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>

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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 border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tet-pLKO-puro lentiviral vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">21915</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS (Tetracycline-free)</td>
			<td style="width:177px;">Omega Scientific</td>
			<td style="width:119px;">FB-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque</td>
			<td style="width:177px;">Sigma</td>
			<td>10771-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human Monocyte Enrichment Kit</td>
			<td style="width:177px;">StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Magnet</td>
			<td style="width:177px;">StemCell</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgeI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3552S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cut Smart buffer</td>
			<td style="width:177px;">NEB</td>
			<td>B7200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:177px;">Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROMEGA Wizard SV Gel and PCR Cleanup System</td>
			<td style="width:177px;">PROMEGA</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:405px;">One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hema 3 stain set for Wright-Giemsa stain</td>
			<td style="width:177px;">Protocol</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293 cells</td>
			<td style="width:177px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XhoI restriction enzyme</td>
			<td style="width:177px;">NEB</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase buffer</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EDTA 0.5 M solution</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Lipofectamine 2000</td>
			<td style="width:177px;">Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">The Big Easy&#34; EasySep Magnet</td>
			<td style="width:177px;">Stem Cell</td>
			<td style="width:119px;">18001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.1% Poly-L-Lysine solution</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Acetate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium butyrate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;M syringe filters</td>
			<td style="width:177px;">VWR International</td>
			<td>28145-479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride(NaCl)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653-5 kg</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dithioerythritol (DTE)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B8255-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol (EtOH)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">792780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tryptone</td>
			<td style="width:177px;">Amresco</td>
			<td style="width:119px;">J859-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td style="width:177px;">Fluka</td>
			<td style="width:119px;">70161-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:177px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ampicillin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline hyclate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-25g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.5 M EDTA pH 8.0</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Falcon Permeable Support for 24 Well Plate with 8.0&#956;m Transparent PET Membrane</td>
			<td style="width:177px;">Becton Dickinson</td>
			<td style="width:119px;">353097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Serum Albumin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton-Tipped Applicator&#160;</td>
			<td style="width:177px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol Hema 3 STAT pack&#160;</td>
			<td style="width:177px;">Fisher Scientific Company</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resolve Immersion Oil, High Viscosity</td>
			<td style="width:177px;">Richard Allan Scientific</td>
			<td style="width:119px;">M4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Penicillin Streptomycin</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">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

Research

JoVE Journal

Cancer Research

12.2K 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

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility in analyzing differential expression of groups of genes in tumor RNA-seq in a way that may be helpful for advancing precision oncology. However, CorEx produces many factors that can be challenging to analyze and connect to existing understanding. To facilitate such connections, we have built a website, CorExplorer, that allows users to interactively explore the data and answer common questions related to its analysis. We trained CorEx on RNA-seq gene expression data for four tumor types: ovarian, lung, melanoma, and colorectal. We then incorporated corresponding survival, protein-protein interactions, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, and heatmaps into the website for association with the factor graph visualization. Here we employ example protocols to illustrate use of the database for comprehending the significance of the learned tumor factors in the context of this external data.

We introduce the CorExplorer web portal, an resource for exploration of tumor RNA sequencing factors found by the machine learning algorithm CorEx (Correlation Explanation), and show how factors can be analyzed relative to survival, database annotations, protein-protein interactions, and one another to gain insight into tumor biology and therapeutic interventions.

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

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

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

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