On-shRNA, 25 &#181;g psPAX, (packaging plasmid), and 5 &#181;g pMD2.G plasmids (envelope expressing plasmid) using transfection reagent. 
		NOTE: psPAX and pMD2.G plasmids are a gift from the Didier Trono lab (Ecole Polytechnique Federale de Lausanne, Switzerland).</li>
		<li>Induce HEK293 cells the next day by changing the medium to DMEM supplemented with 10 mM sodium butyrate and 20 mM HEPES pH 7.2, and culturing for 8 h.</li>
		<li>Wash the cells with phosphate-buffered saline (PBS) and add 25 mL of fresh DMEM medium containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.2. After incubation at 37 &#176;C for 48 h, carefully collect virus-containing medium by pipetting; avoid spills.</li>
		<li>Filter the collected medium through a 0.45 &#181;M syringe filter to remove HEK293 cells. The medium containing viral particles can be used immediately or stored at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Generation of stably transfected cell lines
	<ol>
		<li>Seed HCT116 colon cancer cells and MDA-MB-231 breast cancer cells at 50,000 cells/well in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% Pen/Strep in a 12-well plate. Incubate at 37 &#176;C at 5% CO2 until cells are 60 - 70% confluent.</li>
		<li>Wash cells with PBS and add 1 mL of virus-containing medium to cancer cells and incubate O/N at 37 &#176;C at 5% CO2. As a control, add 1 mL of fresh DMEM medium supplemented with 10% FBS and 1% Pen/Strep to 2 wells with cancer cells.</li>
		<li>Carefully remove virus-containing medium by aspiration. Wash the cells with PBS and change the medium to DMEM with 10% (v/v) tetracycline-free (Tet-free) FBS and 1% Pen/Strep.</li>
		<li>After 72 h, wash cells with PBS and change the medium to DMEM 10% Tet-free FBS 1% Pen/Strep, 1 &#181;g/mL puromycin for selection of virus-transfected cells.
		<ol>
			<li>Depending on the cell line used, adjust the puromycin concentration for optimal selection by testing the range of puromycin concentrations (0.1, 0.5, 1, 2, and 5 &#181;g/mL) in non-transduced cells and choosing the lowest concentration where no control cells survive after 3 days of culture.</li>
		</ol>
		</li>
		<li>Select virus-transduced cells by culturing cells in the presence of puromycin for 3 - 14 days. As controls, prepare two additional wells with non-transduced cells, one with medium containing puromycin and one without puromycin. Observe partial killing of the cells by puromycin in the well with virus-transduced cells compared to control wells. 
		NOTE: Non-transduced cells will not grow in the presence of puromycin.</li>
		<li>Verify the efficiency of the conditional KD in the puromycin-resistant HCT116 colon cancer and MDA-MB-231 breast cancer cells. 
		NOTE: The effective concentration of doxycycline will vary with the cell line used and must be titrated (typically from 100 ng/mL to 2 &#181;g/mL).
		<ol>
			<li>Determine the doxycycline concentration that is not toxic for the cells but effective in downregulating the expression of the protein of interest. In this protocol, 1 &#181;g/mL was successfully used in vitro.</li>
			<li>Culture the cells for 72 h in the presence and absence of 0.1, 1, and 2 &#181;g/mL doxycycline. Verify the level of expression of the downregulated protein (here, PAI-1) in the cell lysate by Western blot analysis. Compare HCT116 and MDA-MB-231 cells conditionally expressing shRNA to scrambled control cells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of conditioned medium
	<ol>
		<li>Seed 1 x 106 of cells stably transfected with doxycycline regulated pLKO-shRNA containing lentiviruses in 10 cm dishes.</li>
		<li>Grow cell lines in Roswell Park Memorial Institute (RPMI) medium with 10% Tet-free FBS and 1% Pen/Strep in the absence and presence of 1 &#181;g/mL doxycycline for 72 h at 37 &#176;C at 5% CO2, adding fresh doxycycline daily. 
		NOTE: RPMI medium is compatible with culture conditions of monocytes. Doxycycline is a light sensitive compound. Protect the cell cultures from light by covering with aluminum foil and avoid working under direct light exposure.</li>
		<li>Collect the medium by pipetting, filter through 0.45 &#181;m filter, and freeze at -80 &#176;C in aliquots.</li>
	</ol>
	</li>
</ol>
2. Isolation of Monocytes from Human Blood
<ol>
	<li>Isolate monocytes from human peripheral blood. 
	NOTE: Human primary monocytes are isolated from 7 mL of white blood cells concentrated in a leukocyte filter obtained from healthy platelet donors. Depending on the donor, one leukocyte filter produces between 1 x 109 and 2 x 109 of peripheral blood mononuclear cells (PBMCs), approximately 10% of which constitute monocytes.
	<ol>
		<li>Prepare the workstation in the laminar flow cabinet.</li>
		<li>Sterilize scissors and the laminar flow cabinet with ultraviolet light (UV) for 30 min.</li>
		<li>Spray the leukocyte filter with 70% EtOH and air-dry inside the laminar flow cabinet.</li>
		<li>Cut both ends of the leukocyte filter with scissors and empty the content of the filter into a 50 mL tube.</li>
		<li>Dilute to 90 mL by adding PBS containing 1% (v/v) FBS (PBS/FBS).</li>
		<li>Prepare 3 x 50 mL tubes with 15 mL density gradient solution. Layer slowly 30 mL of PBS/FBS diluted blood over the density gradient solution using a pipette controller set on gravitational flow to avoid mixing of the layers.</li>
		<li>Centrifuge in a swing rotor at 400 x g at RT without brakes for 25 min.</li>
		<li>Dispose the top layer and transfer the middle layer containing PBMCs to a fresh 50 mL tube.</li>
		<li>Add PBS/FBS up to 50 mL and centrifuge at 120 x g at RT for 10 min. Remove the supernatant containing platelets.Repeat this step two more times.</li>
		<li>Resuspend the pellet in 50 mL of PBS/FBS and count the PBMCs using a hemocytometer. Centrifuge at 120 x g at RT and resuspend the pellet in PBS/FBS containing 1 mM ethylenediaminetetraacetic acid (EDTA) (PBS/FBS/EDTA) to a final concentration of 5 x 107 cells/mL.</li>
		<li>Use a negative selection kit to enrich monocytes by depletion of non-monocytic cells. 
		NOTE: The negative selection kit uses a cocktail of antibodies (CD2, CD3, CD16, CD19, CD20, CD56, CD66b, CD123, and glycophorin A) against T cells, NK cells, neutrophils, B cells, granulocytes, and erythrocytes. The antibody complexes bind to the surface of these non-monocytic cells and to the dextran magnetic particles. When the tube is placed in a magnet, the beads adhere to the walls of the tube and remove the non-monocytic cells from the solution.
		<ol>
			<li>Add 50 &#181;L the antibody cocktail to 1 mL of 5 x 107 PBMCs/mL, mix with a pipette, and incubate for 10 min at RT. Add 50 &#181;L magnetic beads to the PBMCs with the antibody cocktail, mix with a pipette, and incubate for another 10 min at RT.</li>
			<li>Add PBS/FBS/EDTA up to 25 mL and place the PBMC mixture in a magnet that can accommodate a 50 mL tube. Incubate for 10 min at RT. Magnetic beads will adhere to the walls of the tube and sequester the non-monocytic cells from the solution.</li>
		</ol>
		</li>
		<li>Using a 25 mL pipette, remove the solution containing monocytes from the tube in the magnet. Be careful to not touch the sides of the tube with the pipette.</li>
		<li>Centrifuge the solution at 250 x g at RT, remove the supernatant, and resuspend the pellet containing monocytes in RPMI medium containing 10% Tet-free FBS and 1% Pen/Strep.</li>
	</ol>
	</li>
</ol>
3. Boyden Chamber Migration Assay
<ol>
	<li>Prepare 1% (w/v) bovine serum albumin (BSA) solution by dissolving 1 g of BSA in 100 mL of ultrapure water, and filter through a 0.2 &#181;m pore size to sterilize.</li>
	<li>Incubate the porous membrane inserts in 1% sterile BSA solution O/N for improved adherence of macrophages to the membrane. For monocytes/macrophages, use 5 - 8 &#181;m pore size inserts.
	<ol>
		<li>Fill a sterile tissue culture dish with 1% BSA solution and place the inserts in the dish. Apply 200 &#181;L 1% BSA solution inside the inserts.</li>
	</ol>
	</li>
	<li>Wash the inserts twice by placing them in a dish filled with sterile PBS and applying PBS inside the filter.</li>
	<li>Seed 40,000 HCT116 or MDA-MB-231 cells in 0.5 mL RPMI medium containing 10% Tet-free FBS and 1% Pen/Strep per well, in a 24-well plate, in triplicate. Alternatively, place 0.5 mL of previously generated conditioned medium in the well as a source of chemoattractant.</li>
	<li>On the next day, plate 8,000 monocytes in the prepared insert in 0.3 mL volume of the RPMI medium containing 10% Tet-free FBS and 1% Pen/Strep, in triplicate, and incubate at 37 &#176;C at 5% CO2.</li>
	<li>Collect the inserts after 48 h. 
	NOTE: The length of an experiment must be optimized depending on the specific conditions and cells used, by performing a time-course experiment where inserts are collected at different incubation times.
	<ol>
		<li>Shake off the excess medium and precisely swipe the inner surface with a cotton-tipped applicator to remove cells that did not migrate.</li>
		<li>Stain the outer side of the inserts using the Wright-Giemsa method.</li>
		<li>Carefully mount 3 inserts/glass slide in a high viscosity microscope immersion oil, making sure that the side of the filter with migrated macrophages faces up.</li>
	</ol>
	</li>
	<li>Image the slides using a light microscope with 20X&#160;objective. Take pictures of 9 fields/filter. Count migrated macrophages in 9 fields/filter.</li>
</ol>

<ol>
	<li>Ryding, A. D., Sharp, M. G., Mullins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+transgenic+technologies.">Conditional transgenic technologies.</a> J Endocrinol. 171 (1), 1-14 (2001).</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> Nat Rev Clin Oncol. , (2017).</li><li>Franklin, R. A., 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=The+cellular+and+molecular+origin+of+tumor-associated+macrophages.">The cellular and molecular origin of tumor-associated macrophages.</a> Science. 344 (6186), 921-925 (2014).</li><li>Ruffell, B., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+therapeutic+resistance+in+cancer.">Macrophages and therapeutic resistance in cancer.</a> Cancer Cell. 27 (4), 462-472 (2015).</li><li>Sharma, P., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+immune+checkpoint+therapy.">The future of immune checkpoint therapy.</a> Science. 348 (6230), 56-61 (2015).</li><li>Mizuguchi, H., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+adenovirus-mediated+tetracycline-controllable+expression+system.">Characteristics of adenovirus-mediated tetracycline-controllable expression system.</a> Biochim Biophys Acta. 1568 (1), 21-29 (2001).</li><li>Meyer-Ficca, M. L., 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=Comparative+analysis+of+inducible+expression+systems+in+transient+transfection+studies.">Comparative analysis of inducible expression systems in transient transfection studies.</a> Anal Biochem. 334 (1), 9-19 (2004).</li><li>Wiederschain, D., 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=Single-vector+inducible+lentiviral+RNAi+system+for+oncology+target+validation.">Single-vector inducible lentiviral RNAi system for oncology target validation.</a> Cell Cycle. 8 (3), 498-504 (2009).</li><li>Wee, 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=PTEN-deficient+cancers+depend+on+PIK3CB.">PTEN-deficient cancers depend on PIK3CB.</a> Proc Natl Acad Sci U S A. 105 (35), 13057-13062 (2008).</li><li>Chen, Y., Stamatoyannopoulos, G., Song, C. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+CXCR4+by+inducible+small+interfering+RNA+inhibits+breast+cancer+cell+invasion+in+vitro.">Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro.</a> Cancer Res. 63 (16), 4801-4804 (2003).</li><li>Wiznerowicz, M., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+suppression+of+cellular+genes:+lentivirus+vector-mediated+drug-inducible+RNA+interference.">Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.</a> J Virol. 77 (16), 8957-8961 (2003).</li><li>Solari, 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=MYCN-dependent+expression+of+sulfatase-2+regulates+neuroblastoma+cell+survival.">MYCN-dependent expression of sulfatase-2 regulates neuroblastoma cell survival.</a> Cancer Res. 74 (21), 5999-6009 (2014).</li><li>Liao, Y., Tang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+RNAi+system+and+its+application+in+novel+therapeutics.">Inducible RNAi system and its application in novel therapeutics.</a> Crit Rev Biotechnol. 36 (4), 630-638 (2016).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods Mol Biol. 294, 15-22 (2005).</li><li>Jungi, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+chemotaxis+by+a+reversible+Boyden+chamber+eliminating+cell+detachment.">Assay of chemotaxis by a reversible Boyden chamber eliminating cell detachment.</a> Int Arch Allergy Appl Immunol. 48 (3), 341-352 (1975).</li><li>Jung, H. 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=Monoclonal+antibodies+against+autocrine+motility+factor+suppress+gastric+cancer.">Monoclonal antibodies against autocrine motility factor suppress gastric cancer.</a> Oncol Lett. 13 (6), 4925-4932 (2017).</li><li>Park, 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=Effect+of+Sorbus+commixta+on+the+invasion+and+migration+of+human+hepatocellular+carcinoma+Hep3B+cells.">Effect of Sorbus commixta on the invasion and migration of human hepatocellular carcinoma Hep3B cells.</a> Int J Mol Med. , (2017).</li><li>Schildberger, A., Rossmanith, E., Eichhorn, T., Strassl, K., Weber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes,+peripheral+blood+mononuclear+cells,+and+THP-1+cells+exhibit+different+cytokine+expression+patterns+following+stimulation+with+lipopolysaccharide.">Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide.</a> Mediators Inflamm. 2013, 697972 (2013).</li><li>Heil, T. L., Volkmann, K. R., Wataha, J. C., Lockwood, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+peripheral+blood+monocytes+versus+THP-1+monocytes+for+in+vitro+biocompatibility+testing+of+dental+material+components.">Human peripheral blood monocytes versus THP-1 monocytes for in vitro biocompatibility testing of dental material components.</a> J Oral Rehabil. 29 (5), 401-407 (2002).</li><li>Tsuchiya, 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=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>de Almeida, M. C., Silva, A. C., Barral, A., Barral Netto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+human+peripheral+blood+monocyte+isolation.">A simple method for human peripheral blood monocyte isolation.</a> Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).</li><li>Flo, R. 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=Negative+selection+of+human+monocytes+using+magnetic+particles+covered+by+anti-lymphocyte+antibodies.">Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies.</a> J Immunol Methods. 137 (1), 89-94 (1991).</li><li>Grondahl-Hansen, J., 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=Plasminogen+activator+inhibitor+type+1+in+cytosolic+tumor+extracts+predicts+prognosis+in+low-risk+breast+cancer+patients.">Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients.</a> Clin Cancer Res. 3 (2), 233-239 (1997).</li><li>Borgfeldt, C., Hansson, S. R., Gustavsson, B., Masback, A., Casslen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiation+of+serous+ovarian+cancer+from+cystic+to+solid+tumors+is+associated+with+increased+expression+of+mRNA+for+urokinase+plasminogen+activator+(uPA),+its+receptor+(uPAR)+and+its+inhibitor+(PAI-1).">Dedifferentiation of serous ovarian cancer from cystic to solid tumors is associated with increased expression of mRNA for urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1).</a> Int J Cancer. 92 (4), 497-502 (2001).</li><li>Devy, L., 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=The+pro-+or+antiangiogenic+effect+of+plasminogen+activator+inhibitor+1+is+dose+dependent.">The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent.</a> FASEB J. 16 (2), 147-154 (2002).</li><li>Bajou, K., 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=Plasminogen+activator+inhibitor-1+protects+endothelial+cells+from+FasL-mediated+apoptosis.">Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis.</a> Cancer Cell. 14 (4), 324-334 (2008).</li><li>Xu, X., Wang, H., Wang, Z., Xiao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+promotes+inflammatory+process+induced+by+cigarette+smoke+extraction+or+lipopolysaccharides+in+alveolar+epithelial+cells.">Plasminogen activator inhibitor-1 promotes inflammatory process induced by cigarette smoke extraction or lipopolysaccharides in alveolar epithelial cells.</a> Exp Lung Res. 35 (9), 795-805 (2009).</li><li>Ji, Y., 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=Pharmacological+Targeting+of+Plasminogen+Activator+Inhibitor-1+Decreases+Vascular+Smooth+Muscle+Cell+Migration+and+Neointima+Formation.">Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.</a> Arterioscler Thromb Vasc Biol. 36 (11), 2167-2175 (2016).</li><li>Carmeliet, P., 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=Inhibitory+role+of+plasminogen+activator+inhibitor-1+in+arterial+wound+healing+and+neointima+formation:+a+gene+targeting+and+gene+transfer+study+in+mice.">Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice.</a> Circulation. 96 (9), 3180-3191 (1997).</li><li>Cao, C., 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=Endocytic+receptor+LRP+together+with+tPA+and+PAI-1+coordinates+Mac-1-dependent+macrophage+migration.">Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration.</a> EMBO J. 25 (9), 1860-1870 (2006).</li><li>Thapa, B., Koo, B. H., Kim, Y. H., Kwon, H. J., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+regulates+infiltration+of+macrophages+into+melanoma+via+phosphorylation+of+FAK-Tyr(9)(2)(5).">Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr(9)(2)(5).</a> Biochem Biophys Res Commun. 450 (4), 1696-1701 (2014).</li><li>Kortlever, R. M., Higgins, P. J., Bernards, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+is+a+critical+downstream+target+of+p53+in+the+induction+of+replicative+senescence.">Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.</a> Nat Cell Biol. 8 (8), 877-884 (2006).</li><li>Fang, H., Placencio, V. R., DeClerck, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protumorigenic+activity+of+plasminogen+activator+inhibitor-1+through+an+antiapoptotic+function.">Protumorigenic activity of plasminogen activator inhibitor-1 through an antiapoptotic function.</a> J Natl Cancer Inst. 104 (19), 1470-1484 (2012).</li><li>Bennett, S., Breit, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+in+the+isolation+and+culture+of+human+monocytes+that+are+of+particular+relevance+to+studies+of+HIV.">Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV.</a> J Leukoc Biol. 56 (3), 236-240 (1994).</li><li>Geissmann, F., 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=Development+of+monocytes,+macrophages,+and+dendritic+cells.">Development of monocytes, macrophages, and dendritic cells.</a> Science. 327 (5966), 656-661 (2010).</li><li>Bauer, M., Goldstein, M., Heylmann, D., Kaina, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocytes+undergo+excessive+apoptosis+following+temozolomide+activating+the+ATM/ATR+pathway+while+dendritic+cells+and+macrophages+are+resistant.">Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.</a> PLoS One. 7 (6), e39956 (2012).</li></ol>

The authors have nothing to disclose.

Composed of a variety of cell types, the TME is crucial for the development of cancer. To ascertain optimal growth conditions, cancer cells attract monocytes through secretion of chemotactic factors. Here we present a protocol for studying the mechanism of monocyte recruitment by tumor cells in vitro. For this purpose, a combination of inducible gene expression system, purification of primary human monocytes, and as an example of a migration assay, the Boyden chamber assay, is used.
T...

Tumor associated macrophages (TAMs) contribute to tumor development by promoting tumor growth, metastasis, and regulating the immune response2. Cancer cells recruit inflammatory monocytes, which infiltrate tumor and differentiate into pro-tumorigenic TAMs3. Infiltration of the tumor with TAMs correlates with poor clinical outcome and has been linked to the immunosuppressive role of macrophages4,5. However, the mechanisms of the recruitment of macrophages to the tumor are not well explored and a better understanding of the involved pathways is crucial for further advancement of the field and promising therapies. One of the challenges in studying interactions between tumor cells and normal cells in the tumor microenvironment (TME) is the complexity of the mechanisms and cells involved, requiring in vitro approaches that allow dissection of the crosstalk. Here we present a versatile methodology that can be applied to study the paracrine effect of a cancer cell-derived, secreted protein on migration of other cell types like macrophages, in vitro. Using a system where the expression of the shRNA against a cancer cell-derived protein involved in the recruitment of monocytes is under the control of the tetracycline inducible promoter, the paracrine effect of the secreted protein on monocytes is quantitated. In this protocol, cloning of shRNA sequences into the tetracycline regulated vector is presented followed by generation of stable cancer cell lines. Further, the purification of primary human monocytes and a Boyden chamber assay are used to analyze the paracrine effect of a cancer-cell derived protein on migration of monocytes.
Downregulation of protein coding genes is commonly applied with siRNA and shRNA techniques, though not without limitations in its use. The long-term knock-down (KD) of genes may elicit secondary adaptive responses of cells that interfere with experimental results. Lack of temporal control over gene expression makes it challenging to study the dynamic role of a protein over time or the role of a protein crucial for cell survival. This issue is especially important in in vivo settings, where the role of a protein in tumor development and progression might require downregulation of the expression of the protein of interest only after tumor is established. Conditional KD has the advantage of preventing a too early lethal effect of the KD on cells, and to enable analysis of the role of the protein within different stages of tumor growth, while unconditional KD could result in lack of tumor development.
A number of conditional KD systems have been developed to address the limitations of stable KD. The conditional gene expression systems include Ecdysone-inducible overexpression systems, the Cytochrome P-450 induction system1, and tetracycline regulated gene expression systems. The tetracycline-regulated gene expression systems allow control over expression of the shRNA upon addition of the antibiotic tetracycline (or its more stable analogue - doxycycline). In Tet-ON systems, the expression of shRNA is induced in the presence of tetracycline/doxycycline resulting in a gene expression KD, while in Tet-OFF systems, the expression of shRNA is suppressed in the presence of tetracycline resulting in gene expression. A drawback of tetracycline-inducible system is previously reported low levels of expression of shRNA in the absence of doxycycline - so-called leakiness6,7. In the Tet-ON system described here, upon administration of tetracycline, the binding of constitutively expressed tetracycline repressor (TetR) protein to the Tet-responsive element (TRE) sequence within the H1 promoter region of the shRNA of interest is suppressed. This results in expression of shRNA and inhibition of translation of the protein of interest in a tetracycline-dependent manner8,9.
Other available Tet-ON systems include simultaneous knock-in of the TetO sequence between TATA box and proximal sequence element (PSE) and between TATA box and transcription start site developed by Chan et al.10 This system requires less than toxic doses of tetracycline to regulate shRNA expression, however, under a non-induced state, low levels of shRNA are expressed. The Kr&#252;ppel-associated box (KRAB) based Tet-ON system11 includes KRAB, a zinc finger protein, which subjects genes located within a 3 kb range of the KRAB binding site to transcriptional suppression. The chimeric protein tTRKRAB can bind to TetO, and due to the large-range of DNA controllable capacity, TetO do not need to be limited between the transcription start site and the promoter and have low impact on the activity of the promoter. Under the non-induced state, this controllable RNA interference system was reported to show a lower level of leaked expression of shRNA11,12; however, it requires a sequential, two-vector cloning approach. In comparison to previously developed conditional KD systems such as the Ecdysone-inducible overexpression system or Cytochrome P-450 induction system, the tetracycline-regulated system has the advantage of its robustness and reversibility, and therefore is the most routinely used system13. The system used in this protocol has the advantage over the dual TetO knock-in system and KRAB Tet-ON system as it requires straight forward, single vector cloning, allowing quick generation of multiple clones, and it exhibits very low levels of leakiness in the absence of doxycycline.
TME is critical for the development of cancer. To facilitate tumor growth, cancer cells recruit inflammatory monocytes by secreting chemotactic proteins. Recruited monocytes infiltrate the tumor and differentiate into pro-tumorigenic TAMs that contribute to tumor growth and metastasis. In vitro studies of the immune cell recruitment utilize migration assays, with Boyden chamber assay being widely used14,15,16,17. In this assay, the chemoattractant protein source, for example, cancer cells, or a purified protein, is placed in the bottom chamber. Immune cells are placed in the upper chamber separated with porous membrane from the bottom compartment. Cells migrating toward the increasing gradient of chemoattractant, and those found on the lower side of the membrane are stained and counted under the microscope. Here we test the chemoattractant function of Plasminogen Activator Inhibitor 1 (PAI-1) on monocytes by generating stable, inducible cancer cell lines where expression of PAI-1 is regulated by doxycycline addition. We use human primary monocytes in the Boyden chamber migration assay to assess the role of PAI-1 in monocyte migration. Differences between human primary monocytes and widely used monocytic cell lines like THP1 have been reported and include different cytokine expression patterns18; for example, 5 - 10 fold increase in TNF-&#945; expression levels by THP1 cells compared to human monocytes19. THP1 cells are derived from human leukemia monocytic cells, are easy to maintain, and proliferate with an average doubling time of 19 - 50 h20. On the contrary, human monocytes are characterized by a short lifespan in the absence of growth factors. Since monocytes are purified from blood of donors, a fair amount of variability occurs among the individuals and depending on the purification method, contamination with other cell types may occur. Nevertheless, primary monocytes are relevant and it has been recommended to use or confirm the results obtained with the monocytic cell lines using primary monocytes in biological research19. Here we describe a protocol for purification of human primary monocytes from peripheral blood. Alternative methods of monocyte purification include density gradient and adhesion protocols and two step procedure with single gradients of Ficoll-Hypaque followed by a Percoll gradient21. The purity of the monocyte population obtained by those methods ranges between 70 - 90%. The method described here uses a density gradient followed by a negative immune selection22 and enables the purification of human monocytes without direct contact with antibodies, thereby avoiding their accidental activation and resulting in a &#62;95% pure population of monocytes.
The protocol presented here is used for setting up a functional Tet-ON system for gene expression KD in human cancer cell lines to study the chemoattractant effect of the cancer-derived secreted protein PAI-1 on monocytes. PAI-1 is overexpressed by a variety of tumors and its expression paradoxically correlates with poor clinical outcome23,24. The pro-tumorigenic role of PAI-1 is a result of its pro-angiogenic and anti-apoptotic functions25,26. PAI-1 has been shown to contribute to inflammation by promoting the recruitment of macrophages to the site of inflammation27. PAI-1 was shown to promote smooth muscle cellmigration28,29 and to participate in the Mac-1 dependent macrophage migration30. PAI-1 overexpression has been also shown to significantly enhance the recruitment of Raw 264.7 macrophages into B16F10 melanoma tumors31. However, the role of PAI-1 in TAM migration has not been investigated in detail. We use the described protocol to answer the question of whether PAI-1 attracts monocytes to cancer cells. This methodology allows dissection of the crosstalk between tumor and TME by silencing the secreted protein in cancer cells and analyzing the components of the TME.

<table><tbody><tr><td>Tet-pLKO-puro lentiviral vector</td><td>Addgene</td><td>21915</td><td></td></tr><tr><td>FBS (Tetracycline-free)</td><td>Omega Scientific</td><td>FB-15</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>10771-500ML</td><td></td></tr><tr><td>EasySep Human Monocyte Enrichment Kit</td><td>StemCell</td><td>19059</td><td></td></tr><tr><td>EasySep Magnet</td><td>StemCell</td><td>18002</td><td></td></tr><tr><td>EcoRI HF</td><td>NEB</td><td>R3101S</td><td></td></tr><tr><td>AgeI HF</td><td>NEB</td><td>R3552S</td><td></td></tr><tr><td>Cut Smart buffer</td><td>NEB</td><td>B7200S</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>PROMEGA Wizard SV Gel and PCR Cleanup System</td><td>PROMEGA</td><td>A9281</td><td></td></tr><tr><td>psPAX vector</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pMD2.G vector</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>One Shot Stbl3 Chemically Competent&amp;nbsp;E. coli</td><td>Thermo Fisher Scientific</td><td>C737303</td><td></td></tr><tr><td>Hema 3 stain set for Wright-Giemsa stain</td><td>Protocol</td><td>123-869</td><td></td></tr><tr><td>HEK293 cells</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>XhoI restriction enzyme</td><td>NEB</td><td>R0146S</td><td></td></tr><tr><td>Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Ligase buffer</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>EDTA 0.5 M solution</td><td>Thermo Fisher Scientific</td><td>R1021</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>The Big Easy&quot; EasySep Magnet</td><td>Stem Cell</td><td>18001</td><td></td></tr><tr><td>0.1% Poly-L-Lysine solution</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S7670</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma-Aldrich</td><td>B5887</td><td></td></tr><tr><td>0.45 &amp;mu;M syringe filters</td><td>VWR International</td><td>28145-479</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881-500g</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>Sodium Chloride(NaCl)</td><td>Sigma-Aldrich</td><td>S7653-5 kg</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>dithioerythritol (DTE)</td><td>Sigma-Aldrich</td><td>B8255-5g</td><td></td></tr><tr><td>ethanol (EtOH)</td><td>Sigma-Aldrich</td><td>792780</td><td></td></tr><tr><td>tryptone</td><td>Amresco</td><td>J859-500g</td><td></td></tr><tr><td>yeast extract</td><td>Fluka</td><td>70161-500g</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>ampicillin</td><td>Sigma-Aldrich</td><td>A9393-5g</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>puromycin</td><td>Sigma-Aldrich</td><td>P9620</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-25g</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI)</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>0.5 M EDTA pH 8.0</td><td>Thermo Fisher Scientific</td><td>R1021</td><td></td></tr><tr><td>Falcon Permeable Support for 24 Well Plate with 8.0&amp;mu;m Transparent PET Membrane</td><td>Becton Dickinson</td><td>353097</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Cotton-Tipped Applicator&amp;nbsp;</td><td>Medline</td><td>MDS202000</td><td></td></tr><tr><td>Protocol Hema 3 STAT pack&amp;nbsp;</td><td>Fisher Scientific Company</td><td>123-869</td><td></td></tr><tr><td>Resolve Immersion Oil, High Viscosity</td><td>Richard Allan Scientific</td><td>M4004</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr></tbody></table>

conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

Three shRNA sequences were tested for the most efficient KD of PAI-1. For this, shRNA sequences against PAI-1 and scrambled (Table 1) were cloned into Tet-pLKO-puro expression vector following the protocol described above. The HT-1080 fibrosarcoma cell line was stably transfected with generated constructs and the cells were treated with doxycycline for 3 days. The expression of PAI-1 was verified by Western blotting (Figure 2A</strong...

Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

conditional-knockdown-gene-expression-cancer-cell-lines-to-study

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

12.3K Views.  University of Southern California. This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment. 

Video: Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

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 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.

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 ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Immunology and Infection

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

Developmental Biology

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...